Neuroscience Matters by UofTHMB

Neuroscience Matters 10 June 2013

Vol. 1 | No. 1 | Pages 1 - 62

Aging & Neuroplasticity

Restoring Vision for the Blind

Imaging the Whole-Brain

Flipping the Switch and Unlocking the Brainâ&#x20AC;&#x2122;s Full Potential

A Revolution in Neuroscience Research through Retinal Prosthetic Strategy

With Single Neuron Resolution to Identify Novel Functional Networks

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at the University of Toronto ÂŠ 2013

editorin-chief:

Dr. William Ju

Letter Dear Readers, the great fortune of working with some of the brightest young minds from the Iinhave our Neurosciences program at the University of Toronto. Prior to their I ask my students to pick a topic or published research that they Editor: graduation believe will have a significant impact within the next 10 years in the field of

neuroscience. The 2013 class has chosen a very diverse but fascinating group of papers which are highlighted in the current issue of Neuroscience Matters. While only time will tell how accurate the students’ topics have been, I hope that you will enjoy their insights into some of the most interesting aspects of our discipline. Signed, Bill

Neuroscience Matters design & layout editor:

Jenise Chen

Jenise Chen is in a Chemistry and Human Biology: Health and Disease double major and is thus stuck with drawing molecules and formulas throughout her undergraduate career. In order to show off her creative and artistic talents, she has involved herself in many clubs and groups, including the Human Biology Students’ Union as the Webmaster and the Stethoscope magazine as the Layout Designer and Editor. Jenise is proud to have been given this amazing opportunity to take on designing and editing the journal created by Dr. Ju. She had a wonderful time creating the journal and hopes all the readers can enjoy both content and design aesthetics! Happy reading, everyone!

contributors:

HMB420 Class of 2013

Thanks to the following people for contributing their article to this journal! Rashan Amin Ahmed Aslam Ashkan Azimi Amy Chow Daniel. A Dalessandro Alexander Di Giacomo Milko Dvekar Farhiya Elmi Mu Hsuan Ho Nariman Hossein-Javaheri Amaris Hui Hunaid Husain Farhana Islam Sallini Kalachandran

Helen (Bomin) Kim Ekaterina Kouzmina Roxanne Leung Jing Lu Amirah Momen Trevor Morey Marzia Niamah-Hussain Alexa Quach Jelum Raval Leora Sazant Joyce Tang Thomas Wasuita Pavel Yarmak

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Coupling of Excitation and Inhibition in the Striatum Orest Kayder1,2 Human Biology Department, The University of Toronto. Toronto, Ontario CA. 2 Division of Cell and Molecular Biology, Krembil Neuroscience Center, Toronto Western Hospital. Toronto, Ontario CA. 1

Dopaminergic projections from the ventral tegmental area (VTA) and the substantia nigra pars compacta (SNc) are crucial in shaping the striatal output serving important modulatory function for various motivated behaviors. Previous works have suggested the importance of both dopamine (DA) and glutamate co-released from some of the midbrain dopamine neurons1. However, Tritsch et al.2 by using the optogenetic stimulation of the light-activated channel Chennelrhodopsin-2 (ChR2) expressed specifically in SNc neurons report that these populations of dopaminergic subsystems are capable of inducing a short-lasting inhibition of the striatal output through the release of γ-aminobutyric acid (GABA). Remarkably, it has been found that the release of the GABA was independent of VGAT, a vesicular transporter which had been previously shown to mediate the transport of GABA into the synaptic vesicles. Instead, the short-lasting inhibition of the striatal neurons was dependent on the VMAT2 protein, a transporter

which mediates the dopamine secretion, therefore suggesting a potential for co-release between GABA and DA, the neurotransmitters with two physiologically distinct functional behaviors. Furthermore, the use of a conditional knock-out mouse lacking VGAT specifically in the GABAergic neurons while expressing VMAT2 was sufficient to maintain the GABA release, providing a definitive physiological evidence for VMAT2-mediated GABA release by dopaminergic neurons projecting to the striatum. It also suggests that the striatal output is dominated by GABA-dependent inhibition, rather than glutamate-dependent activation. Thus, the unique ability of the dopaminergic neurons projecting to striatum indicate that GABA is perhaps co-released by other dopaminergic neuronal subpopulations, the physiological significance of which is to be determined at the level of striatum and in the other brain areas in vivo.

Key words: GABA; dopaminergic neurons; striatum; ventral tegmental area (VTA); substantia nigra pars compacta (SNc); VGAT; VGLUT2; VMAT2; optogenetic stimulation; striatal projection neurons (SPN) I. BACKGROUND The midbrain dopaminergic system is physiologically important for a number of diseases including motor conditions such as Parkinson’s disease (PD), as well as such that schizophrenia, attention deficit hyperactivity disorder (ADHD), drug abuse and depression3 which arise due to the disturbance of cognitive function. These latter are dependent on the eufunctioning interface between the dopaminergic projections from the substantia nigra pars compacta (SNc) to the striatum. The release of DA is instrumental in defining the striatal output by working through the D1 receptors to stimulate the direct-pathway striatal projection neurons (dSPNs) or via D2 receptors to inhibit indirect-pathway SPNs (iSPNs). A great deal of emphasis has been placed on developing a clear neuroanatomical mapping pattern for the dopaminergic projections, thus setting aside the molecular properties and functional significance of the neuronal networks. The dopaminergic input to the striatum displays a heterogeneous population of neuronal projections from SNc and VTA, which may be differentiated based on several structural parameters, specifically: the ion channels4 and receptors5 expressed on their

surface the patterns of dopamine release6 and the vesicular transporters7. The latter constitute a specific family of proteins which pump the neurotransmitters from the neuronal cytoplasm into the synaptic vesicles for further exocytosis. The released neurotransmitter may function by regulating the flow through the ionic channels on the post-synaptic membrane, therefore modulating the output of the striatal system, or by binding to the autoreceptors located on the pre-synaptic terminal. Given the degree of physiological importance of the dopaminergic projections to the striatum and the ability of the neurons to corelease glutamate and dopamine1, Tritsch et al.2 suggested that the effects of activity in the dopaminergic cells may not be restricted to the roles of the neurotransmitters outlined above. To investigate the functional significance of the dopaminergic network the Cre-recombinase system delivered with the adeno-associated virus (AAV) was targeted specifically at the SNc to express the ChR2. Surprisingly, Tritsch and colleagues’ work shows that both types of SPNs were inhibited following optogenetic stimulation of dopaminergic projections from SNc and VTA, however the inhibitory effects were abolished after application of a GABAa receptor antagonist. In addition, knocking-out of the well-studied

conventional vesicular GABA transporter VGAT did not block GABA’s inhibitory effect on the striatal neurons challenging the current model of GABA release. Moreover the inhibition of VMAT2 was sufficient to abolish the GABA-mediated inhibition suggesting that GABA can be concentrated in the same vesicles with DA, thus providing the basis for regional diversity of dopamine signaling, the better understanding of which may facilitate the quality of therapeutic intervention for disorders as schizophrenia and PD. II. REVIEWED RESEARCHMATERIALS AND METHODS Mice Heterozygous (Slc6a3IRES-Cre/wt) and homozygous (Slc6a3IRES-Cre/ IRES-Cre ) mice were bred with Drd2-EGFP transgenic animals which express EGFP under control of chromosome containing D2 receptor locus allowing to distinguish the direct and indirectpathway SPNs. Conditional deletion of VGLUT2 and VGAT in the DA neurons was achieved by crossing Slc6a3IRES2Cre/wt;Slc17a6lox/ wt and Slc17a6lox/lox animals, or Slc6a3IRES2Cre/wt;Slc32a1lox/wt and Slc32a1lox/lox;Drd2-EGFP mice, respectively. The mice strains were maintained on the mixed background of FVB and C57BL/6. Virus preparation Recombinant AAV encoding an inverted reading frame of the light-gated non-selective cation channel channelrhodopsin-2 (ChR2) under transcriptional control of the EF1α promoter in Cre-containing neurons was used to achieve conditional expression of the ChR2-mCherry fusion protein. The replacement of ChR2 codon variant using Nhe1 and Asc1 restriction sites was used to generate Cre-dependent AAV vectors encoding VGAT, VMAT2 or EGFP. These viral vectors were delivered by means of stereotaxic intracranial injections into the dorsal striatum of the mice (P18-25) anaesthetized with isoflurane. Electrophysiology A microscope (Olympus BX51WI) continuously superfused (2-3 ml min-1) with 32-34°C ACSF was used to visualize cells through a х40 water-immersion objective to identify the highest density of ChR2+ axonal arbors in the striatal regions and EGFP+ iSPNs. The presence or absence of EGFP fluorescence was used to identify iSPNs and dSPNs respectively. Anterior dorsolateral and dorsomedial striatum within 300µm of the callosal-striatal border were targeted for whole-cell voltage and current-clamp recordings. No drugs were used in the bath solutions for wholecell recordings unless otherwise specified. The ChR2-expressing fibers were activated with a wide-field illumination of the recorded cell produced by focusing the brief pulses of light (1ms duration; 6.5-10.mW mm-2) from a 473 nm laser (Optoengine) on the back aperture of the microscope objective. For current-clamp recordings, application of the depolarizing current steps evoking 10-20 Hz trains at regular intervals (10-15 s) either alone or in

combination with a 1 ms flash of blue light was used. A Multiclamp 700B amplifier (Molecular Devices) digitized at 10kHz was used to amplify membrane potentials and currents and to filter lowpass set at 3kHz. Limit of detection for EPSCs and IPSCs was set at 10pA. RESULTS DA neurons directly release GABA onto SPNs The stimulation of the DA neurons leads to a large inhibitory currents at the dSPNs and iSPNs with peak amplitudes (617±78pA, n=29). To understand the nature of the IPSC current responsible for the SPN inhibition, a number of pharmacological antagonists were applied while holding the membrane potential constant at (~0 mV), which is the reversed potential of ionotropic glutamate receptors (fig. 1). IPSCs in SPNs were reduced with SR95531 and biccuculine (both are GABAa antagonists), with no significant difference observed after application of TPMPA (GABAc antagonist) measured relative to ACSF (control) indicating that GABAa receptor mediated inhibition, but not the GABAc receptor inhibition, is responsible for the inhibitory effects on the SPNs. Similarly, the reduction of the IPSCs with the quinpirole (selective D2, D3 agonist) and the abolishment with cadmium (Ca2+ channel blocker), whereas no apparent change in the amplitude of the IPSCs following the application of NBQX/CPP (AMPA and NMDA receptors antagonist) and the D1/2R suggests that the nature of the IPSCs is a Ca2+ dependent release of transmitter other than DA and glutamate. VMAT2 is required for the release of GABA from the DA neurons To find out which vesicular transporter is responsible for packaging the GABA into the vesicles at DA terminals a number of cKOs were designed. The main candidate was VGAT transporter which was previously shown to be a GABA vesicular transporter in the non-dopaminergic neurons; however the specific deletion of it in the DA neurons did not result in the change of the amplitude of the currents compared to control (fig. 2a,b) suggesting that VGAT is not responsible for the vesicular transport of the GABA in the DA neurons. To challenge the idea that GABA is being produced inside the vesicles through decarboxylation reaction of glutamate which is loaded into the vesicles by the VGLUT2 (glutamate vesicular transporter), the VGLUT2 knock-out was produced. Though, no difference was observed between VGLUT2 cKO and the control (fig 2a,c), suggesting that GABA vesicular transport is VGLUT2 independent. To find an alternative GABA vesicular transporter, reserpine, an irreversible blocker of the VMAT2 (monoamine transporter including DA) was applied to the nigrostriatal nigrostriatal DA neurons leading to the abolishment of the IPSC (fig. 2d), suggesting that it mediates the GABA dependent inhibition of the SPNs. To investigate if VMAT2 can serve as a functional substituent for the cells endogenously

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expressing VGAT, a rescue experiment with introduction of VMAT2 into the VGAT cKO was produced (fig. 3), which showed that VMAT2 alone is sufficient to maintain the release of GABA.

Figure 1 (Above). Mean (n=3-18) IPSC amplitudes in the DA neurons following application of antagonists compared with control(ACSF). The abolishment of the current with the antagonists corresponds to the importance of these processes in the inhibition of SPNs. Taken from Tritsch et al., 2012.

have the capacity to mediate the release of GABA from the dopaminergic neurons. SIGNIFICANCE OF THE WORK

Figure 3 (Below). IPSCs in dSPNs evoked by ChR2 (1ms, blue) stimulation of iSPNs in the presence of SR95531 (control, pink) or its absence (red) indicating the sufficiency of VMAT2 to maintain GABA release. Taken from Tritsch et al., 2012.

Figure 2 (Right). Representative IPSCs (red) and EPSCs (grey) evoked in striatal DA neurons upon stimulation of the ChR2 (1ms, blue) for a) control; b) VGAT cKO (Slc6a3IRES-Cre/wt;Slc32a1lox/lox); c) VGLUT2 cKO (Slc6a3IRES-Cre/wt;Slc17a6lox/lox); d) VMAT2 antagonist reserpine. Taken from Tritsch et al., 2012

DISCUSSION There is a great interest in the field of neuroscience pertaining to the influence that DA neurons have on the striatal projections. One of the functions of the DA neurons is to induce EPSPs on the post-synaptic striatal neurons8, however the work by Tritsh et al.2 shows that GABA mediated inhibition dominates over DA mediated excitation in the striatum, which are co-released and modulate the activity of the striatal output in mechanistically and temporally distinct ways. The activation of the GABAa receptors on the SPNs is possible through the direct release of GABA from the DA neurons, or through the recruitment of the interneurons which secrete GABA. However, the short latency between the induction of the IPSC with light and the actual IPSC, in tandem with the fact that the arrival of EPSCs is synchronous with IPSCs stand in favor of the direct release of GABA by the DA neurons9. Further evidence in support of the direct release comes from the idea that DA neurons from substantia nigra pars compacta express the mRNA for GAD6510, therefore have the potential to synthesize the GABA neurotransmitter. The key to understanding the mechanism of GABA release is through determining the vesicular transporter responsible for packaging the neurotransmitter into the synaptic vesicles. The

most probable candidate for the vesicular transport of GABA was VGAT, which has been previously shown to be responsible for GABA packaging in the non-dopaminergic neurons11. However, the release of GABA turned out to be VGAT independent, as was confirmed with the conditional knock-out model (fig. 2a,b). An alternative mechanism has been proposed to suggest that GABA is being produced through the decarboxylation reaction from glutamate inside the vesicles. Therefore, a cKO of the VGLUT2, which is responsible for packaging glutamate, was produced in order to test the hypothesis (fig. 2a,c). Similarly as with VGAT, the GABA release was shown to be VGLUT2 independent. The search for an alternative vesicular transporter leads to examination the role of the VMAT2 which is responsible for DA packaging. The application of the reserpine results in the abolishment of the IPSCs at the striatal level suggesting that GABA release is VMAT2 dependent. Given these considerations, the functional significance of the VMAT2 was raised to a new level by examining if its function alone would be sufficient for packaging the GABA neurotransmitter in the cells that endogenously do not express the vesicular transporter of interest. The established combination of VGLUT2 and the conditional knock-out of VGAT were sufficient to maintain the endogenous levels of GABA release (fig. 3). Therefore based on the combinatorial data derived from different experimental paradigms the VMAT2 transporter appears to

It is hard to underestimate the functional significance of the monoaminergic neurons in the different circuits of the brain. The authors2 demonstrate that the adult DA neurons influence neuronal activity in striatum in two different, molecularly and functionally distinct ways. Moreover the finding provides an insight into the degree of neurophysiological diversity within the dopaminergic system, presenting evidence that DA neurons projecting to striatum secrete DA and GABA, the neurotransmitters with functionally distinct behaviors. This may further the insights into differences in plasticity and synaptic connectivity between subpopulations of dopaminergic neurons which is important for the understanding of their various physiological roles. However, there are several confounding factors which halt the direct transition to the clinical studies at the current point of time. Tritsch and colleagues challenge the existing model that dopaminergic neurons work through increasing the probability of striatal output, by providing a solid evidence of a potential for phasic inhibition. Furthermore, it provides evidence in regards to the heterogenic nature of the dopaminergic projection to the striatum, therefore emphasizing the importance for better differentiation and classification of the DArgic subsystems. Although, the current finding does not result in a breakthrough in our understanding of the synaptic integration between the nigrostriatal pathways and the striatal output, it provides the ground for many more questions than answers, serving the basis of great potential for further research, highlighting the importance of the work in the field of neuroscience. An insight into the physiology of the dopaminergic neurons should change the way researchers think about the dopaminergic neurons, which may be successfully translated into the improvement of current therapeutic practices. FUTURE DIRECTIONS Even though the optogenetics was instrumental in identifying the process, it presents a caveat for the study in light of its massive synchronous stimulation. Therefore the same experimental paradigm may further be exploited through more selective targeting of various dopaminergic subpopulations. Furthermore, a greater effort might be necessary to prove that the neurotransmitter which potentiates the GABAa, and is dependent on the molecular complex requiring VMAT2 for the vesicular transport is in fact the GABA as suggested by the authors, since hypothetically it could be an analog of the neurotransmitter. Additionally, the insight into the mechanism of how GABA is synthesized in the DA neurons may be instrumental in the elucidation of further studies. Interestingly,

the VMAT2 has been previously shown to be implicated in the vesicular transport in non-dopaminergic neurons12 therefore further research would be necessary to understand the potential role that GABA may play in the population of neurons, which could have been previously overlooked. Perhaps, the most interest of the researchers would be kept to the understanding of the physiological significance that GABA plays in the subsystems of DAergic neurons which were examined. Although the complexity of the question should not be disregarded, since the neurophysiological basis for the pattern of DA release changes based on different behavioral contexts, therefore the differences in the pattern of GABA release may be anticipated13. Additional layer of complexity may be anticipated from examining the secretion of the DArgic projections during the different stages of development. Even though the study provides further evidence that there are certain differences in the electrophysiological and modulatory properties of DA neurons, a combined general map that would differentiate the diversity of the dopaminergic neurons by recognizing anatomical, molecular and functional diversity has not been elucidated and will be of great interest for development. Currently, the dopaminergic system of interest has been implicated in the number of conditions such that schizophrenia, drug abuse, Parkinson’s’ disease, ADHD3 therefore it bears a great potential for the transition to the clinical studies and establishing better pharmacological agents with reduced prevalence of severe sideeffects. REFERENCES 1. Hnasko, T. et al. Neuron 65, 643-656 (2010). 2. Tritsch, N. X., Ding, J. B. & Sabatini, B. L. Nature 490, 262-266 (2012). 3. Bjorklund, A. & Dunnett, S. Trends Neurosci. 30, 185-187. 4. Lammel, S. et al. Neuron 57, 760-773 (2008). 5. Ford, C. P., Williams, J. T. & Mark, G. P. J. Neurosci. 26, 2788-2797 (2006). 6. Stefani, M. R. & Moghaddam, B. J. Neurosci. 26, 8810-8818 (2006). 7. Stuber, G. D., Hnasko, T., Britt, J. P., Edwards, R. H. & Bonci, A. J. Neurosci. 30, 8229-8233 (2010). 8. Gerfen, C. R. & Surmeier, D. J. Annu.Rev. Neurosci. 34, 441-466 (2011). 9. Tecuapetla, F. et al. J. Neurosci. 30, 7105-7110 (2010). 10. Gonzalez-Hernandez, T. et al. Eur. J. Neurosci. 13, 57-67 (2001). 11. Wojcik, S. M. et al. Neuron 50, 575-587 (2006). 12. Yelin, R. & Schuldiner, S. FEBS lett. 377, 201-207 (1995). 13. Bassareo, V. et al. J. Neurosci. 22, 4709-4719 (2002).

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Is an extrasynaptic pool of glutamate receptors a requirement for LTP? Farhana Islam and Ahmed Aslam1 Human Biology Department, The University of Toronto. Toronto, Ontario CA.

Perhaps one of the most important challenges in Neuroscience is to identify the mechanism of learning and memory in the human brain. An astonishing feature of the mammalian brain is its ability to learn and store a large amount of information by modifying underlying synaptic connections1. The process through which information storage is achieved is known as Long Term Potentiation (LTP)1. The cellular mechanism of LTP that underlies memory formation is well understood at the level of glutamatergic synapses in the hippocampal region2. This involves short-lived, strong neuronal activity between two neurons that induces long lasting changes in their synaptic function1. LTP in hippocampal CA1 neurons involves the trafficking of GluA1-GluA2-containing AMPA-type glutamate receptors from the extrasynaptic to the postsynaptic region1. It was previously believed that the expression of cytoplasmic carboxy-tail (C-tail) of the GluA1 subunit of

AMPA receptors was required for the trafficking1. Granger and colleagues performed single-cell molecular replacement to introduce modified GluA1 subunits to selective neurons of the CA11. Their experimental work aimed to identify the minimum necessary requirement of the GluA1 C-tail for the preferential trafficking of AMPAR to the post-synaptic density zone during LTP1. The findings showed that the C-tail is not a requirement for the induction of LTP2. In fact, LTP was normal in CA1 pyramidal neurons that lacked GluA1 subunits or AMPARs1. Therefore, the results of this study were astonishing in comparison to the previously assumed model of LTP1. The only manipulation by which LTP was severely altered was due to the absence of extrasynaptic reserve pool of glutamate receptors1. These findings provide new insights into the mechanism of LTP induction, and shift the focus from subunit composition to the synapse itself1.

Key words: Long-Term Potentiation (LTP), hippocampal CA1 neurons, GluA1 C-tail, AMPARs, extrasynaptic receptors, kainite receptors, GluA2, neuroplasticity I. BACKGROUND Long term potentiation (LTP) is essential for the storage of experience in the brain2. This phenomenon, fundamental for neuroplasticity, modifies neuron-to-neuron connections to produce long lasting changes in synaptic strength2. The cooperation of two types of glutamate receptors is fundamental for the process of LTP: AMPA receptors (AMPARs) and NMDA receptors (NMDARs)2. At the CA1 glutamatergic synapses of the hippocampus, where LTP has been well studied1, the primary role of AMPAR is to depolarize the postsynaptic neuron by permitting the entry of Na+, when bound to glutamate released by the activated presynaptic neuron2. This allows the removal of the Mg+ plug from the NMDA channel and permits the influx of second messenger Ca2+ ions initiating biochemical processes that ultimately change the composition of the synapse and contribute to strengthening the synaptic connection2. One biochemical process that is initiated by Ca2+ second messengers is the insertion of more AMPARs into the post-synaptic density zone (PSDZ)1,2. This increase in the number of AMPARs in the PSDZ relative to basal conditions initiates LTP induction and causes the post-synaptic neuron to be more responsive to the pre-synaptic neuron, thereby enhancing synaptic response1. However, the mechanism of rapid AMPAR insertion into the PSDZ during LTP has not been fully elucidated1.

AMPARs are hetero-oligomeric molecules that are composed of different combinations of four unique subunits: GluA1, GluA2 GluA3, and GluA41,3. The subunit composition of an AMPAR receptor determines its influence on synaptic depolarization1,3. The existing model of AMPAR insertion into the PSDZ posits that in the CA1 pyramidal neurons of the hippocampus, under basal conditions, most AMPARs are composed of GluA2-GluA3 subunits1–3. However, when an LTP stimulus is provided, it puts in motion events that lead to preferential insertion of GluA1-GluA2 AMPARs from extrasynaptic pools into the PSDZ1–3. Study by Zamanillo and colleagues have shown that GluA1 knockout mice demonstrated impaired LTP in the CA1 compared to wild-type mice, suggesting that GluA1 is required for LTP4. Furthermore, Meng and colleagues analyzed LTP in knock-out mice deficient in GluA2-GluA3 and found that these mice are severely impaired in basal synaptic transmission but demonstrated normal levels of LTP, indicating that these subunits are essential for basal constitutive trafficking but are not essential for the induction of LTP5. On the basis of these findings, there is a consensus that the differential trafficking behavior of AMPARs is mediated by the carboxy-terminal tails (C-tails) of the individual subunits, such that LTP is mediated by the preferential insertion of the GluA1 into the PSDZ via its C-tail interactions1.

However, despite the consensus that GluA1 is essential for LTP, there has been no evidence to show that the GluA1 C-tail is absolutely necessary in the preferential trafficking of GluA1 to the PSDZ1. The present paper reviews the experimental work by Granger and colleagues to determine the minimum necessary requirement of the GluA1 C-tail for LTP and to identify crucial protein interactions that permit its preferential recruitment from extrasynaptic reserves1. To accomplish their objectives, Granger and his team performed single cell genetics to replace all endogenous AMPARs in selective neurons of the CA1 with mutated GluA1 C-tails containing systematic truncations1. Their findings not only demonstrate that the GluA1 C-tail is not necessary for LTP, it also challenges the current model of LTP and the core processes that underlie synaptic plasticity1. II. MATERIALS AND METHODS GluA1 C-Tail Truncations GluA1 C-tail truncations were made by overlapping extension PCR1. GluA1(ΔC) contained the full truncation of the C-tail ending in amino acid 812. Two complimentary truncations were generated: GluA1(Δ824) terminated at amino acid 824 and GluA1(ΔMPR) had its proximal region excised1. Replacement GluA1 subunits was confirmed by GFP epifluorescence1. To screen for surface expression of various mutated GluA1 in CA1 neurons, surface rectifications in wild-type neurons were performed1.

were no longer expressed by neurons containing Cre and resulted in the complete absence of AMPARs1,6. This provides an AMPARnull background for the expression of mutated GluA1 subunits1. Surrounding neurons that is not transfected with Cre express GluA subunits as normal1. The number and composition of NMDARs at the synapse remain unchanged1,6. Cre-expression was confirmed when all glutamate-evoked EPSC was eliminated in somatic outside-out patches of Grial-3fl/fl CA1 neurons that had been transfected with Cre1. This effect is rescued by co-expression of GluA1 full-length C-tail1. To confirm the absence of endogenous GluA receptors, rectification from GluA1replacement neurons were obtained1. Neuronal Transfection Embryos were temporarily removed from pregnant Grial-3fl/fl mice and injected with plasmid DNA containing the replacement GluA1, GluA2, or GluK1 plasmid via 35 V pulse electroporation1. After electroporation, the embryos were returned to the abdomen of the pregnant mice, and sacrificed on post-natal day 17-20 for LTP recording1. Sparse biolistic transfection of Cre, GluA1, and KAR subunits were confirmed using GFP epifluorescence1. Electrophysiology In acute P17-20 hippocampal slices, stable baseline AMPAR excitatory post-synaptic currents (EPSCs) was recorded for 3-5 minutes simultaneously from control and GluA1, GluA2, or GluK1-replacement neurons1. LTP was induced in the CA1 by 2 Hz stimulation for 90 seconds while clamping the cell at 0 mV1. All slices were maintained in artificial cerebrospinal fluid during recording1. Rectification was calculated mathematically based on the amplitude of the AMPA EPSC from 0 to +40mV and from -70 to 0 mV1. RESULTS

Figure 1. Various GluA1 C-tail truncations. GluA1 contains full-length C-tail, Δ824 is excised up to amino acid 824, ΔMPR contains the excision of a proximal region, and ΔC contains full a C-tail truncation1. Taken from Granger et al, 2013.

Single-Cell Genetics Conditional knock-out (KO) mice, Grial-3fl/fl, were generated using site-specific Cre-Lox recombination1. Grial-3fl/fl mice contained genes coding for GluA1, GluA2, and GluA3 flanked by loxP sites1. Cre DNA recombinase was then transfected in selective neurons of the CA1 hippocampal pyramidal neurons1. loxP sequences contain binding sites for Cre recombinase1,6. Cre catalyzes DNA recombination and allows for the deletion of undesired target DNA sequence1,6. In this manner, genes coding for GluA subunits

GluA1 C-tail is not required for LTP AMPAR EPSC was absent after an LTP stimulus in Grial-3fl/fl CA1 neurons expressing Cre alone, which indicates that endogenous GluA subunits were not expressed in these selective neurons1. Transfection of full-length GluA1 C-tail in Cre-expressing Grial-3fl/fl CA1 neurons exhibited normal LTP, which confirmed that the intact GluA1 subunit is sufficient1. GluA1(Δ824) and GluA1(ΔMPR) expressed LTP similar to the neighbouring control neurons1. Transfection of GluA1(ΔC) with GluA2 to produce a more natural AMPAR heteromers exhibited LTP comparable to controls1. The results were contradictory to previous findings that stated that LTP stimulus triggers the trafficking of GluA17,8. This suggested that no part of the C-tail was required for LTP.

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Neuroscience Matters to GluA1 AMPAR1. KARs differ in their structural characteristics because they contain no sequence homology with GluA1 and consist of different auxiliary subunits1,9. Grial-3fl/fl neurons were modified to express GluK1 subunit of KARs to identify the absence of AMPAR on LTP generation1. The results revealed that the KARs, when overexpressed in Grial3fl/fl CA1 neurons in the absence of endogenous AMPARs, showed LTP generation indistinguishable from control neurons1. To confirm that LTP is not induced via a separate mechanism in the KAR-expressing neurons, ACET, a GluK1 antagonist, was applied at the end of the experiments1. EPSCs was entirely abolished with ACET administration confirming that LTP observed in KARexpressing neurons was mediated entirely by KARs1.

Figure 2. EPSC in Grial-3fl/fl neurons expressing (a) GluA1, (b) GluA1(Δ824), (c) GluA1(ΔMPR), (d) GluA1(ΔC)+GluA2. LTP was indistinguishable from control cells in all conditions. Taken from Granger et al., 2013.

GluA1 subunit is not required for LTP and GluA2 is sufficient for LTP The researchers on finding that the absence of C-tail was not significant for the production of LTP, they next investigated whether the absence of GluA1 subunits affected LTP in CA1 neurons1. GluA2 was transfected instead because it differs fundamentally from GluA1 in terms of absence of phosphorylation sites and protein-protein binding sites that are present in GluA1 subunit1. Furthermore, GluA2 have limited C-tail homology to GluA1 subunit which affect its role in C-tail trafficking1,6. Recording with neurons expressing GluA2 subunits in place of GluA1 showed parallel findings with the wild-type Grial-3fl/ fl neurons that expressed GluA1 AMPAR subunit only1. LTP recorded was indistinguishable from the control neuronal cells.

Figure 3. EPSC in Grial-3fl/fl neurons expressing GluA2 only. LTP was indistinguishable from control cells. Taken from Granger et al., 2013.

Kainate receptors can substitute GluA1 to generate LTP Kainate receptors (KARs) expressed GluK1 subunits which are an entirely different class of fast, ionotropic receptors compared

Figure 4. EPSC in Grial-3fl/fl neurons expressing GluK1 only. LTP was indistinguishable from control cells. EPSC was diminished with ACET administration. Taken from Granger et al., 2013.

DISCUSSION The existing model of LTP in hippocampal CA1 neurons posits that AMPARs containing GluA1-GluA2 subunits is preferentially trafficked and inserted into the PSDZ by GluA1 C-tail interactions3,7,10. However, Granger and colleagues have provided evidence to refute this assumption and has shown that LTP does not require the GluA1 C-tail or the GluA1 subunit, but depends on the availability of a reserve pool of extrasynaptic glutamate receptors regardless of subunit composition1. The most unexpected finding was that LTP was induced in KAR-expressing CA1 pyramidal neurons that do not contain AMPARs, which demonstrates that even neurons lacking AMPA can undergo LTP provided the availability of an alternative pool of fast, ionotropic glutamate receptors1. However, the role of GluA1 C-tail in modulating LTP cannot be completely ruled out1. Study by Lee and colleagues have demonstrated that mice that have serine 831 and 845 phosphorylation sites on the GluA1 C-tail mutated to alanine by gene knock-in techniques, show complete absence of LTP and long-term depression (LTD), as well as deficits in spatial learning

tasks11. Likewise, Makino and colleagues have shown that the S831 and S845 sites can lower the threshold for LTP induction and increases the likelihood of synaptic plasticity12. Although, GluA1 C-tail interactions may not cause the preferential insertion of AMPARs into the PSDZ, it may still have modulatory effects on synaptic plasticity1,11,12. Given that the GluA1 C-tail is not required for LTP, the authors questioned whether the GluA1 subunit itself is a requirement for LTP1. Although the GluA2 subunit lacks phosphorylation sites and protein binding sites of GluA1, the results have shown that GluA2 homomers expressed in Grial-3fl/fl CA1 neurons exhibited intact LTP indistinguishable from control cells1. However, the finding that has been most astounding was that, even with the complete absence of AMPARs, LTP is intact in CA1 neurons when fast, ionotropic KARs are expressed instead1. This is indeed astounding because AMPARs have previously known to be tightly linked with LTP induction and synaptic plasticity1. Although the current findings have demonstrated that GluA1 subunits nor AMPARs are essential for LTP to occur, multiple previous studies have shown that the deletion of GluA1 can alone impair LTP1. As a result, the authors of the current paper postulated a new model for AMPAR insertion during LTP that is able to reconcile both previous findings and the present experimental findings1. The proposed model is that LTP requires a large, reserved pool of extrasynaptic glutamate receptors regardless of subunit composition1. The exocytosis of AMPARcontaining endosomes into the PSDZ causes the strengthening of the synaptic connection between the two neurons as the number of AMPARs on the PSDZ responding to the presynaptic signal is increased1. When surface receptors required for LTP is experimentally depleted in a neuron, the same conditions deplete the extrasynaptic pool of these surface receptors available for inclusion into the PSDZ, thereby causing impaired LTP1. Hence, LTP can be described an increase in the density of fast, ionotropic excitatory receptors in the PSDZ from extrasynaptic reserves, which causes enhanced synaptic connection regardless of subunit composition or type of glutamate receptor1. A limitation of the present study is that although intact EPSCs are observed in Grial-3fl/fl CA1 neurons transfected with modified GluA1, or GluA2 and GluK1 subunits in in vitro hippocampal slices, this may not translate into intact behavioural responses in vivo. The behavioural outcome of the outlined modifications are not known. It may be that in vivo KARs or GluA2 homomers may not be sufficient for information storage in the brain, and GluA1 may be fundamentally necessary as observed in earlier studies4,11.

SIGNIFICANCE OF THE WORK LTP involves the rapid increase in the strength of neuron-toneuron connection, which is essential for the formation of learning and memory2. Changes in AMPAR density in the PSDZ provides a basis for enhanced synaptic depolarization that is evident in LTP1,2. The data presented in this study suggests that the existing model for the role of AMPAR in LTP induction needs to be reconsidered1. The finding that LTP necessitates an extrasynaptic pool of excitatory surface receptors that do not necessarily have to contain GluA1 subunits provides an important new insight into the molecular mechanism underlying synaptic plasticity1. This study shifts the focus of LTP from receptor subunits, to alterations in the synapse itself, specifically the PSDZ and its ability to trap receptors from extrasynaptic pools1. It also implies that the current understanding of LTP is incomplete and there is still much to be elucidated1. FUTURE DIRECTIONS The fundamental question posed by the authors of this paper still remains to be answered: what are the specific interactions that cause the preferential insertion of GluA1 AMPARs at the synapse during plasticity? The identification of these specific interactions with AMPARs may be fundamental to understanding the synaptic plasticity alterations that underlie memory and learning in the brain1. It is also important to test the model that is proposed by the authors. If it is true that simply the presence of an extrasynaptic pool of surface receptors is sufficient for LTP, than in neurological disorders that are characterized by dysfunctional AMPARs or an abnormality in its biochemical processes, replacement therapy with other receptors may circumvent associated impairments. Updating the existing model is also necessary to elucidate the process of synaptic plasticity and its role in learning and information storage in the brain. REFERENCES 1. Granger, A. J., Shi, Y., Lu, W., Cerpas, M. & Nicoll, R. A. LTP requires a reserve pool of glutamate receptors independent of subunit type. Nature 493, 495–500 (2013). 2. Rudy, J. W. The Neurobiology of Learning and Memory. (Sinauer Associates, Incorporated Publishers, 2008). 3. Derkach, V. A., Oh, M. C., Guire, E. S. & Soderling, T. R. Regulatory mechanisms of AMPA receptors in synaptic plasticity. Nature Reviews Neuroscience 8, 101–113 (2007). 4. Zamanillo, D. et al. Importance of AMPA Receptors for Hippocampal Synaptic Plasticity But Not for Spatial Learning. Science 284, 1805–1811 (1999). 5. Meng, Y. Synaptic Transmission and Plasticity in the Absence of AMPA Glutamate Receptor GluR2 and GluR3. Neuron 39, 163–176 6. Lu, W. Subunit Composition of Synaptic AMPA Receptors Revealed by

Neuroscience Matters a Single-Cell Genetic Approach. Neuron 62, 254–268 (2009). 7. Malinow, R. AMPA receptor trafficking and synaptic plasticity. Annual Review of Neuroscience 25, 103–126 (2002). 8. Boehm, J. Synaptic Incorporation of AMPA Receptors during LTP Is Controlled by a PKC Phosphorylation Site on GluR1. Neuron 51, 213– 225. 9. Contractor, A. Kainate receptors coming of age: milestones of two decades of research. Trends in Neurosciences 34, 154–163 (2011). 10. Esteban, J. A. AMPA receptor trafficking: a road map for synaptic plasticity. Mol. Interv. 3, 375–385 (2003). 11. Lee, H.K. Phosphorylation of the AMPA Receptor GluR1 Subunit Is Required for Synaptic Plasticity and Retention of Spatial Memory. Cell

Neuroscience Matters 112, 631–643 12. Makino, Y., Johnson, R. C., Yu, Y., Takamiya, K. & Huganir, R. L. Enhanced synaptic plasticity in mice with phosphomimetic mutation of the GluA1 AMPA receptor. PNAS 108, 8450–8455 (2011).

Received April 5, 2013; revised Month, ##, 200#; accepted Month, ##, 2013. Address correspondence to: Farhana Islam, farhana.islam@mail.utoronto.ca; Ahmed Aslam, ahmed.aslam@mail.utoronto.ca Copyright © 2013 Dr. Bill JU, Human Biology Program

Uncovering the Molecular Basis of Memory: Are Histone Modifications the Answer? Amy Chow1 Human Biology Department, The University of Toronto. Toronto, Ontario CA.

Post-translational modifications of histones have recently been implicated in memory formation and consolidation. An increase in histone acetylation correlates with improved memory and histone deacetylase inhibitors are known to enhance memory. While the role of acetylation has been relatively well established, it is unknown how other forms of modifications affect memory. This review focuses on how methylation may serve as another crucial modulator. Histone methylation shares some links to histone acetylation, but has more variety – such that different patterns of methylation on different residues of the histone tail can have drastically different effects on transcriptional activation and repression. Recent findings reveal that histone methylation

is necessary for memory formation and consolidation, through analyzing different methylation patterns after fear conditioning training and knocking out a specific histone methyltransferase. As such, histone modifications are not only part of the mechanism in the formation and consolidation or memories, but also may constitute the physical basis of memory in the form of a histone code. This review explores this new hypothesis in memory research and considers future steps to be taken in compiling a better understanding of the role histones play in memory, as well as their potential to be used in future treatments of neurological disorders that involve memory deficits.

Key words: histone methylation; histone acetylation; memory; consolidation; histone code

Despite extensive research on the molecular mechanisms of how memories are formed, consolidated and retained, memory traces are still poorly understood. Long-term memory is known to require transcription and de novo protein synthesis1, which strengthens neuronal networks and facilitates network rearrangement to accommodate different patterns of activation. Proteins such as CamKII are involved, and trigger signaling cascades that result in transcriptional changes. Cellular and proliferation factors such as CREB, c-Fos, zif268 and BDNF produce changes at the synaptic level and sustain neuronal growth.

compact manner in order to fit into each cell nucleus. DNA is wrapped around a histone core, which is made of 8 subunits: two each of histone 2A, 2B, 3 and 4. All of these subunits have tails (composed of a chain of amino acids), which can be subject to post-translational modifications. Different modifications change how accessible DNA is to transcriptional machinery and thus play a vital role in determining gene expression. For instance, histone acetyltransferases (HAT) add an acetyl group to a residue, which has been correlated with transcriptional activation2. Conversely, histone deacetylases (HDAC) remove an acetyl group from a residue, which has led to transcriptional repression alongside corepressors2.

In attempting to understand the molecular basis of memory, it is crucial to understand the underlying gene expression and transcriptional regulation. DNA is packaged in a highly

With regards to memory, recent work has revealed that increasing histone acetylation on certain residues of the histone tail enhances memory and synaptic plasticity3,4,5,6, while histone deacetylation

I. BACKGROUND

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has been correlated with memory deficits7. In a task where rats were to identify novel objects among previously seen objects, histone acetylation levels were increased in the hippocampus after training and correlated with better memory performance8. In addition to this, CREB-binding protein, critical for long-term memory and synaptic plasticity, is known to be a HAT. Along those lines, administration of a H3-specific HAT inhibitor interfered with the learning of conditioned food aversion in snails, which could be rescued by the addition of a HDAC inhibitor9. Similarly, HDAC inhibitor Trichostatin A enhanced LTP10 and Class II specific HDAC inhibitor sodium butyrate (NaB) enhanced the formation and persistence of long-term memory for weak stimuli9. These pieces of evidence suggest that histone acetylation is required for memory. However, the role of other post-translational modifications, such as methylation, play in memory and synaptic plasticity has not been extensively investigated. Histones are methylated by histone methyltransferases (HMT) and effects depend on which residue is methylated and its methylation pattern (ie. monomethylation, dimethylation or trimethylation)11. II. REVIEWED RESEARCH: MATERIALS AND METHODS To study the role histone methylation plays in memory formation, Gupta and colleagues investigated the levels of histone methylation in the hippocampus of adult rats12. Using a contextual fear conditioning paradigm, rats were put in a new context and shocked, which produced a fear memory. Rats would demonstrate forming and consolidating this fear memory by increased freezing behaviour when placed in the same context when tested 1 hour or 24 hours after training. Histone methylation patterns were studied at two loci, which have been correlated with particular patterns of gene expression. Trimethylation of histone 3 at lysine 4 (H3K4) is a known marker for transcriptional activation, whereas dimethylation of histone 3 at lysine 9 (H3K9) is associated with transcriptional repression. Histones were extracted from CA1 of the hippocampus, both 1 hour after training and 24 hours after training. Anti-H3K4me3 and anti-H3K9me2 antibodies were used to isolate the methylated H3 histones from the protein extract from the cell and protein concentrations were determined by Western blot. DNA immunoprecipitation was used to study the binding of DNA binding proteins on promoters upstream of the BDNF and zif268 genes. To ensure that any changes in methylation patterns were not due to the circumstances surrounding the fear conditioning task, a latent inhibition paradigm was used. By allowing the rat to explore the context prior to administering a shock, the rat does not form an association between the context and the shock.

In addition, the role of HMT was assessed by comparing HMT knockouts with controls. Single and double mutant mice for two genes that are essential in the H3K4-specific HMT complex (eed and Mll) were used and subjected to the contextual fear conditioning paradigm. The amount of freezing behaviour was assessed for a measure of fear memory formation. RESULTS H3K4 trimethylation levels in the CA1 of the hippocampus were found to have increased by 50% 1 hour after training, only in the rats that were subjected to the fear conditioning training, as compared to controls that were only exposed to the context or completely naïve to the experiment. These levels returned to baseline levels 24 hours after training, to comparable levels as naïve or context-exposed rats (see Figure 1). H3K9 dimethylation levels were increased 300% relative to naïve controls 1 hour after training in both groups of rats that were exposed to the context alone and those that underwent fear conditioning training. After 24 hours, these levels were reduced to 50% of baseline levels for both groups when compared to naïve controls (see Figure 2). These changes in methylation could not be attributed to the circumstances surrounding fear conditioning training, as rats that did not pair the shock with the context (under latent inhibition) did not show increased levels of H3K4 trimethylation as rats that were fear conditioned. The levels of H3K4 trimethylation in the latent inhibition group were similar to naïve controls (see Figure 3). Reduced freezing behaviour was correlated with mice deficient in H3K4-specific methyltransferase, with greatest impairment in the double mutants (see Figure 4). The Mll gene appears to play a greater role than the eed gene in reducing freezing behaviour. Trimethylation of H3K4 was increased around the promoters of BDNF and zif268.

Figure 1. Levels of trimethylated histone H3K4 (H3K4me3) in CA1 of a rat hippocampus. Left: 1 hour after training, rats exposed to fear conditioning showed increased levels of H3K4me3 compared to naïve controls and rats exposed to only the context. Right: H3K4me3 levels of the fear conditioned group returned to levels similar to controls 24 hours after training. Western blots are visualized above. (Graph from Gupta et al, 2010)

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Neuroscience Matters

Neuroscience Matters DISCUSSION

Figure 2. Levels of dimethylated histone H3K9 (H3K9me2) in CA1 of a rat hippocampus. Left: 1 hour after training, elevated levels of H3K9me2 were seen in rats that were exposed to fear conditioning as well as context alone. Right: 24 hours after training, H3K9me2 levels in those two groups were significantly reduced compared to naïve controls. Western blots are visualized above. (Graph from Gupta et al, 2010)

Figure 3. Latent inhibition paradigm used to control for the effects of stress in response to the shock. Left: Rats trained under the latent inhibition paradigm showed significantly less freezing behaviour than rats that were fear conditioned. Right: Latent inhibition rats did not show increased levels of H3K4me3 as compared to fear conditioned rats. (Graph from Gupta et al, 2010)

Figure 4. Single and double mutants for H3K4-specific methyltransferase genes eed and Mll. Double mutants show the least amount of freezing compared to single mutants of eed or Mll and wild-type. The single Mll mutant also showed significant reduction in freezing compared to the single eed mutant or wild-type rats. (Graph from Gupta et al, 2010)

The findings of this experiment indicate that histone methylation plays an important role in memory formation and proper longterm consolidation of contextual fear memories. Several molecular correlates of memory formation were revealed. Trimethylation of H3K4 may be responsible for associative fear learning, encoding both the context and shock. This histone modification may activate RNA polymerase II and cause subsequent histone acetylation at other promoter sites, particularly at zif268 and BDNF promoters13. Modification of these specific promoter sites may make them more accessible to transcriptional machinery, to enhance gene expression. Similarly, H3K9 dimethylation may encode for context, as H3K9 dimethylation was increased when the rats were placed in the context alone or trained under the fear conditioning paradigm. This suggests that both transcriptional activation through H3K4 trimethylation and repression by H3K9 dimethylation are important for the formation or consolidation of contextual fear memories. Furthermore, the H3K4-specific HMT appears to be crucial for forming or consolidating memory, as its absence was correlated with deficits in long-term contextual fear memory. Following up on this study, Kerimoglu and colleagues14 have continued to study the role of this particular methyltransferase in memory formation. Several different behavioural tests were used to determine whether different forms of hippocampus-dependent memory required histone methylation of H3K4. The authors found that Mll2 HMT knockouts did not show a significant preference for novel objects when tested both 5 mins after and 24 hours after the initial exposure in a novel object recognition paradigm, compared to control mice14. Similarly, Mll2 knockouts did not produce as much freezing behaviour under a fear conditioning paradigm and required a longer escape latency to find the hidden platform in a Morris water maze task, both when tested 24 hours after initial training14. These Mll2 knockouts were fully viable and did not show any phenotypical developmental defects. Nevertheless, since developmental problems cannot be completely ruled out, these researchers repeated this experiment and knocked out the Mll2 gene using the Cre-lox system by adeno-associated virus injection into the dentate gyrus of adult mice. Similar results were found and thus the authors concluded that loss of this crucial HMT resulted in poor memory formation or consolidation in the hippocampus14. These converging lines of evidence suggest that histone methylation at H3K4 is required for both forming shortterm memory and consolidating for long-term memory. Criticisms Gupta and colleagues only analyzed what effect histone methylation had on the promoters of zif268 and BDNF based on a priori reasoning that they have been previously implicated

in memory. Future experiments should make use of unbiased sequencing techniques to elucidate other genes involved and what roles they play in cellular processes. For instance, the use of DNA microarray analysis by Kerimoglu and colleagues allowed for the identification of 161 different genes that showed altered expression in the midst of synaptic strengthening in the dentate gyrus14. The loss of the H3K4-specfic HMT resulted in a downregulation of a set of genes that are also known to affect synaptic function and memory formation14. For instance, NF-κB activating protein like (Nkapl) was downregulated, which has a role in regulating synaptic plasticity and memory15. Other genes include synaptic proteins such as syntaxin binding protein 2 (stxbp2), synaptophysin-like 2 (sypl2), and factors that prolong neuronal survival (antioxidant OXR116) and neurogenesis (E2F1; Cooper-Kuhn et al., 2002). SIGNIFICANCE OF THE WORK As one of the first papers showing evidence that histone methylation is involved in contextual learning, it highlights that epigenetics is at the core of the processes of encoding and memory consolidation. Histone modifications play a significant role in the mechanism of storing memory, as signaling cascades trigger histone methylation at a transcriptional start site (TSS), which is required to recruit histone acetylation enzymes needed to activate transcription. This produces new proteins required for synaptic plasticity and changes the efficacy of synaptic connections across a neuronal network. While histone modifications may be the end result of signaling cascades, these molecular tags may also potentially serve as the physical locus where memory is stored in the brain. Memories may be encoded in the brain literally in the pattern of histone modifications in what is termed the histone code17. This pattern of histone modifications produce certain gene expression profiles that may underlie a memory. The dynamic, rapid and reversible nature of histone modifications makes it a good candidate to be a form of transient storage particularly in the hippocampus, and possibly other areas of the brain. FUTURE DIRECTIONS As researchers begin to realize the important role epigenetics plays in memory and consolidation, future research should tackle other post-translational modifications and evaluate their roles. The arrival of better real-time sequencing techniques allows for easier identification of what genes are involved in memory processes. For example, ChIP-sequencing provides an unbiased method of identifying what DNA sequences DNA-binding proteins interact with, without any prior knowledge of any specific binding sequences. However, since histone acetylation or methylation may play a role

in a variety of functions in the cell beyond just memory, the use of non-specific HDAC inhibitors or HAT/HMT enhancers may lead to adverse side effects. In fact, one study showed that longterm HDAC inhibitor use led to impairments in contextual fear learning in mice18. The role histones play in maintaining memory traces likely involves a delicate balance of cellular processes. Greater specificity is required, especially if histone modifications are only required at particular loci in the epigenome to improve memory. This area of research will drive the development of more class-specific inhibitors or enhancers to improve memory. This line of work also sheds light on the use of histone modification enzymes as possible treatments of various brain disorders. Many disorders involve problems at the various levels of cellular regulation: from gene mutations to changes in gene expression and changes in epigenetic regulation. The work in histone acetylation has led to the use of HDAC inhibitors to enhance memory in disorders that involve memory impairments. For instance, reduced expression of CBP in the hippocampus as a result of Huntington’s disease results in reduced histone H3 acetylation. Administration of HDAC inhibitor TSA was able to improve memory deficits in mouse models19, serving as a potential treatment for cognitive impairments in this disorder. In addition, administration of a class I specific HDAC inhibitor improved contextual fear memory for Alzheimer’s disease mouse models20. By studying methylation processes further, we can begin to understand how to use this knowledge to treat brain disorders that involve disordered histone methylation. In addition, there has been some evidence that HDAC inhibitors also causes increased H3K4 trimethylation and decreased H3K9 dimethylation12, suggesting there may be a link between histone acetylation and methylation. Thus, continuing to explore the area of histone methylation will serve as a compliment to understanding histone acetylation, and ultimately a more complete picture of the role histone modifications play in the formation and consolidation of memory. REFERENCES 1. Kandel ER. The molecular biology of memory storage: a dialogue between genes and synapses. Science, 294, 1030 –1038 (2001). 2. Peleg S, Sananbenesi F, Zovoilis A, Burkhardt S, Bahari-Javan S, AgisBalboa RC, Cota P, Wittnam JL, Gogol-Doering A, Opitz L, SalinasRiester G, Dettenhofer M, Kang H, Farinelli L, Chen W, Fischer A. Altered histone acetylation is associated with age-dependent memory impairment in mice. Science, 328, 753–756 (2010). 3. Peixoto, L. & Abel, T. The role of histone acetylation in memory formation and cognitive impairments. Neuropsychopharmocology Reviews, 38, 62-76 (2013). 4. Li B, Carey M, Workman JL. The role of chromatin during transcription. Cell, 128, 707–719 (2007). 5. Shi Y, Whetstine JR. Dynamic regulation of histone lysine methylation by demethylases. Mol Cell, 25, 1–14 (2007). 6. Shilatifard A. Molecular implementation and physiological roles for histone H3 lysine 4 (H3K4) methylation. Curr Opin Cell Biol, 20, 341–

Neuroscience Matters 348 (2008). 7. Guan JS, Haggarty SJ, Giacometti E, Dannenberg JH, Joseph N, Gao J, Nieland TJ, Zhou Y, Wang X, Mazitschek R, Bradner JE, DePinho RA, Jaenisch R, Tsai LH. HDAC2 negatively regulates memory formation and synaptic plasticity. Nature, 459, 55– 60 (2009). 8. Levenson JM, O’Riordan KJ, Brown KD, Trinh MA, Molfese DL, Sweatt JD. Regulation of histone acetylation during memory formation in the hippocampus. J Biol Chem, 279, 40545– 40559 (2004). 9. Danilova AB, Grinkevich LN. Failure of Long-Term Memory Formation in Juvenile Snails Is Determined by Acetylation Status of Histone H3 and Can Be Improved by NaB Treatment. PLoS ONE, 7(7): e41828 (2012). 10. Vecsey CG, Hawk JD, Lattal KM, Stein JM, Fabian SA, Attner MA, Cabrera SM, McDonough CB, Brindle PK, Abel T, Wood MA. Histone deacetylase inhibitors enhance memory and synaptic plasticity via CREB: CBP-dependent transcriptional activation. J Neurosci, 27, 6128–6140 (2007). 11. Gupta-Agarwal S, Franklin AV, Deramus T, Wheelock M, Davis RL, McMahon LL, Lubin FD. G9a/GLP histone lysine dimethyltransferase complex activity in the hippocampus and the entorhinal cortex is required for gene activation and silencing during memory consolidation. J Neurosci, 32, 5440 –5453 (2012). 12. Gupta, S., Kim, S. Y., Artis, S., Molfese, D. L., Schumacher, A., Sweatt, J. D. & Lubin, F. D. Histone methylation regulates memory formation. The Journal of Neuroscience, 30(10), 3589-3599 (2010). 13. Lubin, F. D., Roth, T. L., & Sweatt, J. D. Epigenetic regulation of BDNF gene transcription in the consolidation of fear memory. The Journal of Neuroscience, 28(42), 10576-10586 (2008). 14. Kerimoglu, C., Agis-Balboa, R. C., Kranz, A., Stilling, R., BahariJavan, S., Benito-Garagorri, E. & Fischer, A. Histone-Methyltransferase MLL2 (KMT2B) Is Required for Memory Formation in Mice. The Journal

Neuroscience Matters of Neuroscience, 33(8), 3452-3464 (2013). 15. Ahn HJ, Hernandez CM, Levenson JM, Lubin FD, Liou HC, Sweatt JD. c-Rel, an NF-κB family transcription factor, is required for hippocampal long-term synaptic plasticity and memory formation. Learn Mem, 15, 539 –549 (2008). 16. Durand M, Kolpak A, Farrell T, Elliott NA, Shao W, Brown M, Volkert MR. The OXR domain defines a conserved family of eukaryotic oxidation resistance proteins. BMC Cell Biol, 28, 13 (2007). 17. Wood, M. A., Hawk, J. D. & Abel, T. Combinatorial chromatin modifications and memory storage: A code for memory? Learn. Mem, 13, 241-244 (2006). 18. Adachi M, Autry AE, Covington III HE, Monteggia LM. MeCP2mediated transcription repression in the basolateral amygdala may underlie heightened anxiety in a mouse model of Rett syndrome. J Neurosci, 29, 4218–4227 (2009). 19. Giralt, A., Puigdellívol, M., Carretón, O., Paoletti, P., Valero, J., ParraDamas, A., & Ginés, S. Long-term memory deficits in Huntington’s disease are associated with reduced CBP histone acetylase activity. Human molecular genetics, 21(6), 1203-1216 (2012). 20. Kilgore M, Miller CA, Fass DM, Hennig KM, Haggarty SJ & Sweatt JD. Inhibitors of class 1 histone deacetylases reverse contextual memory deficits in a mouse model of Alzheimer’s disease. Neuropsychopharmacology, 35, 870–880 (2010).

Insight into the Plasticity of Brain-Sex: A Novel Gender State Alexander Di Giacomo1 and Daniel A. Dalessandro1 Human Biology Department, The University of Toronto. Toronto, Ontario CA.

Traditional conceptualizations of sex and gender are becoming less viable in explaining burgeoning incidences of intersex conditions. Bigender states, or Alternating Gender Incongruity (AGI) as per Ramachandran and Case, are exemplary of such a condition. These individuals experience spontaneous, conscious alternations between genders. They also report experiencing phantom genitals in their switches and great frequencies of ambidexterity. In their characterization of bigender individuals, Ramachandran and Case present individuals who reside in a transient sex and gender state, making them an ideal demographic to understand what contributes and constitutes normative psychosexual and body-sex

development. The experience of “bigenderism” underscores the importance of moving towards more dynamic conceptualizations of what constitutes gender and biological sex. Moreover, it questions what exactly constitutes the link between gender and body-sex, how these switches come about, the validity of a bipolar framework to understanding gender, and the potential role of cerebral lateralization in this unique population. On a more macro level, information regarding this condition can grant additional insight into other associated intersex conditions, such as gender identity disorder, as well as therapeutic interventions for any concurrent psychiatric conditions

BACKGROUND Sex and gender are traditionally considered to be representative of a single, invariable state of an individual; where sex refers to the anatomical differences between males and females, while gender is regarded as the societal and cultural differences.1 In accordance with this archaic model, it predicts that an individual born as an anatomical male [sex] will consciously interpret their society and culture from a stereotypical male perspective [gender], and vice-versa.1 This model is quite problematic in that it implies the following: firstly that sex and gender are in accordance with one another at all times and secondly that there can be no “mixing” of sex gender states. For example, an anatomical male cannot occupy a female gender state, or vice versa.1 Precedent in the scientific literature has only recently been able to qualitatively challenge this traditional definition.2 Analysis of homo- and transsexual individuals has revealed that sex and gender states are not homogenous.3,4 For example, a transsexual individual can anatomically be considered a “female” [sex] but self-identify as being male [gender].4 Clearly, there is a grey-area that remains to be uncovered, which will only come from the study of sex and gender as separate, dynamic entities. An individual’s sex-gender state is influenced constantly by four affecters: external morphology, sexual orientation, “body-sex” image, as well as gender identity.5 In traditional conceptualizations of sex and gender, these affecters are believed to work in a uniform, unidirectional fashion, creating a congruency between sex and gender identity.5 However, more contemporary definitions are orienting towards an understanding that these affecters function in a dynamic way, each contributing variable magnitudes. This contributes to an inherently dynamic sex-gender sate—a framework far different from what is traditionally understood. Using this dynamic model, one can envision hundreds of possible (and uncharacterized) discrete gender states all resulting from this inconsistent mixing of affecters. The scope of the present review will focus specifically on one gender state characterized under the constraints of this new model: “bigender”.1,5 As per the model, this gender state falls under the transgender “umbrella”. Individuals who are bigender are classified as experiencing conscious, transient switches between gender states, presumably due to varying affecter influence. This suggests that an individual’s sex-gender state is dynamic, due to variable magnitudes among affecters. Unfortunately, this is the extent of what is known about the bigender state. The nature of these affecter switches is yet to be characterized: for example, what triggers these switches between sex-gender states, as well as whether or not all affecters switch simultaneously or independently of one another, remains a topic for research.

In lieu of this, recent work by Ramachandran and Case has focused on better characterizing the bigender switches using self-report questionnaires.5 They propose that the involuntary nature of the bigender switches have an underlying anatomical basis; and interactions between anatomical and highly subjective socio-cultural factors account for the high variability within the bigender state.5 Although cursory, their work is specifically focused on laying a firm theoretical framework for the bigender state, allowing for more specific future work into the nature of bigender switches.5 REVIEWED RESEARCH MATERIALS AND METHODS Participants were recruited via an online forum for individuals who identify as bigender. Recruited participants were subjected to rigorous screening in order to determine the nature and frequency of gender switches. In order to determine plausible modalities that may facilitate gender switches, subjects were given the Edinburgh Handedness Survey. The purpose of this was to determine if bigender individuals were comparable to previously observed associations between handedness and gender.6 Subjects in the sample group also completed self-reports regarding the intensity and experience of phantom sex organs reported to occur with their gender switches. RESULTS Of the 600 users and contributors to the bigender website, 39 completed the full survey. Confounding individuals in the same group were removed, such as those with Multiple Personality Disorder (DID), as discerned by a clinician. This was primarily due to the fact that these individuals reported unconscious switches between gender states alongside personality. Of the 32 remaining participants, 11 were anatomically female and one identified as intersex (strictly for reasons of androgynous facial appearance). These participants then provided insight into their switches between gender states. Some exemplary reports regarding the nature of their bigender experience include insightful and thought-provoking accounts: “I still have the same values and beliefs, but a change in gender is really a change in the filter through which I interact with the world and through which it interacts with me.” In terms of reports of fantom body parts, individuals in Ramachandran’s study5 often reported concurrent cycling of phantom genitalia. 21/32 respondents reported experiencing phantom genitals; that is, genitals characteristics of their non-

Neuroscience Matters biological sex. These experiences were rated as moderate in strength. Bigender individuals in this study also exhibited a higher than expected incidence of ambidexterity: 6/28 individuals selfreported as ambidextrous.5 Interestingly, one respondent also selfreported being ambidextrous compared to 9 cissexual controls. DISCUSSION Bigender individuals spur much speculation as to what could possibly cause such a powerful and frequent change in gender. As per Ramachandran and Case, the unique nature of bigender individuals present a unique condition (which they have termed Alternating Gender Ingruity; AGI) that entertains multiple theoretical frameworks, such as: (1) gender switches as resultant of alternating hemispheric dominance, with which also switches “gender-typical” cognition and emotion; (2) hemisphere switching may be related to alternating sympathetic/parasympathetic patterns of emotional reactivity; (3) unusual sensory/parietal body map development, wherein there are competing, gendered body maps.5 In addition to spurring speculation regarding what could explain these bigender switches, bigender individuals also challenge the efficacy of our current understandings regarding sex and gender. The qualitative data collected by Ramachandran and Case5 challenge typical understandings of what constitutes sex and gender. More specifically, bigender individuals stand outside what is typically understood to contribute to gender development and gender constancy in the traditional framework. Bigender individuals and their random, frequent switches between gender states greatly underscores the importance of a more dynamic understanding of what constitutes sex, gender, and the bodybrain image. Clearly, in order to better orient oneself as to what underlays understandings of “maleness” or “femaleness,” one requires a more dynamic, or perhaps “plastic” understanding of normative gender development. Results from this demographic also sheds light on the bridge that connects components of the internal and external self (i.e. gender and body-sex). For example, it is possible (as per the authors) that both bigender switches and the experience of phantom genitalia are resultant of gendered body maps competing for sensory input. Thus, perhaps the link that connects the experience of “maleness” in terms of gender and biological sex is contingent on proper body-map development in the parietal lobe (the same can be said regarding “femaleness”). This offers precursory information regarding the nature of what constitutes the connection between brain and body. Moreover, it also sheds light on a potential region of interest in establishing the connection between body- and brain-sex.

Neuroscience Matters As seen in the results, bigender individuals have a higher than expected incidence of ambidexterity. This offers a potential avenue of understanding the nature of switches in the bigender state. Namely, it could be the case that ambidextrous individuals, who may share more information between hemispheres, may also switch between gender states5. However, until a larger sample group is recruited, this conjecture remains speculative. Of course, there are also shortcomings to this study. First and foremost, bigender states are still being understood. That is, there is still much more to understand until this condition is properly characterized and understood. As a result of this, investigators are limited to small sample sizes SIGNIFICANCE OF THE WORK The immediate implications of this work are multi-faceted. Firstly, social acceptance of any novel gender states requires thorough characterization. The present work, although cursory, will undoubtedly lead to further work investigating the influences of societal and cultural environments of gender. This will lead to a better understanding of the individual characteristics of bigender individuals, leading to de-stigmatization. With further studies of this gender state, also comes the design of better treatment paradigms for other comorbid mental illnesses, such as depression and social anxiety. The amount of productivity lost in society due to mental illness is staggering. Even more concerning is the ubiquity of such illnesses in the population of bigender individuals. A better understanding of the bigender state, and overall acceptance will lead to external funding for programs aimed toward treatment of the mental illnesses in these populations. Most important to consider is the ramifications of gender studies in general on our understanding of novel gender states. It was previously alluded that the high variability in affecter magnitude and localization can possibly lead to the characterization of many new (other than bigender) discrete gender states. The bigender state was discovered from studying transsexual populations. Therefore the implications of further examining bigender populations will lead to the discovery of further gender states; because social acceptance can only be achieved through a meticulous study of the unknown. As a final implication, the results from this study question what constitutes sex and gender development. Bigender individuals are seated in a rare, “transitional” zone of human sexuality. As such, they have the capability to shed light on how genes, hormones, and culture interact during brain development. This has far reaching implications in terms of understanding normative psychosexual development. In addition, deeper understanding of this condition

may help illuminate other associated intersex conditions, such as gender dysphoria. CONCLUSIONS It is clear from the data presented that there are distinct features characteristic of the bigender state. Although the data qualitative, it most effectively sheds light on the subjective subsets of gender. In particular, several are of interest: the comorbidity of the bigender state with handedness, the high frequency of the gender switches, as well as the high variability in the respondents’ self-reported characteristics of their gender states. With respect to handedness, this finding is interesting in that it gives support to the hypothesis of hemispheric localization of gender.6-8 It is easier to imagine that cisgender individuals will have their affecters localized in a single hemisphere in the brain; in individuals with left-hemisphere localization, their affecters will result in a consistent male gender state, whereas the opposite case will result in a female gender state. In bigender individuals, the high incidence of ambidexterity supports that they have affecters localized in each hemisphere of the brain, explaining the “switches” in their self-identified gender state.5 Furthermore, the consistently high frequency of these gender switches in bigender individuals suggests that the affecters alternate quite rapidly. The most significant finding is the subjectivity of what each respondent considers their “male” and “female” gender states. This gives some support to the concept that affecters switch independently of one another, as opposed to cohesively. To this extent, the high variability in both the frequency of switching as well as the characteristics of the individuals’ gender state is attributed to the individual variance in affecter magnitude. FUTURE DIRECTIONS There are certainly more questions posed than answered by these findings. The most predominant of which is what, exactly, triggers the variability in the affecters. Variation in an individual’s environment will undoubtedly result in affecter variation. Further studies can look at altering the composition of a specific environmental factor in a controlled environment to better determine the cause of the bigender switch. It would also be advantageous to design quantitative studies that complement the aforementioned qualitative results. For one, functional magnetic resonance imaging (fMRI) studies are the most important to conduct in order to conclude the hemispheric localization of affecters. For example, specific questions pertaining to each affecter can be asked in conjunction with fMRI imaging of bigender individuals to accurately trace activity.

Looking at the role of physiological and genetic factors in bigender individuals are also required. For example, male and female genders are characterized by specific hormone levels. It would thus be interesting to investigate the correlation between estrogen and testosterone in bigender individuals, for example, during female and male gender states, respectively. Finally, information derived regarding this intersex demographic can challenge barriers associated with attaining funding for research. More specifically, the more a condition such as AGI5 can be characterized, the more recognition studies of this nature can attract both funding and individuals who feel they fit features of this condition. This can contribute to greater sample sizes, allowing for the use of perhaps more parametric means of analysis. In a similar vein, with this greater recognition also comes further characterization of this and potentially other intersex conditions, allowing for more complete understandings of both the underlying modalities of intersex conditions and the individuals who experience them. However, until this barrier is surpassed, this research remains cursory. REFERENCES 1. Prince, V. Sex vs. Gender. International Journal of Transgenderism 8, 29-32 (2005). 2. APA Office of Public and Member Communications. Answers to Your Questions About Transgender Individuals and Gender Identity. Washington, DC: Author. 3. Clements, K et al. The Transgender Community Health Project: Descriptive Results. San Francisco Department of Public Health (1999). 4. Ramachandran VS, McGeoch, PD. Occurrence of Phantom Genitalia After Gender Reassignment Surgury. Medical Hypotheses 69, 1001-1003 (2007). 5. Case, LK, Ramachandran VS. Alternating Gender Incongruity: A New Neusopsychiatric Syndrome Providing Insight into the Plasticity of Brain-Sex. Medical Hypotheses 78, 626-631 (2012). 6. Savitz J, Van der Merwe L, Solms M, Ramesar R. Lateralization of hand skill in bipolar affective disorder. Genes Brain Behav 2007;6(8):698–705 7.Walletin M. Putative Sex Differences in Verbal Abilities a n d Language Cortex: A Critical Review. Brain Language 108, 175183 (2009). 8. Clements AM, Rimrodt SI, Abel JR, et al. Sex Differences in Cerebral Laterality of Language and Visuospacial Processing. Brain Language 98 150-158 (2006). 9. Werntz DA, Bickford RG, Bloom FE, Shannahoff-Khalsa DS. Alternating Cerebral Hemispheric Activity and the Lateralization of Autonomic Nervous Function. Human Neurobiology 2, 39-43 (1983).

Received April 2nd, 2013; revised Month, ##, 200#; accepted Month, ##, 2013. Address correspondence to: Daniel Dalessandro <daniel. dalessandro@utoronto.ca.>

Neuroscience Matters

Down regulations of Insulin Activate Retrograde Signaling to Suppress Food Intake Farhiya Elmi1 Human Biology Department, The University of Toronto. Toronto, Ontario CA.

Throughout the last three decades obesity has been a major concern to public health. It can be understood as a medical state in which the quantity of energy ingestion surpasses energy usage. Some of the well known comorbidities that are presented alongside obesity are cancer, diabetes, cardiovascular disease and depression. Health officials in the past and present use various media outlets as a means to communicate to the growing population the seriousness and fatality of this disease. In schools, physical education teachers are required to be trained in order to instruct their students regarding healthy food choices as well as healthy living options. In the science realm many researchers have observed brain patterns and neuronal mechanism that underlie these feeding habits. For these obese individuals food becomes the ‘drug’ of choice. In the past a lot of focus has been placed on the ventral tegmental area (VTA). The VTA is known as the region of the brain that is implemented in the reward system. It is in this region where the dopamine neurons

reside. Dopamine is an important neurotransmitter as it reinforces behaviors such as feeding, food prediction, sex and motivation etc 8 . It is also shown to play a role in drug addiction. Although in the past insulin has been recognize to play a role in the inhibition of feeding habits, its underlying mechanism was still a matter of the unknown. However, Gwenael Labouebe and colleagues in a recent paper, which is also the focus of this paper, revealed new insights regarding the role of insulin in the ventral tegmental area1. They proposed in their research paper after performing several experiments that insulin induces long term depression (LTD) of the excitatory neurons which synapse onto the VTA dopamine neurons through the means of endocannabiniods1. In addition to the inhibition of these neurons, these researchers also concluded that insulin also reduces anticipatory actions and inclinations for food associated signals1.

Keywords: Obesity; Insulin; ventral tegmental area; dopamine; long term depression; glutamate; endocannabinoids. INTRODUCTION The discovery of the insulin hormone in 1922 by researchers Charles Best and Frederick Banting at the University of Toronto has notably changed the way diabetes is perceived. Prior to the discovery made by Banting and Best, patients’ sufferings from diabetes had the inevitable prognosis of fatality. However, the revolutionary impact of this hormone is not limited to its role in the glucose uptake of the cells. It has also been shown to play several imperative roles in other parts of the body. Newly emerging studies have demonstrated the role of insulin in the brain and its promising therapeutic roles. The ability of insulin to cross the brain’s blood barrier (BBB) has assisted researchers to further observe its role in the brain and the various mechanisms that it is involved in. With the brain expressing insulin receptors, researchers use this as a means to alter and study the various effects of the insulin (ligand) - receptor interaction. Recently, intra-nasal insulin has been shown by some researchers to play a role in alleviating depression symptoms. Insulin has been shown to have therapeutic effects in Alzheimer disease 2. Furthermore, the introduction of insulin into the VTA has the ability to hinder opioid induced feeding habits 3. Aside from opioid feeding, insulin plays a role in regular feeding habits. Following the intake of food, insulin is released into the brain and acts through the down regulation of its tyrosine kinase receptor (TKR). This is return

sends out satiety signals to the brain1,4. Insulin is also believed to fulfill this role by acting on the VTA dopamine neurons. The ventral tegmental area, also known as VTA, plays an imperative role in the brain. The VTA contains these dopamine neurons and projections to the nucleus accumbens which form the reward circuitry of the brain. The release of dopamine in this region reinforces behaviours such as feeding, food prediction, sex and motivation etc 8. It is also shown to play a role in drug addiction. Neurons which synapse onto the VTA dopamine neurons can stimulate long term potentiation (LTP) or long term depression (LTD) 1. Although insulin is understood to play role in this area to suppress feeding behaviour, the underlying mechanism that took place remained unknown. Several studies in the past have suggested various models of the function of insulin in the VTA. Recently researchers were able to demonstrate through the mechanism of retrograde signaling insulin can induce LTP of the glutamageric neurons, which act on the VTA1. This in return has an impact on feeding behaviour as well as anticipatory behaviour1. RESULTS The affect of insulin induced LTD primarily took place at excitatory neurons1 In order to determine whether insulin depressed excitatory synaptic transmission of the VTA neurons, Gwenael Labouebe and

colleagues using an electrode on brain slices of mice, stimulated excitatory post synaptic current (EPSC) from various glutamate neurons which act on the VTA 1. Next they applied an insulin bath solution and it was observed that insulin caused LTD of the stimulated EPSC1. They also wanted to observe if insulin required its receptor to carry out its affect 1. Hence, by introducing a toxinpicropodophyllotoxin (PPP) they were able to block the activity of the insulin receptor1. Here they observed that when the receptor was occluded, insulin induced LTD did not drastically vary from the control1. Furthermore, from their results they also observed that insulin did not affect GABA neurons and their impact on the VTA neurons. Therefore, they concluded that insulin induced long term depression of the VTA neurons had the most affect on the excitatory (glutamatergic) neurons1. The process of LTD required retrograde endocannabinoid signaling and binding of ligand to CB receptor1 In this section of their paper the authors were aiming to understand how the down regulation of insulin modulated LTD of the excitatory neurons1. By performing other experiments they were able to determine that the underlying mechanism of insulin was not influenced by AMPAR trafficking1. However, they observe that the binding of insulin to its receptor and the down regulated activity lead to the synthesis of the endocannabinioids such as 2-arachidonylglycerol (2-AG) and Anandaminde1. These endocannabinoids in return crossed the lipid bilayer and bind to the cannabinoid receptor 1 (CB-1) on the presynaptic neuron1. Once the endocannabinoid binds to the G-coupled protein receptor, it leads to the blockage of calcium channels and the outflow of posstassium. This in return interferes with vesicle transmission; in this case, blocking the release of glutamate neurotransmitters1. The activity of CB-1 receptors has been demonstrated in other studies including its role in the hippocampus by initiating depolarized induced suppression of inhibition (DSI) 5. In this study the affect of CB-1 receptor mediated depolarization induced suppression of excitation (DSE). The paradigm of DSE functions by inhibiting presynaptic release of glutamate. An important finding that the authors also concluded is that although CB-1 receptor activation is essential for the course of LTD, this activation is not required for its maintence1. The effects of LTD were attenuated after applying AM251; CB-1 receptor antagonist 1. The role of insulin in predicting food behaviours1 Interestingly, when mice were fed a meal high in fat, the LTD activity of insulin was halted1. The researchers rationalized this result as being due to an increase rise in endogenous insulin and this in return blocked the effect of the administered insulin1. The researchers were aware of the normal activity of rats prior to food intake from past experiments. Furthermore, they were also aware that rats would increase their mobility before the intake of food and that this was correlated to the rise in dopamine levels in the VTA1. However, once they administered Intra- VTA insulin to

these mice, they noticed a reduction in their movements1. The mice spent less time crossing over, rearing and tunneling- all related to anticipatory food behaviour1. SIGNIFICANCE AND CONCLUSION Prior to this study, there were proposed models of the function of the insulin in the VTA, but not using endocannabiniods as a model. Researchers both in the health and neuroscience realm can utilize the results of this study to their advantage with this mechanism now in mind. Not only is it imperative to understand how insulin can induce LTD via endocannabinoids to suppress feeding habits, but it can also serve as a model to understand the brain functions of obese individuals. With the rising levels of obesity, public health has become a major concern. Some of the well documented pathological illnesses that follow obesity include cancer, diabetes and cardiovascular diseases. However, newly emerging studies have correlated obesity with depression; a serious mental illness that affects most people 6. If we can help reduce obesity levels by administering intranasal insulin and repairing damage to the VTA through this process, then we can also assist in reducing the incidence of depression. Hence, the neuronal pathway of insulin in the VTA can serve to contribute as a way to medicate pathological diseases such as obesity and depression. It is a less invasive treatment with minimum to no side effects. Furthermore, this model of insulin inducing LTD in the VTA by means of retrograde signaling can serve as an additional model for neuronal plasticity and shape the way we perceived it1. However in this paper, the failure to mention the potential use of fatty acid amide hydrolase inhibitor (FAAH inhibitor) can be viewed as a shortcoming, as FAAH inhibitor prolongs the activity of the endocannabinoids by blocking their breakdown7. FAAH inhibitor has also been shown in the past to be affective in medicating some psychiatric and neurological disorders including anxiety, depression and has been shown to play a therapeutic role in obesity. Finally, in the next ten years with this model (insulin induced LTD) in mind, we will be able to see profound changes in obesity levels such as controlling addictive food behaviours and an overall decrease in obesity and depression levels. REFERENCES 1. Labouebe, Gwenael et.al. Insulin induces long-term depression of ventral tegmental area dopamine neurons via endocannabinoids. Nat Neurosci. 16, (2013). 2. Mebel, Dmitry et.al. Insulin in the ventral tegmental area reduces hedonic feeding and surpresses dopamine concentration via increased uptake. Euro Journ of Neurosci. 36, 2336-2346 (2012). 3. Young Jou, Lee et. al. Alzheimer’s phenotypes induced by overexpression of human presenilin 2 mutant proteins stimulate significant changes in key factors of glucose metabolism. Mol Med Rep. (2013). 4. A. Christine, Konner et.al. Role for insulin signaling in catecholaminergic neurons in control of energy homeostasis. Cell Metabol.13(6), 720-728 (2011).

Neuroscience Matters 5. Zachariou, Magarita et.al. A biophysical model of endocannabinoidmediated short term depression in hippocampal inhibition. PLoS One. 3, (2013). 6. J,Thormann et.al. Obesity and Depression: An Overview on the complex interactions of two diseases. Fortschr Neurol Pscyhiatr. 81 (3), 145-53 (2013). 7. Blankman JL and Cravatt BF. Chemical probes of endocannabinoid metabolism. Pharmacol Rev. 19; 65 (2), 849-71 (2013). 8. Blum, K et.al. Dopamine genetics and function in food intake and substance abuse. J Genet Syndr Gene Ther. 10; 4 (121) (2013).

Neuroscience Matters Received April, 20123; revised Month, 04, 2013; accepted Month, 04, 2013. Address correspondence to: Farhiya Elmi; farhiya.elmi@mail. utoronto.ca Copyright © 2013 Dr. Bill JU, Human Biology Program

Monophosphoryl Lipid A as a Potential Treatment for Alzheimer’s Disease Mu Hsuan Ho1 Human Biology Department, The University of Toronto. Toronto, Ontario CA.

Alzheimer’s disease is a neurodegenerative disease characterized by degeneration of cognitive and behavior function over time, which ultimately leads to death. It is the most common form of dementia worldwide. Patients affected by Alzheimer’s disease have poor prognosis, and no complete cure is available with the current field of medicine. Alzheimer’s disease pathology includes the accumulation of amyloid β protein and the formation of neurofibrillary tangles in the brain. Strong inflammation mediated by microglia also synergistically affects the progression of Alzheimer’s disease. However, previous studies have shown that microglia activation plays a role in the clearance of amyloid β protein through phagocytosis. Toll-like receptors (TLRs) on the surface of microglia are primarily responsible for this activation. As a result, recent studies have been focusing the activation of the innate immune system to treat Alzheimer’s disease in preclinical models. Hence, the identification of a suitable ligand

to trigger a sustained but mild inflammation becomes important. Monophosphoryl lipid A (MPL) is a modified and detoxified form of lipopolysaccharide that can be isolated from Salmonella Minnesota, a specific Gram- negative bacterium. With the characteristic of launching a low and sustained immune response while retains several immunomodulatory properties of LPS, MPL has been used widely as numerous successful and safe vaccines. This review paper shows that MPL stimulation in-vitro activates TLR4 and its downstream signaling for an immune response. This leads to an up-regulation of amyloid β clearance through microglia phagocytosis. Moreover, MPL intraperitoneal injection in an Alzheimer’s disease mouse model, APP(swe)/PS1(M146L), leads to an enhancement in amyloid β clearance and improvement in cognitive functions. The data suggest that MPL could potentially be a useful treatment for Alzheimer’s disease patients in the future.

Key words: Alzheimer’s Disease; amyloid β; innate immunity; inflammation; microglial cells; phagocytosis; Toll-like receptor 4 (TLR4); Monophosphoryl lipid A (MPL) I. BACKGROUND Alzheimer’s disease is currently the most common form of dementia. Loss of neurons occurs in the cortex, which resulting in atrophy of the affected regions1. Slow developed symptoms are key clinical features, which interfere with memory, thinking, and behavior over the course of the disease1. The prognosis of Alzheimer’s disease is poor, and patients typically have symptoms worsen over time that eventually causing death. The incidence of Alzheimer’s disease positively correlates with increasing age. Pathology of Alzheimer’s disease is characterized by the deposition of amyloid β plaque and the formation of neurofibrillary tangles at the parenchymal area of the brain1. Currently, there is no cure

for this disease, but various treatments are available as the research continues. It is generally believed that inflammatory processes mediate by the microglial cells in the brain plays a role in the progression of Alzheimer’s disease. Microglial cells are a type of glia cell resides in the brain and spinal cord. Upon activation, these cells scavenge metabolic wastes through phagocytosis and regulate immune responses by releasing cytokines. Recent studies showed that activation of microglial cells can enhance the clearance of amyloid β via phagocytosis, which may be beneficial for Alzheimer’s patients2. Toll-like receptors (TLRs), a class of immunoreceptors, have key functions in the innate immune system3. Previous studies

showed that these TLRs on the surface of microglia interact with amyloid β proteins, which trigger downstream signaling cascades to launch immune responses3,4. Specifically, activation of TLR2, TLR-4, and TLR-9 with agonist increases amyloid β protein clearance in mice models4. On the other hand, transgenic mice with deficiency in TLR-2 and TLR-4 are linked to a lack of amyloid β protein clearance, which resulting in an extra accumulation of amyloid β proceeds by cognitive tasks impairment3. As a result, screening for a suitable ligand to trigger TLR activation can potentially be a treatment to enhance amyloid β clearance in Alzheimer’s disease patients. Lipopolysaccharides (LPS) are large molecules embedded on the outer wall of Gram-negative bacteria; these molecules are endotoxins that can elicit a strong immune response. Monophosphoryl lipid A (MPL) is a chemically detoxified form of LPS isolated from Salmonella Minnesota with various modification on its structure5. It possesses properties such as at least 100-fold less pyrogenic, but still induces many similar immunomodulatory responses relative to LPS6. These characteristics allow MPL to be used extensively as safe vaccine adjuvants in humans5. The activation of innate immune responses has been tested in preclinical models as a possible treatment for Alzhiemier’s disease7. Here, the author Michaud et al. hypothesized that MPL is a suitable ligand to trigger a chronic and sustained mild inflammation, hence achieving a consistent clearance of amyloid β to treat Alzheimer’s disease8. II. REVIEWED RESEARCH MATERIALS AND METHODS NF-κB/AP-1 Activation Assay HEK293 cells were prepared for in-vitro NF-κB/AP-1 Activation Assay in this review paper. Cells were transfected with vectors expressing TLR4, MD-2, and CD14. Different concentrations of MPL, LPS, PBS, anti-TLR4, and Pam3CSK4 (anti-TLR2) were used to stimulate the cells for 5 hours under FBS-free condition. The expression levels of NFκB/AP-1 were measured using realtime PCR. E. coli Beads Phagocytosis Assay BV-2 microglia were stained with phalloidin/DAPI and incubated for 18 hours with 1 μg/mL of MPL, LPS, and PBS using six-well plates. Fluorescein-coated E. coli K-12 BioParticles were used to replace the previous medium, and incubated for 2 hours at 37°C. Morphology of the microglia cells were observed under a fluorescence microscope. Fluorescence intensity was measured and recorded for each of the condition.

Amyloid β Phagocytosis Assay BV-2 microglia were stimulated with 1 μg/mL of MPL, LPS, and PBS for 18 hours. 1 μg/mL of fluorescent amyloid β oligomers were used to incubate under a FBS-deprived culture medium for 3 hours at 37 °C. Transgenic Mouse Line An Alzheimer’s Disease mouse model, APP(swe)/PS1(M146L), was generated from the C57BL/6J mice that harbored the human presenilin and amyloid β precursor protein. Only 3 months old males were used in this experiment. MPL, LPS, and PBS were administered through intraperitoneal injection to different mice one a week, for a total of 12 consecutive weeks. The cognitive function of the mice was examined with repeated trials of T water maze. RESULTS In-vitro Assays The NF-κB/AP-1 Activation Assay revealed that MPL drives a distinct TLR4 stimulation from LPS. Real-time PCR showed that there is a lower NF-κB/AP-1 activation in MPL stimulated cells relative to LPS stimulated cells; there is also no detection of NFκB/AP-1 activation upon MPL stimulation of TLR2 (see Figure 1). Furthermore, fluorescence microscope images revealed that microglial cells appeared to undergo morphological changes upon MPL and LPS stimulation relative to PBS control; microglial cells appear to be amoeboid shape under non-activated PBS condition, and elongated shape when activated via MPL and LPS (see Figure 2). E. coli Beads Phagocytosis Assay and Amyloid β Phagocytosis Assay showed MPL and LPS stimulation leads to phagocytosis of beads and amyloid β; data indicated strongest response of phagocytosis for LPS, medium response for MPL, and least response for PBS control (see Figure 3). In-vivo experiment APP(swe)/PS1(M146L) transgenic mice injected with MPL showed a significant enhancement of the amyloid β plaque clearance from the brain tissue section, and improvement of cognitive functions; LPS injection revealed no clearance but an increase in amyloid β plaque deposition relative to PBS, and no improvement in any cognitive function (see Figure 4).

Neuroscience Matters

Neuroscience Matters some promising methods for therapeutic treatments. However, there are several limitations from this set of experiments. First, APP(swe)/PS1(M146L) mouse model may not perfectly reflect human Alzheimer’s Disease conditions. For example, despite the transgenic mice were capable of expressing amyloid β proteins, there was no evidence of any neurofibrillary tangles shown in the brain tissue sections. Moreover, the control base line of amyloid β clearance was not compared with the original wildtype C57BL/6J mice. Last, cognitive functions examined using T water maze with different mice were mentioned, but no actual data was provided.

Figure 1. (top) NF-κB/AP-1 Activation Assay. TLR4 activation measured through the relative expression of NF-κB/AP-1 with different concentrations of MPL, LPS, and PBS. Anti-TLR4 at 2 different concentrations is used as a positive control. (bottom) TLR2 activation measured through a relative expression of NFκB/AP1 with different concentration of MPL. Pam3CSK4 (Anti-TLR2) is used as a positive control.

IMAGE NOT AVAILABLE. Figure 2. Microglial cells stained with phalloidin (green) and DAPI (blue). PBS non-activated microglial cells remained under amoeboid shape. LPS and MPL activated microglial cells changed to elongated form.

Figure 3. E. coli Beads Phagocytosis Assay and Amyloid β Phagocytosis Assay. (A) Microglial cells with no beads, beads only, beads with MPL, and beads with LPS; degree of phagocytosis measured with fluorescence intensity. (B) Image of fluorescence microglial cells with internalized beads; cytosol, nucleus, and beads, shown in red, blue, and green respectively. (C) Microglial cells with no Aβ, Aβ only, Aβ with MPL, and Aβ with LPS; degrees of phagocytosis measured with fluorescence intensity. (D) Image of fluorescence microglial cells with internalized Aβ; cytosol, nucleus, and Aβ shown in green, blue, and red respectively.

Figure 4. (Bottom) (A,B,C) Brain tissues (red) and the distribution amyloid β plaques (white dots) upon stimulation of PBS, MPL, and LPS for 12 weeks. (D,E) The relative number and plaque load of amyloid β plaque. (F) Western blot analysis of the amyloid β protein; actin is used as a housekeeping positive control protein.

DISCUSSION The two major findings from the data supported the hypothesis that TLR4 agonist, MPL, induces phagocytosis of amyloid β and elicits an inflammatory response considerably lower than the one generated by LPS. In addition, chronic administration of MPL significantly improved Alzheimer’s Disease-related pathology by restoring cognitive functions and reducing levels of amyloid β plaques in size and number. First, in-vitro assay confirms the role of MPL as an agonist of TLR4 and not TLR2 to trigger the downstream signaling of NF-κB/AP-1. LPS, on the other hand, triggered both TLR4 and TLR2 with a stronger downstream signaling cascade. This was potentially due to the structural difference between LPS and MPL, which induced different conformational changes of TLR49. Presumably, this altered the recruitment of distinct adaptor proteins to the TLR4 intracellular domain, thus providing different responses9. Next, despite the fact that in-vitro phagocytosis assays showed LPS is superior to MPL in the phagocytosis of both E. coli beads and amyloid β, this was not true when the setting is brought to a long term in-vivo condition. Surprisingly, chronic LPS administration in mice exacerbated the amyloid β plaque load. An explanation is that a stronger immune response launched by LPS significantly accelerated the phagocytosis of amyloid β plaques at first, but excessive and sustained inflammation resulted in the tolerance and desensitization that reduced the clearance of amyloid β plaques over time. Therefore, MPL was the superior candidate that triggered a lower level immune response that consistently stimulated the clearance of amyloid β plaque chronically. SIGNIFICANCE OF THE WORK The preclinical data from this review paper opened up opportunities for many future studies on microglial cells and their correlation with Alzheimer’s disease. These data also suggested

FUTURE DIRECTIONS While MPL seems to be very promising for treating Alzheimer’s disease, further research is required to understand other possible consequences MPL could elicit. Research should focus on other what other immunoreceptors could potentially be activated by MPL, and screen for any collateral damage due to inflammations in the brain. Moreover, MPL has not been tested in non-human primates or clinical settings10. Hence, future studies should also focus on in-vivo experiments of MPL in non-human primate models, and later proceed to human patients. REFERENCES 1. Querfurth H.W. & LaFerla F.M. Alzheimer’s disease. N Engl J Med 362(4), 329-344 (2010).

2. Wyss-Coray T. Inflammation in Alzheimer disease: Driving force, bystander or beneficial response? Nat Med 12(9), 1005–1015 (2006). 3. Tahara K. et al. Role of toll-like receptor signaling in Abeta uptake and clearance. Brain 129(11), 3006–3019 (2006). 4. Reed-Geaghan E.G., Savage J.C., Hise A.G., Landreth G.E. CD14 and toll-like receptors 2 and 4 are required for fibrillar Abeta-stimulated microglial activation. J Neurosci 29(38), 11982–11992 (2009). 5. Casella C.R., Mitchell T.C. Putting endotoxin to work for us: Monophosphoryl lipid A as a safe and effective vaccine adjuvant. Cell Mol Life Sci 65(20), 3231–3240 (2008). 6. Garçon N., Van Mechelen M. Recent clinical experience with vaccines using MPL and QS-21-containing adjuvant systems. Expert Rev Vaccines 10(4), 471–486 (2011). 7. Shaw A.C., Joshi S., Greenwood H., Panda A., Lord J.M. Aging of the innate immune system. Curr Opin Immunol 22(4), 507–513 (2010). 8. Michaud J.P. et al. Toll-like receptor 4 stimulation with the detoxified ligand monophosphoryl lipid A improves Alzheimer’s disease-related pathology. PNAS 110(5), 1941-1946 (2013). 9. Park B.S. et al. The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex. Nature 458(7242), 1191–1195 (2009). 10. Fiala M., Veerhuis R. Biomarkers of inflammation and amyloid-beta phagocytosis in patients at risk of Alzheimer disease. Exp Gerontol 45(1), 57–63 (2010).

Molecular Mechanisms of Stress Induced Memory Consolidation Nariman Hossein-Javaheri1 Human Biology Department, The University of Toronto. Toronto, Ontario CA.

Strong exposure to stress can impair memory consolidation and retrieval. Some levels of stress however, may improve consolidation of hippocampal dependent memories. Stress activates corticosterone pathway where it stimulates gluconeogenesis, activating anti-stress and anti-inflammatory responses in rodents. Activity of these receptors to some extent, can help generation of new memories. Little is known about the underlying molecular mechanism of this pathway. Examining changes in the activity or expression of proteins that are generally important for consolidation of memories, CaMKIIα, TrKB, ERK,

Akt, PLCγ, BDNF and CREB, may provide some insights on how glucocorticoid receptors improve memory consolidation. Furthermore, activity of glutamatergic receptors involved in memory consolidation, NMDA or AMPA, may also be affected in response to stress. In this review, overall changes in the activity of these proteins and receptors in response to stress were investigated.

Key words: Stress; Inhibitory-avoidance task; glucocorticoids; CaMKIIα; brain-derived neurotrophic factor (BDNF); cAMP response element-binding protein (CREB); Amnesia

Neuroscience Matters INTRODUCTION Exposure to high levels of stress or chronic stress can impair memory consolidation. During stress, corticosterone is released in rodents and acts as an anti-inflammatory, immunosuppressant substance and binds to glucocorticoid receptors (GR). Activation of GRs in the hippocampus, reduces the overall activity of N-methyl-D-aspartate (NMDA) receptors and decreases hippocampal synaptic plasticity1. Not only consolidation but also retrieval of memories are affected under stressful conditions2. Although high activity of glucocorticoid receptors seems to be destructive for memory functioning, mild stress can enhance overall activity of the memory system3,4. It is proposed that perhaps this is an adaptive mechanism in order to encode emotional or important memories which are associated with some levels of stress. This could explain why GRs are expressed mainly in the hippocampus, amygdala and pre-frontal cortex5. The molecular pathway explaining how corticosterone can enhance memory functioning is incompletely understood. In vitro experiments suggest that glucocorticoid action can enhance α-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)- GluA2 receptor trafficking and facilitate stress learning6. Furthermore, GRs enhance activity of MAPK-Egr1 allowing synaptic plasticity both in vivo and in vitro studies7. Stress can lead to AMPAR trafficking, synaptic plasticity and long-term potentiating (LTP) but the effects of GRs on proteins that are dominantly responsible for LTP have remained elusive. Multiple proteins are expressed during memory consolidation from which CaMKIIα, CREB and BDNF have significant importance. These proteins however are mainly activated in the pathway regulated by NMDA receptors during memory encoding8. In this review, fear memory induced by the activity of glucocorticoid receptors and possible effects of stress on the expression of CaMKIIα, TrKB, ERK, Akt, PLCγ, BDNF and CREB in hippocampal neurons was investigated9. REVIEWED METHODS Animals and behavioral training: Adult rats (8-9 weeks old) were trained on inhibitory avoidance task, receiving a foot shock of 0.6mA or 0.9mA for 2seconds . In order to examine possible effects of glucocorticoid receptors on the formation of new memories, these receptors were antagonized by injecting RU38486, dissolved in 5% DMSO and 1xPBS, bilaterally into dorsal hippocampi. Two groups of rats were trained where one was injection by RU38486 and the other, received only the vehicle. Injections were made 15 minutes before or immediately after training. Memory retrieval was tested 1 hour, 2 days and 7 days after training. Biochemical analysis: For biochemical studies, the same procedure was performed but rats were trained in three groups using 0.9mA shock to elicit a more traumatic memory. First group was injected with RU38486, Second group received an injection of the vehicle alone. The third group, called the naive group,

Neuroscience Matters was not trained but was injected with the antagonist. Rats were then sacrificed 30minutes or 20hours after training. Tissue was fixed with 10% formalin. To verify proper placement of cannula implants, brain slices were examined under a light microscope. Dorsal hippocampi were then dissected in a dissection buffer followed by homogenization. After filtration, synaptosomes were obtained by centrifuging the filtrate at 1000g. Western blot was then carried out for the obtained sample. Protein concentrations were determined by using Bio-Rad protein assay. After running the SDS-PAGE gel, membranes were dried, reactivated and washed before getting blocked by 3% milk and TBS. They were then incubated with the desired primary antibodies for 24hours. Samples were washed after and treated with the proper secondary antibody, washed and incubated with detection reagents. To obtain data, membranes were exposed to densitometric analysis. Statistical analysis: In order to control the significance of data, one-way ANOVA test was performed.

It is important to know however that only BDNF and no other neurotrophic factors (NGF, NT-3) could rescue memory retention. Furthermore, it was observed that BDNF can only rescue RU38486 induced amnesia and cannot treat memory impairment induced by propranolol. Clearly, BDNF is required for GR dependent long term memory consolidation according to the behavioral data. At the synaptic level, authors were able to find that BDNF can rescue the molecular impairments caused by RU38486. Biochemical analysis revealed that the overall activities of pCREB, Arc, pCaMKIIα, GluA1, pERK1/2, pTrkB, pAkt and pPLCγ after BDNF treatment were increased. Therefore BDNF is essential for inhibitory avoidance long-term memory formation according to both biochemical and behavioral data. In conclusion, glucocorticoid receptors induce memory consolidation through CaMKIIα-BDNF-CREB pathway.

RESULTS Behavioral testing based on inhibitory avoidance task, revealed that blocking glucocorticoid receptors with RU38486, can completely disrupt memory retention while injection of the vehicle alone, had no effect. This antagonist, affected long-term memory formation very rapidly but did not influence short term memories. Furthermore, the intensity of foot shock had no effect on memory recall. Thus, it is possible to conclude that overall activity of hippocampal GRs is required for the formation of new memories while short term recall is not affected. Biochemical analysis in the three experimental, control and naive groups suggested that blocking GRs significantly decreased phosphorylation and expression of proteins responsible for longterm potentiation. Overall, a decrease in concentrations of pCREB, Arc, pCaMKIIα, GluA1, pERK1/2, pTrkB, pAkt and pPLCγ was observed in synaptosomes. The expression levels however, were greater in the trained, experimental group in comparison to the naive group. Changes in phosphorylation were rapid and in order to see if observed changes were dependant on genomic or non-genomic regulation, RNA synthesis was inhibited using actinomycin D prior to training. actinomycin D did not affect training-related phosphorylation suggesting that changes were genomic independent. Many proteins affected in the pathway, TrkB, ERK1/2, Akt and PLCγ, constitute to the cellular response to BDNF. Hence, authors proposed that activity of CaMKIIαBDNF-CREB is necessary for GR induced memory formation.

Strong evidence support the hypothesis that mild excitations of glucocorticoid receptors, enhance memory formation. Exogenous corticosterone (cortisol in humans) can also improve memory functioning4. The main emphasis of this review paper, was on the expression of brain derived neurotrophic factor (BDNF) in response to stress and how it can recruit CREB. BDNF is a stress responsive gene. It increases the number of dendritic spines and enhances memory function through activation of CREB10. BDNF and CREB can get activated by excitation of glucocorticoid receptors in the hippocampus which is mediated by phosphorylation of TrkB 11,9. The whole pathway however, depends on the initial activity of cAMP where it regulates BDNF function in mature hippocampal neurons by modulating the signaling and trafficking of its receptor TrkB12. Exogenous glucocorticoids however, can reduce the activity of cAMP by activation of β2-adrenergic receptors2 . It has been argued however that since only BDNF, and no other neurotrophin, rescued the amnesia induced by GR blockage, and since this effect did not extend to β-adrenergic receptors, activity of BDNF is highly selective and limited to GRs9. In chronic stress, BDNF does not act as a memory consolidator, instead, it causes downregulation of GRs13 hence, less CREB expression. It is perhaps explainable how certain levels of glucocorticoids under mild stress can enhance learning while chronic or high levels of stress impair memory functioning3. These findings can be clinically applicable in rare conditions in which glucocorticoid receptors are inactive, saturated or require bypass. Or, perhaps BDNF can be used in treatment of chronic stress and mood disorders.

In order to prove their claim, BDNF was antagonized in rats trained in the inhibitory avoidance task. Testing was performed according to the previous procedure. Blocking the BDNF significantly reduced long term memory retention while short term memory was unaffected. Furthermore they were able to find that RU38486 induced amnesia can be rescued with BDNF treatment.

This experiment was performed with great control but some aspects of it that were slightly elusive. As mentioned previously, authors indicated that the observed effects were nongenomic. CREB phosphorylation and activation, allows immediate early gene expression of c-fos, BDNF and other neuropeptides. Hence, the effects cannot be completely genomic independent14.

DISCUSSION

Furthermore, It would have been interesting to examine memory retention in both adult and younger rats. BDNF expression and hippocampal neurogenesis can change significantly during different stages of development. In early life, BDNF levels are higher but it decreases with age. Early high levels however, should respond greater to stress, allowing better memory consolidation in a stressful environment. Or perhaps, stress exposure causes downregulation of glucocorticoid receptor through activity of BDNF and suppress consolidation. Greater investigation is required to reach a conclusion on this matter. In addition, in a new experiment, an agonist for glucocorticoid receptor could be used instead of an antagonist to see if performance on the inhibitory avoidance task is enhanced. An antagonist, blocks the activity of a receptor but an agonist may activate certain pathways regulated by GR which may or may not improve performance or memory consolidation. REFERENCES 1- Wiegert, O., Pu, Z., Shor, S., Joels., M., Krugers, H., Glucocorticoud receptor activation selectively hampers N-methyl-D-aspartate receptor dependent hippocampal synaptic plasticity in vitro, Neuroscience 135, 403-411 (2005). 2- Schutsky, K., Ouyang, M., Castelino, C.B., Zhang, L., Thomas, S.A., Stress and Glucocorticoids Impair Memory Retrieval via 2 Adrenergic, Gi/o-Coupled Suppression of cAMP Signaling, J Neurosci 31, 1417214181 (2011). 3- Schilling, T.M., Kolsch, M., Larra, M.F., Zech, C.N., Blumenthal, T.D., Frings, C., Schachinger, H., For whom the bell (curve) tolls: Cortisol rapidly affects memory retreival by an nverted U-shaped dose - response relationship, Psychoneuroendocrinology 38, http://dx.doi.org/10.1016/j. bbr.2011.03.031(2013). 4- van Marle, H.J., Hermans, E.J., Qin, S., Overeem, S., Fernandez, G., The effect of exogenous cortisol during sleep on the behavioral and neural correlates of emotional memory consolidation in humans, Psychoneuroendocrinology 38, http://dx.doi.org/10.1016/j. bbr.2011.03.03 (2013). 5-Roozendaal, B., 1999 Curt P. Richter award. Glucocorticoids and the regulation of memory consolidation. Psychoneuroendocrinology 25, 213–238 (2000). 6-Conboy, L., Sandi, C., Stress at learning facilitates memory formation by regulating AMPA receptor trafficking through a flucocorticoid action, Neuropsychopharmacology 35, 674-685 (2010). 7-Revest, J.M., Di Blasi, F., Kitchener, P., Rouge-pont, F., Desmedt, A., Turiault, M., Tronche, F., Piazza, P.V., The MAPL pathway and Egr1mediate stress-related behavioral effects of glucocorticoids, Nat Neurosci 8, 664-672 (2005). 8- Adams, J.P., Dudek, S.M., Late-phase long-term potentiation: getting to the nucleus, Nat Rev Neuroscie 6, 737-743 (2005). 9- Chen, D.Y., Mukku, D.B., Pollonini, G., Alberini, C.M., Glucocorticoid receptors recruit the CaMKIIa-BDNF-CREB pathways to mediate memory consolidation, Nat Neurosci 15, 1707-1714 (2012). 10- Luine ,V., Frankfurt, M., Interactions between estradiol, BDNF and dendritic spines in promoting memory, Neuroscience, http://dx.doi. org/10.1016/j.bbr.2011.03.031 (2012). 11- Alboni, S., Tascedda, F., Corsini, D., Bennatti, C., Caggia, F., Capone,

Neuroscience Matters G., Barden, N., Blom, J.M., Brunello, N., Stress induced altered CRE/ CREB pathway activity and BDNF expression in the hippocampus of glucocorticoidreceptor-impaired mice, Neuropharmacology 60, 13341346 (2011). 12- Ji.Y., Pang, P.T., Feng, L., Lu, B., Cyclic AMP controls BDNFinduced TrkB phophorylation and dendritic spine formation in mature hippocampal neurons, Nat Neuroscie 8, 164-172 (2005). 13-Chiba, S., Numakawa, T., Ninomiya, M., Richards, M.C., Wakabayashi,

Neuroscience Matters C., Kunugi, H., Chronic restrain stress causes anxiety-and depressionlike behaviors, downregulates glucocorticoid receptor expression, and attenuates glutamate release induced by brain-derived neurotrophic factor in the prefrontal cortex, Prog Neurophsychopharmacol Biol Psychiatry 39, 112-119 (2012). 14- Jeanneteau, F., Garabedian, M.J. & Chao, M.V. Activation of Trk neurotrophin receptors by glucocorticoids provides a neuroprotective effect. Proc Natl Acad Sci USA 105, 4862–4867 (2008).

INDUCING NEUROGENESIS: New path to recovery Ekaterina Kouzmina , & Ashkan Azimi1 Human Biology Department, The University of Toronto. Toronto, Ontario CA.

Currently, neurogenesis is a widely studied phenomenon, with a high interest in adult neurogenesis. This interest was sparked by the idea that adult neurogenesis may be used for treating neurodegenerative disorders or cortical and spinal cord injuries. Wang et al., wanted to check whether a commonly used drug, metformin, can stimulate adult neurogenesis and what behavioural effects it would have. They confirmed that metformin induces neurogenesis both murine and human neural precursor cultures via the aPKC-CBP pathway. There are two isoforms of aPKC (ζ and ί) necessary for the induction of neurogenesis both in culture and in vivo as they play different roles. Metformin-induced neurogenesis occurs both in the olfactory bulb and hippocampus. In particular, hippocampal neurogenesis enhances spatial memory by making it easier to update new information. These

findings have great implications for future research; metformin is an FDA-approved drug, meaning that if further experiments show that it has neuroprotective properties which subsequently produce significant behavioural improvements in patients, it can go into the market without undergoing excessively prolonged clinical trials. Since, these properties have not yet been proven, further research should focus on seeing metformin effects on stimulating neurogenesis of specific cell types, as well as trying combinatory treatment methods (such as using metformin coupled with other drugs). Once positive effects are seen in those treatments, the next stages would be to perform clinical trials in patients with neurodegenerative disorders (such as dementia) or those who acquired cortical damage as a result of environmental insults to the CNS (such as traumatic brain injuries, TBI).

Key words: neural stem cells, neurogenesis, adult neurogenesis, aPKC-CBP, metformin, spatial memory, Protein Kinase C I. BACKGROUND Neurogenesis is truly an astonishing phenomenon. It is a process that was originally thought to be exclusive to embryonic development and that it was lost by a great deal postnatally. It was not until Altman in 1962 that, by applying Tritiated thymidine post-lesion, observed new-born neurons1. This changed how we view the brain and discredits the previous dogma about the plasticity (or rather the lack of plasticity) in the adult brain. Now, knowing that brain is creating new cells, the search for the origin of these new cells started. In 1969, the identification of the rostral migratory stream (RMS), a pathway connecting the subventricular zone (SVZ/VZ) to the olfactory bulb (OB) showed that neural precursor cells (NPCs) - consisting of neural stem cells and progenitor cells - travel down the RMS and eventually terminate in the OB as interneurons2. So, the mammalian adult brain has some regenerative capacity;

however, is it enough to result in a noticeable functional recovery after injury? Unfortunately, this pool of cells is not efficient in repopulating the lost and damaged cortical mass, namely due to very limited neurogenesis in the mammalian brain and glial scars mostly occupy the damaged site. The reasons for this could be due to different extracellular environment in the CNS, the factors being produced by the support cells (glial cells), which now are of much of a greater value to us than before – when they were previously considered to be just the ‘glue’ holding the brain together and nothing more. However, the question remained; how is it possible to override this endogenous process and allow for regeneration of CNS postinjury? Huge advancements have been made over the years. Series of experiments in 2007 demonstrated the combinatory effects of erythropoietin (EPO) and EGF in cortical regeneration poststroke as well as some functional recovery in the rats3. These results were promising with one big caveat; using growth factors, well-

known mitogens, will increase the rate of tumorigenesis which can be detrimental to patients in a clinical setting. Hence, it is crucial to find substitute biologics (such as drugs) that are deemed safe to use. Take Metformin for instance, it is FDA-approved and is used as treatment for type II diabetes. II. REVIEWED RESEARCH MATERIALS AND METHODS4

located on curtains surrounding the maze. Swim speed, distance traveled, and escape latency were measured by a tracking device. After 5 days of training, memory probe test was done on the mice. Immediately after the probe test, the reversal training started (with the platform moved to another location) for two days. After the two-day training period, another probe test was given. RESULTS

Animals. In the reviewed article, the authors used CD1 mice at 2 months of age for their in vivo/behavioural studies. Cell Culture. Cortical precursors used for analysis were dissected out of the CD1 embryos at embryonic days 11.5-12.5. More than 400 cells/condition, in at least 8 randomized fields of view were counted per experiment. In utero electroporation. This technique was done at E13/E14 in CD1 mice. Quantification was done in 3-4 fields of view per 20micron coronal section.

Roles of isoforms aPKC has two isoforms: ɩ and ζ, which Wang et al. focused on. First of all they showed that both of these isoforms were important for neurogenesis. They used shRNA (small/short hairpin RNA) to knock down the expression of the specific isoform in culture of radial precursor cells extracted from cortices of embryonic mice (E12.5). Knocking out either isoform did not have any effect on the cell death or cell division, but the amount of newborn neurons was decreased (as marked by βIII tubulin in Figure 1A). Notably, the decreased neurogenesis was rescued by expressing the respective isoform of human aPKC (Figure 1B/C).

Maintenance and Differentiation of hESCs. The hESC were maintained in HESC medium and were depleted of feeder cells prior to differentiation neuralized embryoid bodies (EBs) were generated by culturing small cell aggregates with different factors. EBs were then cultured for another 12 days after which they were dissociated and re-plated with bhFGF. BrdU Labeling. Mice were BrdU-treated for SVZ and hippocampal analysis. For hippocampal BrdU-labeling experiments, in one set of experiments, 100 mg/kg BrdU i.p. injections and 200 mg/kg metformin or PBS injections were applied daily for 3 days. For another 9 days, metformin/PBS alone was injected daily. The sacrificed mice were perfused with 4% PFA. After fixing the brains, the hippocampus was sectioned at 20 μm. In another set, mice were given 200 mg/kg metformin or PBS injections for 7 days, with the BrdU injection on day 7 (analyzed 24 hours after). These brains were also used for SVZ analysis. For the behavioural analysis, BrdU injections were made on days 5, 6, and 7 and analyzed as above after 38 days. In a set for the TMZ/behavior experiment, the mice were injected with BrdU on day 38 and analyzed 24 hours later. For SVZ analysis, mice were injected 5 times with 60mg/kg i.p BrdU at 3-hour intervals, prior to daily 200 mg/kg metformin/PBS injections over the span of 21 days. The brains were sectioned at 14 μm. Morris Water Maze. The authors made daily metformin/saline injections (200mg/kg) in two-month-old females C57/129J t. Some subjects were also injected with 25 mg/kg TMZ or 10% DMSO. In addition, some were injected with BrdU. Mice were trained on the hidden platform under the water, which was made opaque with nontoxic white paint. Extra-maze visual cues were

Figure 1. A) The number of newborn neurons was significantly decreased when either isoform of aPKC was knocked down. B) Knock-down of murine aPKCɩ was rescued by expressing human form of aPKCɩ. C) Knock-down of murine aPKCζ was rescued by expressing human form of aPKCζ. (Wang et al., 2012) *p < 0.05, **p < 0.01

Authors further showed that aPKC is expressed on the apical side of precursors lining ventricles of day 12 embryonic mice. Differential knock down of the isoforms using in utero electroporation of shRNA shined the light on the particular roles that each isoform plays in neurogenesis. aPKCɩ was found to be important for maintenance of radial precursor population, in terms of not allowing these cells to turn into basal progenitors. This was shown by the fact that when aPKCɩ was knocked down, the number of radial precursor cells decreased significantly, but the number of basal progenitors increased significantly. This means, that the radial precursors differentiated in to basal progenitors instead of neurons. In contrast, aPKCζ was assigned a direct role of helping radial precursors differentiate into neurons. This was shown by the fact that when the ζ isoform was knocked down, the number of new neurons significantly decreased, while the number of radial precursors significantly increased. This means that the radial

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precursors simply could not differentiate into neurons. Metformin Activates aPKC and Promotes Neurogenesis The authors were interested in the pathway activated by metformin in the cortical precursor cells. Knowing that the aPKC-CBP pathway is activated in the liver cells by metformin, they screened for threonine403/410 phosphorylation, which confers aPKC kinase activity. They cultured precursors for one day, exposing them to metformin for 15 minutes, after which the lysate was probed for phosphorylation of the aforementioned residues on the aPKC proteins. They observed a reproducible increase in phosphorylation. They further analyzed the effects of metformin on the precursor survival, by looking at Cascapse-3 activity; metformin did not affect cell survival. They also observed that metformin increased the number of ßIII-tubulin-positive cells (marker for neurons) by two folds. This increase was observed with a decrease of Pax6/Sox2-postive cells, biological markers for neural stem cells. Up to this point, the authors showed that metformin activates aPKC-CBP and that it is involved in neurogenesis. They now wanted to link neurogenesis and aPKC-CBP pathway activity; is metformin-induced neurogenesis regulated by the aPKC-CBP pathway? They were able to show that metformin was unable to induce neurogenesis in the cell culture transfected with either the aPKC and/or CBP shRNA. The authors also looked into the effect of metformin on genesis of glial cells (astrocytes as well as oligodendrocytes). They observed an increase in both GFAP (astrocyte) and A2B5 (oligodendrocytes). All the experiments for this section were in vitro; the authors tested the validity of these findings in vivo. The authors performed in utero electroporation of embryos, while treating the moms with metformin. They observed a small but significant increase in neurons. Neurogenesis in human neural precursors The experimenters wanted to test whether metformin had an effect on neural precursors. They generated forebrain neural precursors from human embryonic stem cells. These neural precursors were cultured and subjected to several treatments. First of all, the myristoylated aPKC inhibitor was used to see the effects of getting rid of the aPKC isoforms. Similar to knockdowns in murine culture, cell death and division were not affected. Yet, neurogenesis was significantly decreased. In contrast, when the forebrain precursor culture was subjected to metformin, the amount of newborn neurons was substantially increased (Figure 2). Additionally, experimenters subjected the culture to aPKC inhibitor and metformin at the same time. The results showed that the presence of inhibitor eliminated increase in neurogenesis that was caused by metformin (Figure 2). This part of the experiment showed that metformin acts similarly on murine and human radial precursors using aPKC pathway in order to increase neurogenesis.

mice (CBP+/+) had the expected increase in neurogenesis upon metformin treatment (Figure 3). Yet, the heterozygous mice treated with metformin did not differ from heterozygous mice treated with saline in the numbers of newborn neurons (Figure 3). This showed that the heterozygous group was haploinsufficient as both copies of CBP were needed for enhanced neurogenesis, which in turn confirmed that metformin acts via aPKC-CBP pathway in vivo.

Figure 2. Metformin injections significantly increased neurogenesis in mice, compared to saline. This effect was eliminated by injecting metformin together with an aPKC inhibitor. (Wang et al., 2012) *p < 0.05, ***p < 0.001

Metformin Promotes Neurogenesis from Adult Forebrain NPCs (both OB and SVZ) Up to this point, the authors were focused on the effects of metformin on embryonic precursors. They wanted to test whether their hypothesis would hold true in the adult brain. They looked at NPCs in the SVZ. First, they confirmed the presence of aPKC in the adult forebrain using immunohistochemistry, confirming the presence of aPKC in both the nucleus and the cytoplasm in neurospheres assay. They reconfirmed this in vivo (immunostaining coronal sections). Since it is practically common knowledge that these NPCs will ultimately end up as olfactory bulb interneurons, the authors raised the question of metformin-induced olfactory neurogenesis. Using BrdU labeling, the authors observed a significant increase in BrdU-positive, NeuN-positive olfactory neurons. They also looked at the effects of metformin on SVZ NPCs. Another set of BrdU labeling was performed, demonstrating that metformin significantly increased the number of BrdU-labeled cells that were also positive for Ki67 (proliferation marker). Hippocampal neurogenesis Adult neurogenesis does not only occur in the olfactory bulb, but also in hippocampus. Wang et al. wanted to test the effects of metformin on this type of neurogenesis as well. Adult mice were injected with metformin as well as neuronal and progenitor cell markers (in order to see the effects). Murine dentate gyri were examined and analyzed upon sacrificing the animals. The analysis showed that metformin-injected mice had significantly more newborn neurons than control mice (injected with PBS). At the same time, there was no difference in the number of cells in the precursor pool. Authors also wanted to examine whether the action of metformin occurred via the CBP pathway. They used normal mice (CBP+/+) and heterozygous mice (CBP+/-). The mice from both groups were treated with metformin at the age of 7 months and were later sacrificed for analysis. The results showed that the homozygous

Figure 3. Number of newborn neurons increased in homozygous CBP+/+ when treated with metformin, but not in heterozygous CBP+/(haploinsufficient) mice. (Wang et al., 2012) *p < 0.05

Metformin Enhances Spatial Memory Formation The authors, having just shown the role of metformin in hippocampal neurogenesis, were interested in the behavioural consequences of such event. Do these newly made neurons partake in spatial memory function, or do they essentially have no functional merit? To test this, the authors made use of the famous Morris water maze task with two phases, acquisition and the reversal phases. The authors noticed that metformin-treated group acted superiorly to the control group not in the acquisition, but in the reversal task, where they had to relearn the placement of the hidden platform. After the completion of training, the authors confirmed that improvement in the task performance is associated with increased number of neurons in the dentate gyrus, by looking at the number of BrdU/NeuN-positive cells. In the next step, they wanted to establish causality between metformin-induced hippocampal neurogenesis and enhanced spatial learning. In order to do so, they decreased the NPC pool by selectively killing replicating cells in the brain, using temozolomide (TMZ) and observed whether this inhibited metformin-induced improvement of spatial memory. This experiment showed that TMZ hindered metformin’s action to induce enhanced learning on the reversal task. After the behavioural scoring was done, the authors wanted to make sure that this decrease in spatial learning was due to lower number of neurons in the dentate gyrus and not an unwanted side-effect of the TMZ treatment. They achieved this by BrdU-pulse labeling showing approximately a 50% reduction in BrdU-positive cells in the TMZ-treated group, when compared to the control group.

SIGNIFICANCE OF THE WORK In the context of CNS repair with any real hope of clinical application, this paper is one of the forerunners in the field. Let us expand on the current techniques used in laboratory (not clinical) regenerative medicine. The core idea behind repairing the nervous system is to replace lost and damaged cells. Countless CNS-related disorders are problematic since they eventually result in cortical loss; dopaminergic neurons in Parkinson’s Disease, cholinergic neurons in Alzheimer’s Disease, and motor neurons in Amyotrophic Lateral Sclerosis are just a few examples. So how is this cell replacement done? It can be done in two main ways; in vitro alteration and transplantation back into the patient/ damaged subject, or in vivo manipulation. The former, explained another way, is basically altering the differentiation state of the stem cell to the cell type of interest and then transplanting it back into the subject, hoping it will integrate into the circuitry and be functional. This method is useful, because it allows for greater control when pushing the neural lineage; however, there is the risk of autoimmune response, cells dying before getting to the desired destination, or inappropriate/lack of integration into the CNS. The latter option, the endogenous repair route, is far more complex. However, it will be a much more efficient and safer way if we could successfully harness the body’s endogenous proliferation and differentiation machinery. Rather than exogenously modify cells and transplant them back in the body, what the authors of this paper are offering is a pharmacological approach to in vivo endogenous repair, using metformin. What is beneficial about using metformin is the fact that it is FDA-approved which makes it a lot quicker for it to be used on clinical trials, compared to other newly-discovered/synthesized biologics (which would expectedly take much longer to be deemed safe and approved). FUTURE DIRECTIONS So what would be the next step in the grand scheme of things? The authors have successfully established the role metformin plays in neurogenesis and subsequent enhancement of spatial memory. The authors make the point that hippocampal neurogenesis is of high relevance to this paper, since it is a structure that is quite vulnerable in disease state (examples, Alzheimer’s Disease and ischemic stroke). Something that this paper offers is a rather well defined molecular pathway (aPKC-CBP). Further research on this pathway can allow for better adjustment of neurogenesis; this fine tuning would hypothetically allow for specific and directed regeneration. In addition, there are other drugs that have been found to play a role in this endogenous repair. CyclosporinA (CsA) has been shown to increase cell survival both in vivo and in vitro in mice, without affecting cell cycle fate and differentiation profile5. The combinatory use of CsA and metformin would be a possible treatment option, with CsA expanding the NSC pool and metformin deriving the differentiation to create more neurons.

Neuroscience Matters REFERENCES 1. Altman, J. (1962). Are New Neurons Formed in the Brain of Adult Mammals?. Science, 135(3509), 1127 – 1128. 2. Altman, J. (1969). Autoradiographic and histological studies of postnatal neurogenesis. IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb. The Journal of Comparative Neurology, 137(4), 433 – 57. 3. Kolb, B. et al. (2007). Growth factor-stimulated generation of new cortical tissue and functional recovery after stroke damage to the motor cortex of rats. Journal of Central Blood Flow & Metabolism, 27, 983 – 997. 4. Wang, J. et al. (2012). Metformin activates an atypical PKC-CBP pathway to promote neurogenesis and enhance spatial memory formation. Cell Stem Cell,11, 23-35. 5. Hunt, J., Cheng, A., Hoyles, A., Jervis, E., & Morshead, C. (2010). Cyclosporin A Has Direct Effects on Adult Neural Precursor Cells. The Journal of Neuroscience, 30(8), 2888 – 2896.

Neuroscience Matters Received March 30, 2012; revised Month, ##, 200#; accepted Month, ##, 2013. Address correspondence to: Ekaterina Kouzmina (pttkatherine@ gmail.com) and Ashkan Azimi (ashkan.azimi@mail.utoronto.ca). Copyright © 2013 Dr. Bill JU, Human Biology Program

The future of brain imaging: Achieving whole-brain imaging with single neuron resolution to identify novel functional networks via modified light-sheet microscopy Roxanne Leung, Trevor Morey, & Thomas Wasuita1 Human Biology Department, The University of Toronto. Toronto, Ontario CA.

Comprehensive mapping of brain activity has become a major focus internationally, as many countries compete to elucidate the mysteries of the human brain, from the culprits behind mental illnesses to the processing of human perception. One significant barrier to realizing these goals has been the extent of electrophysiological and neuroimaging techniques, which are informative, but often provide a tradeoff between the details of high resolution and the systems-wide big picture. In this review, we describe a new method that was developed by Ahrens and Keller at Howard Hughes Medical Institute; a modified lightsheet microscopy platform that allows whole-brain imaging at

the level of individual neurons every 1.5 seconds. To apply this new technique and prove that it is a contender for the next steps in neuroimaging, the authors identified two novel functional circuits that were previously unknown: the hindbrain oscillator and the hindbrain-spinal circuit. This group shows a unique way of functionally imaging all of the neurons within a living brain at single cell resolution. Their technique opens a new realm of possibilities for neuroscientists and may be the first step to creating a complete functional map of the brain, which would allow researchers to gain greater insight into how neural activity leads to complex behaviors.

Key words: light sheet microscopy, zebrafish embryo, imaging, fluorescence, functional circuits, whole-brain, GCamP5G, hindbrain oscillator, hindbrain-spinal circuits INTRODUCTION In recent months, society has become increasingly interested in brain research. With an aging population faced with neurodegenerative diseases, mental illnesses given significant media coverage, and questions of higher-level cognitive processes such as learning, perception, and pain being raised, many world leaders have called for an intensified effort to be placed on neuroscience research. The European Union, China, United States, and Canada are some of the major players in this international quest to understand the enigmatic brain, and many parties have announced increased investments, despite the extensive budget

cuts imposed on other fields in science. The overarching goal of each party can be generalized as one ambitious aim: a systemswide imaging or mapping of the entire brain with single neuron resolution in order to functionally define neural networks that lead to behaviours or diseases.1 Despite the advancements and variety of brain research techniques, there currently is no method of pursuing, let alone achieving, such an endeavour. Thus, a significant next step to accomplish this goal is to develop novel techniques that overcome the barriers of current brain imaging techniques. Currently, there are a variety of informative tools used in neuroscience. Electrophysiological

studies have been established for quite some time now, with the ability to detect electrical activity across single ion channels, individual neurons, and multiple neurons within vicinity of each other. These techniques have allowed some functional networks, such as sensory-motor interactions, to be identified2. Imaging studies, both histological and anatomical, have also contributed significantly to our understanding of the function of specific cell types or brain regions. On one end, fluorescence microscopy has helped identify different cell types or the properties of synapses3. On the other, functional magnetic resonance (fMRI), diffuse tensor imaging (DTI), positron emission tomography (PET), and computed tomography (CT) imaging have been instrumental in assessing the gross brain regions involved in numerous processes and disease states.4 However, a significant limitation with all of these techniques is the tradeoff between resolution and obtaining the big picture. In order to obtain single neuron resolution, the scope of view is very limited. In order to observe whole-brain activity, the detail is lost. Here we describe a novel technique developed by Ahrens and Keller at Howard Hughes Medical Institute, in which single neuron resolution was obtained during whole-brain imaging of activity in a zebrafish embryo. Furthermore, the application was used to identify two previously unknown functional circuits. The technique employed was an adapted form of light-sheet microscopy (SiMView) with genetically modified GCamP5G calcium ion channels in all neurons to indicate neuronal activation. Light-sheet microscopy traditionally utilizes laser beams to create planar light sheets within a specimen. The planar light sheets cause fluorescence in the area of interest, which is captured by a camera perpendicular to the light-sheet. Improvements to the current SiMView platform allowed the authors to achieve a comprehensive, high-resolution image of the whole larval zebrafish brain with the capability of identifying functional networks.3

by investigating changes in fluorescence intensity (Fig 1). Using this method, it was possible to detect action potentials, and even distinguish between neighboring neurons firing simultaneously (Fig 1b, c). Correlations across brain areas and single neurons Being able to visualize whole-brain activity every 1.3 seconds, the authors were interested in looking at co-activated areas in the larval zebrafish at rest, in hopes of defining previously unknown functional neuronal networks. The first step was to divide the brain into 11 areas (Fig 2) and look at the average fluorescence in each of these areas over a period of 30 minutes. Average activity was then correlated between all brain areas, revealing several interesting patterns. Midbrain areas showed high correlation levels to each other, as did several hindbrain and forebrain areas. Midbrain area activity was also significantly correlated to hindbrain areas. What the authors found curious was the lack of correlation between forebrain areas to both the mid- and hindbrain. Due to the confirmed presence of motor neurons in the forebrain, correlated activity was expected in these regions.6

RESULTS Single-cell functional imaging of a larval zebrafish brain In order to improve the imaging rate of the SiMView light sheet microscopy platform5, the authors used a new volumetric imaging protocol, installed new detectors, and eliminated electronic overhead. These changes effectively increased the original imaging rate by tenfold, covering a volume of 800 by 600 by 200 cubic micrometers every 1.3 seconds. Such an area encompasses the entire larval zebrafish brain and is the first instance of whole-brain functional imaging in a vertebrate species. In addition, the albino slc45a2 mutant, which lacks pigmentation on the outer portion of the eyes, was used for the experiment and allowed imaging of the previously inaccessible inter-ocular brain region. As a result, single-neuron resolution was attained for approximately 80% of the zebrafish brain. Along with this anatomical analysis of single neurons, the authors also distinguished between individual cells

Ahrens & Keller, Nat Meth (2013). Figure 1 | (a) single-cell functional activity, visualized by fluctuations in fluorescence intensity. Both dorsal and ventral views are shown at different time points during imaging. Each panel shows the entire zebrafish brain, which was recorded in 1.3 seconds. (b) Enlarged representation of the highlighted regions in part a, showing individual neighboring cells activated together. (c) Neuronal activity in a single slice from part b superimposed on the anatomical cell map (above) and alone (below).

Neuroscience Matters

Neuroscience Matters functional neuronal circuit. Using this method, two functional networks were identified in the larval zebrafish brain. First, the anterior hindbrain showed consistently high correlations in activity with the reference trace, with the ipsilateral side showing strong positive correlation, and the contralateral side exhibiting strong negative correlation to the trace (Fig 3a). The same pattern was also observed bilaterally in the inferior olives, suggesting these areas were also somehow functionally related. Since these neuronal populations were showing anti-phase oscillating activity, the authors referenced them as the “hindbrain oscillator”. Using a second, consistent reference trace, the authors also identified a second network of highly correlated activity in neurons extending down into the spinal neuropil (Fig 3f), calling it the “hindbrainspinal circuit”.

Ahrens & Keller, Nat Meth (2013). Figure 2 | (a) Representation of the zebrafish brain segmented into 11 distinct anatomical regions. (b) Average activity in each of the 11 brain areas obtained from changes in fluorescence intensity over time. (c) Correlation matrices of the brain areas in three separate zebrafish, and an average matrix of correlation between six zebrafish is shown in (d). (e) Average correlations between general areas (hindbrain, midbrain and forebrain) in the six zebrafish.

In order to investigate this apparent lack of correlation, the authors looked at 300 habenula neurons — located in the forebrain — and correlated their individual activity to the average activity in the hindbrain, by using fluorescence levels. As expected, it was found that some populations of habenula neurons showed strong positive correlations to the hindbrain reference trace, while others showed strong negative correlations. This explains the results seen in the previous experiment, since the average activity of the entire population of habenula neurons resulted in no correlation. Another observation made was that several of these individual forebrain neurons showed much slower, oscillating activity than neurons in other brain regions. Since this repeating low-frequency neuronal activity is quite poorly understood, even in mammals, the authors decided to look for associated brain areas that exhibit similar activation patterns. Characterizing two distinct functional networks In order to identify slow, correlated circuit activity across the brain, the authors divided the zebrafish brain into supervoxels, and selected the top 2,000 neurons with low-frequency activity, based on a power spectrum of all supervoxels in the brain. This population was then organized into a correlation matrix, and an average reference trace was manually selected. The time-varying activity of a single voxel was then correlated to all of the other supervoxels in the correlation matrix, and then mapped back onto the three dimensional zebrafish brain image to visualize a

Ahrens & Keller, Nat Meth (2013). Figure 3 – (a) Hindbrain oscillator circuit defined by voxels highly correlated with the chosen reference trace, with positive correlations shown in green, and negative correlations shown in magenta. Blue arrows identify the anterior hindbrain population, and white arrows identify the inferior olive neurons. (f) Spinal-hindbrain neuronal circuit defined by voxels positively correlated with a second reference trace (green). Here, the hindbrain oscillator is shown in magenta. Note: both networks are shown in single-cell resolution.

DISCUSSION Ever since the first neuron was discovered scientists have been looking for a way to watch single cells fire action potentials within a live brain. First came the advent of fMRI, a tool that researchers could use to infer neural activity from hemodynamic changes within the living brain7. However, this technique cannot provide the resolution to watch a single neuron fire. In this paper, Ahrens and Keller present a new method of light-sheet microscopy used in concert with genetically engineered zebrafish larvae to allow scientists to image individual neuronal activation across the entire brain of an organism simultaneously. By using their improved version of SiMView light-sheet microscopy, they were able to detect the fluorescent changes that occur when calcium enters the neuron during an action potential, by causing a conformational

change in the genetically engineered GCaMP5G calcium indicator. These fluorescent changes were then correlated across the brain allowing the researchers to define previously unidentified neural circuits within the zebrafish brain. They identified a new circuit that originates in the hindbrain and activates neurons within the spinal neuropil, as well as a circuit that creates phasic activity between the anterior hindbrain and the inferior olivary nucleus. SIGNIFICANCE OF THE WORK The ability to image single neurons across the entire brain of an organism opens many new doors for the field of neuroscience. Functionally mapping the brain has been a long-standing goal for scientists, and the ability to watch every individual neuron within a brain fire will certainly move us closer to finding the neural substrates that underlie different behaviors. It also allows researchers to view the oscillations of activity that occur within the brain. While this group illustrated an oscillation between the anterior hindbrain and the inferior olivary nucleus in a zebrafish, oscillating and synchronous activity found within the brain has previously been shown to play an important role in sleep and memory for humans8. Having the ability to study these behaviors at the level of single neurons may allow researchers to gain a deeper insight into how memories are formed and stored. This new technique also creates a strong argument for the zebrafish to be used more frequently as a model organism for systems neuroscience. While there may be other model vertebrate organisms that are closer to humans on the evolutionary tree, none of them possess the transparent body that is necessary for this technique to be used. FUTURE DIRECTIONS While Ahrens and Keller present an exciting new technique for studying neural activity across the entire brain with single cell resolution, it is merely just a stepping stone within the golden age of discovery in neuroscience. The development of single neuron imaging within a functional brain opens many doors for the study of behavior. In future experiments, researchers may use this technique to investigate zebrafish while performing a particular task or confronted with various stimuli. These neural networks can then be studied further in higher organisms.

This work will also inspire other researchers to create new techniques that can be used in non-transparent model organisms, such as mice, non-human primates, and eventually humans. One key challenge that must be overcome is the development of a method to detect single action potentials that does not require genetic engineering, so that scientists can view single neuron activity across the entire human brain. Once accomplished, we will not only be able to develop a structural maps of the brain in combination with pre-existing MRI and DTI techniques, but also develop a more accurate functional map of the brain based on neural network activity. With a complete map of the human brain researchers will be able to gain more valuable insight into how the brain creates various behaviors. REFERENCES 1. Potter, M. Brain mapping project aims to help treat brain disorders. Toronto Star (2013). 2. Koyama, M., Kinkhabwala, A., Satou, C., Higashijima, S. & Fetcho, J. Mapping a sensory-motor network onto a structural and functional ground plan in the hindbrain. Proc. Natl. Acad. Sci. USA 108, 1170—1175 (2011). 3. Ahrens, M.B. & Keller, P. J. Whole-brain functional imaging at cellular resolution using light-sheet microscopy. Nat. Meth., advanced online publication (2013) 4. Kimbereley, T.J. & Lewis, S.M. Understanding neuroimaging. J. Am. Phys. Ther. Assoc. 87, 670—683 (2007). 5. Tomer, R., Khairy, K., Amat, F. & Keller, P.J. Quantitative high-speed imaging of entire developing embryos with simultaneous multiview lightsheet microscopy. Nat. Meth. 9, 755–763 (2012). 6. Ahrens, M.B. et al. Brain-wide neuronal dynamics during motor adaptation in zebrafish. Nat. 485, 471–477 (2012). 7. Friston, K.J., et al. Event-related fMRI: Characterizing differential responses. Neuroim. 7, 30-40 (1998). 8. Steriade, M., et al. Thalamocortical oscillations in the sleeping and aroused brain. Science 262, 679-685 (1993).

Received March 30, 2012; revised April, 05 2013; accepted Month, ##, 2013. Address correspondence to: Roxanne Leung, rocksee.vi@gmail. com; Trevor Morey, morey.trevor@gmail.com, Thomas Wasiuta, tom.wasiuta@gmail.com. Copyright © 2013 Dr. Bill JU, Human Biology Program

Neuroscience Matters

Neuroepigenetics: rejuvenate cognition with DNA methylation Jing Lu1 Human Biology Department, The University of Toronto. Toronto, Ontario CA.

Aging-related cognitive decline has been the prime focus of many researchers in the last decade. It is a particularly important field in cognitive neuroscience in long term due to the ever-increasing aging population in many societies. Much effort was devoted to study the structural and functional changes in the brain during normal aging process as well as in neurodegenerative diseases, but the progress are rather slow. In addition, the underlying molecular mechanisms that influence such physiological changes still remained unknown. A recent study demonstrated that DNA methylation, a kind of epigenetic modification catalyzed by DNA methyltransferases (DNMTs), might provide insights for understanding molecular and cellular mechanisms of brain aging process. Dnmt3a, one of the subtypes of Dnmts, have two isoforms (Dnmt3a1 and Dmnt3a2) that function very differently and may regulate many cell processes. Dnmt3a2 in particular was shown to

localize to euchromatin. Some previous evidence suggested that aging is associated with demethylation in nervous system. Indeed, Dnmt3a2 level was decreased in the hippocampus and cortex of aged mice. Overexpression of Dnmt3a2 using recombinant adenoassociated viral construct in aged mice improved the cognitive ability, whereas its Kockdown using short hairpin RNA in young adult mice resulted in memory deficits. Subsequent molecular study also showed a correlation between Dnmt3a2 expression and synaptic plasticity related genes such as Arc and Bdnf. Together, the study showed a novel role of DNA methylation in memory formation, and provided a causal link between Dnmt activities and aging-related cognitive decline. This finding might provide a new therapeutic strategy where cognitive deficits can be selectively recovered by drugs that target Dnmt3a2.

Key words: age-related cognitive decline, DNA methylation, DNA methyltransferases, epigenetics, memory formation, neurodegenerative diseases. I. BACKGROUND Aging is associated with gradual decline in cognitive ability that results in dementia, including Alzheimer’s disease1. Age-related cognitive decline is a particularly important issue in a society with increasing aging population because it creates greater financial burden on the health care system3. In fact, dementia and Alzheimer’s disease are currently a few of the leading causes of morbidity and disability2,3. As a result, understanding normal aging process that lead to dementia and neurodegenerative diseases have been the prime focus of many researchers over the past decade. Although some progress was made in elucidating the cellular and molecular mechanisms of age-related cognitive decline, limited information is known about factors that influence such changes1,2. Study has been showing that maintaining adequate level of social and physical activities in elderlies reduces the risk of dementia. However, current understanding of aging process have not reach the level that allow the development of effective therapeutic treatments 2. Neuroepigenetics is a fast growing field of research which focuses on the long –lasting effect of covalently modifying DNA or nuclear proteins without changing the DNA sequence itself 2 . The role of epigenetics in animal development and cell differentiation has already been extensively investigated, but only recently are

those mechanisms implicated in memory formation and synaptic plasticity 4. Epigenetic modification can be found on histones or DNA itself. Histones are proteins that pack DNA in the nucleus into chromatin. There are two general states for chromatin, heterochromatin and euchromatin. In heterochromatin, DNA is tightly wrapped around histones so that transcription factors cannot access gene promoters. On the other hand, DNA is loosely wrapped in euchromatin and it allows active gene transcription2,4. In epigenetic modification, histones will be covalently tagged with different chemical groups to control the packaging of DNA. DNA itself can also be covalently modified to alter gene expression. Among different kinds of modifications, DNA methylation seems to play a particularly important role in development and cognition4. DNA methylation is typically found on the cytosine base of CpG di-nucleotides, and in general it induces gene silencing4. Many human cognitive disorders have been associated with epigenetic modifications. One example would be the Fragile X Syndrome, in which patients usually have expanded CGG and CCG tri-nucleotides that result in hypermethylation of DNA and thus, gene silencing. DNA methylation is catalyzed by a class of enzymes called DNA methyltransferases (DNMTs). There are three different kinds of DNA mehtylatransferases, Dnmt1, Dnmt3a and Dnmt3b, and

they are responsible for regulating gene activity in various cell processes and developmental stages5. Some evidence suggests that DNA methylation decrease as animal age and this include genes found expressed in the nervous system5. Other studies have found that CpG methylation can be modified by synchronous neuronal activity6, and Dnmt1 and Dnmt3a double knockout mice display deficits in learning and memory and abnormal hippocampal plasticity7. A question still remains as to whether there is a causal link between the DNA methylation status and the aging-related cognitive decline. Answers to this question might be able to provide implications for potential therapeutic strategy. II. REVIEWED RESEARCH MATERIALS AND METHODS Subject Male C57B1/6 mice (3-month-old and 18-month-old) were used for experiments during the study. Two age groups were selected for comparison. Total RNA expression in different treatment group was quantified using qRT-PCR5.

and were removed. Either1 hour or 24 hour after conditioning, mice were placed back to the same context, but this time without a foot shock. Freezing behavior was scored during training and testing5. Object-place recognition test To further confirm the robustness in behavioral consequence of overexpression/Knockdown, mice were also tested with objectplace recognition test, a memory paradigm that involve less adverse stimulus. The paradigm contained two objects in a field with visual cues on the arena wall. Mice were allowed to explore the objects during the training session, and they were removed. After 24 hour, mice were returned to the same field except that one of the two objects was displaced from the original location. Time spent exploring the two objects was measured5, and preferences for the objects were calculated in percentage.

rAAV Mouse hippocampus is infected with recombinant adenoassociated viral constructs through stereotaxic injection. In overexpression studies, the viral vector contained hemagglutinin epitope-tagged Dnmt3a2 (HA-Dnmt3a2) under the control of CamK2a promoter. Control vector had CamK2a promoter fused with GFP. ShRNA was used to Knock-down Dnmt3a2 and it recognizes 5’UTR regions of the Dnmt3a2 sequence. ShRNA is expressed under the control of U6 promoter. Western blot was used to analyze the efficacy of rAAV construct5. Kainic acid administration To investigate activity-dependent expression of Dnmts, kainic acids where injected (saline as control) to induce epileptic seizure. Animals that exhibited characteristic seizure behavior such as rigid posture were used in further analysis5. Behavioral paradigms Several memory tests were used to test the behavioral effect of injected viral constructs5. Trace fear conditioning During initial training, mice were placed into a chamber, and a foot shock was administered 15s after a tone was played. Mice were then taken out of the chamber after conditioning. For testing, mice were placed in a modified context while the same tone was played. Freezing behaviors were measured and scored during the training and testing for comparison5. Contextual fear conditioning Mice were placed into chambers where they received foot-shock,

Figure 1. Schematic diagram of the experiments. (a) Aged mice were injected with Dnmt3a2 overexpression construct or control. After behavioral training paradigm, mice with overexpression construct performed better in the task. (b) Young adult mice were injected with shRNA Dnmt3a2 knockdown construct or control. Mice that were injected with Knockdown constructed displayed memory deficits.

III. RESULTS Gene transcripts for different Dnmts in hippocampus were quantified in both young and aged mice. Two isoforms of Dnmt3a, Dnmt3a1 and Dnmt3a2, were both reduced. Dnmt3a1 and Dnmt3a2 were products from alternative promoters in the same gene loci5. It was shown previously that Dnmt3a1 is typically associated with heterochromatin, whereas Dnmt3a2 is localized to euchromatin. Further investigation revealed that Dnmt3a2 expression behaved like immediate-early gene that was activated by NMDA receptor but this effect is not observed with Dnmt3a1. Additional manipulation also demonstrated the partial dependence of Dnmt3a2 expression on calcium signaling2,5. Based on the expression pattern during age-related cognitive

Neuroscience Matters decline and following activity stimulation, it can be hypothesized that Dnmt3a2 might be the underlying mechanisms leading to cognitive impairments in aged mice. To test whether the reduction of Dnmt3a2 expression in aged mice have a functional implication, aged mice were treated with recombinant adeno-associated virus that contain HA-Dnmt3a2, allowing overexpression5. Results showed that increasing Dnmt3a2 level in aged mice produced higher level of DNA methylation in hippocampus compared to control. In addition, mice treated with HA-Dnmt3a2 displayed better behavioral response 2,5, when tested using a hippocampal-dependent task called trace fear conditioning. The improvement in test scores was only observed for long-term memory, howver. To further confirm the effect of overexpression, mice were trained and tested using object-place recognition paradigm, another independent spatial memory task. The result confirmed the previous finding from trace fear conditioning. Hence, restoring Dnmt3a2 in aged mice improve their cognitive abilities5. To examine the role of Dnmt3a2 in long-term memory formation, a different rAAV construct were administered to the hippocampi of young adult mice to see whether depleting Dnmt3a2 level in cognitively normal mice would impair memory formation. A short hairpin RNA targeting Dnmt3a2 was used in the construct. Control rAAV(no shRNA) and treatment construct were stereotaxically injected into young adult mice. Dnmt3a2 kockdown did not affect dendritic morphology of neurons in the hippocampus. Both contextual and trace fear conditioning tests showed that shRNA treated mice displayed significantly less freezing behavior compared to control mice5. Again, the effect of rAAV construct was seen only in long-term memory formation but not in short-term memory formation5. Further analysis using object-place recognition test on long-term memory confirmed the result. These findings suggest that decreasing Dnmt3a2 levels in the hippocampus is able to impair long-term memory formation2,5. Additional tests were performed to investigate whether decreasing the Dnmt3a2 level reduce the expression of synaptic plasticity genes. Among the examined gene candidate, Dnmt3a2 Knockdown only affected Arc and Bdnf expression level5, but not other plasticity genes. IV. DISCUSSION The study demonstrated a novel role of Dnmt3a2 in long-term memory formation and its causal relationship with the agerelated cognitive decline. Dnmt3a2 level decreases as mice age, but restoring its level were sufficient to recover their cognitive ability. Altered expression of Dnmt3a2 influences the ability of mice to form long term memories, but not short-term memories. Moreover, Dnmt3a2 expression pattern was also correlated with

Neuroscience Matters changes in some synaptic plasticity gene transcripts, suggesting its mediatory function in cognition. Interestingly, there was a novel discovery of an additional effect of DNA methylation on downstream gene expression. Finally, the recovery of cognitive ability in aged mice after overexpression of Dnmt3a2 might implicate a potential drug target for future therapeutic treatment of dementia. One striking finding from the study was the relationship of DNA methylation with their downstream effect. The notion of decreasing DNA methylation reduces gene expression seemed somewhat counterintuitive given the previously assumed role of DNA methylation in gene silencing2. On the other hand, other studies have shown that activity-dependent methylation of Arc promoter resulted in a reduction in its gene transcripts2,8. It might be possible that DNA methylation and its downstream effect are dependent on specific genes that are affected at a given time. In other words, the difference between the effect of DNA methylation that activate or repress genes might be due to complex interplay among Dnmts, their specific targets and other dynamic signaling pathways. If this assumption were true, then Dnmts could be acting as mediators for the dynamic changes during memory formation and aging. Another puzzle that arose from the conclusion is whether there is a mechanistic link between synaptic plasticity and Dnmt3a2 mediated long-term memory2. Experimental results demonstrated that altering Dnmt3a2 level did not produce any effect on shortterm memory. However, results suggested a correlation between synaptic plasticity genes expression and Dnmt3a2 expression, and alteration in synaptic plasticity gene expression did not change the dendritic morphology. Then, how does DNA methylation affect synaptic plasticity and long-term memory formation on a molecular level without affecting synaptic structures and shortterm memory? Future experiment that uses bisulfite sequencing might be able to give us a clue as it allows us to directly examine, on a genome scale, the specific molecular correlated that changes as a result of Dnmt3a2 knockdown2. RT-PCR can also be done in conjunction to show the changes in gene expression level. An additional issue to address is the role of Dnmt3a1 in relation to Dnmt3a2. Since both isoforms are produced by the same gene loci5, altering the expression level of Dnmt3a2 using shRNA like those used in the study might potentially alter the expression of Dnmt3a1. Analyzing the interaction between two isoforms might also provide insights to the role of Dnmt3a2 in gene activation and repression2. One more interesting aspect to consider is the Dnmt activities in other brain regions. Oliveira et al. showed in their previous study that a similar decrease in Dnmt3a2 level was also found in mice cortex5. Furthermore, other studies suggested that remote memory might be storaged in different cortical regions, and DNA methylation might be playing a role in maintaining this kind of memory9. Given that epigenetic modification can produce long-lasting effect and be passed onto cell progenies, it is possible

that different DNA methylation patterns are mediating the memory formation in the cortex. Therefore, further manipulation of Dnmt3a2 levels in other brain region might provide insights to memory consolidation 2,9. V. SIGNIFICANCE OF THE WORK Epigenetic modification have long been examined in developmental biology, however, its role in memory formation and cognition has yet to be established. The study by Oliveira et al. introduced a novel and interesting role of Dnmt3a2 in age-related cognitive decline, and they provided a new perspective to the physiological function of DNA methylation, namely their potential gene-activating role. The result contributed to a fast growing field that focuses on neuroepigenetics and its function in the aging brain. Improving cognitive ability by increasing Dnmt3a2 levels might not only be an exciting therapeutic treatment strategy, but also a important route toward understanding neurodegenerative diseases in the future. REFERENCES

2. Susan C Su & Li-Huei Tsai. DNA methylation in cognition comes of age. Nat Neurosci, 2012:1061–1062 3. Todd, S. et al. Survival in dementia and predictors of mortality: a review. Int J Geriatr Psychiatry. 2013 Mar 22 4. Levenson, J.M, Sweatt, J.D. Epigenetic mechanisms in memory formation. Nat Rev Neurosci. 2005;6(2):108-18. 5. Oliveira,A.M. et al. Rescue of aging-associated decline in Dnmt3a2 expression restores cognitive abilities. Nat Neurosci. 2012;15(8):1111-3. 6. Day, J.J, Sweatt,J.D. Epigenetic mechanisms in cognition. Neuron. 2011;70(5):813-29. 7. Feng J. et al. Dnmt1 and Dnmt3a maintain DNA methylation and regulate synaptic function in adult forebrain neurons. Nat Neurosci. 2010 Apr;13 (4):423-30 8. Penner, M.R. et al. Age-related changes in Arc transcription and DNA methylation within the hippocampus. Neurobiol Aging. 2011;32(12):2198-210. 9. Miller, C.A.,Sweatt, J.D. Covalent modification of DNA regulates memory formation. Neuron. 2007;53 (6):857-69.

1. Grillo, F.W. et al. Increased axonal bouton dynamics in the aging mouse cortex. Proc Natl Acad Sci. 2013, Mar 29.

Anatomical Plasticity of Adult Brain Is Titrated by Nogo Receptor 1 Amaris Hui1 and Amirah Momen1 Human Biology Department, The University of Toronto. Toronto, Ontario CA.

Experience-dependent anatomical plasticity is generally reduced with age – the molecular basis for age-related suppression of dendritic spine and axonal varicosity turnover may lie in the increased expression of Nogo Receptor 1 (NgR1) in the adult after maturation of cortical myelin. In this study, the authors used conditional and traditional gene knockouts of ngr1 in adolescent and adult mice to examine the role of NgR1 in mediating anatomical plasticity of the somatosensory cortex. They postulated that the presence of NgR1 and its ligand Nogo-A function beyond axon growth inhibition as an activity-dependent ‘molecular switch’, limiting age and experience-dependent synaptic dynamics thought to be crucial for forming new memories and sensory maps. Overall, ngr1 null mice experienced double the spine and axonal varicosity turnover rate as control mice, and adult ngr1

knockouts aged P26 to P180 demonstrated similar synaptic turnover as adolescent mice. NgR1 deletion only affected turnover rate and tenacity, but did not affect spine density or morphology. Adolescent-like anatomical plasticity was restored even in aged P360 mice by conditional gene deletion of ngr1. In behavioural tests, a reduced threshold effect in environmental stimulation adequate for driving high spine turnover in adult ngr1-/- mice was observed compared to heterozygous controls. NgR1 knockouts learned the Rotarod task in fewer trials and outperformed control mice in latency to fall. Finally, fear extinction was augmented in ngr1 KO mice, but fear memory formation itself was unaffected. In brief, loss of NgR1 leads to reactivation of juvenile levels of cortical synaptic plasticity and its study is important in our understanding of brain aging and repair mechanisms.

Key words: Nogo receptor 1 (NgR1), Nogo-A, dendritic spine turnover, axonal varicosity, anatomical plasticity, aging, two-photon microscopy, Rotarod, fear conditioning, barrel field cortex, enriched environment.

Neuroscience Matters I. BACKGROUND It is generally accepted that in aging, the adult human brain becomes less anatomically dynamic in response to sensoryenriched environmental experience.1 In adolescence, enriched environmental stimulation increases transient spine formation and synaptic tenacity.2 Though this is a recent area of debate brought about by Grillo et al3, dendritic spine turnover and dynamics of axonal boutons are thought to slow and stabilize in rates of formation and elimination past a critical period between adolescence and adulthood, affecting the rate of learning novel motor skills, fear conditioning, and formation of new sensory maps. Until recently, Nogo-A and its receptor Nogo Receptor 1 (NgR1) have mainly been implicated as CNS myelin-associated inhibitors of axonal regeneration post spinal cord injury, contributing to the non-permissive CNS environment for endogenous repair.4 Its signaling cascade for inducing neurite retraction is well-known, involving co-activation of p75(NTR), LINGO-1, and TROY to elicit activation of the RhoGTPase-ROCK pathway, which in turn causes collapse of actin cytoskeletal filaments within growth cones.5 However, current research has suggested that both Nogo-A and NgR1 are constitutively expressed pre- and post-synaptically at the dendritic spines and axonal varicosities of hippocampal, cerebellar, and cortical neurons – slowing excitatory synaptic turnover and affecting morphology of dendritic spines. Reduced Nogo ligand expression in cerebellar Purkinje cells correlated with larger dendritic trees and increased strength of synaptic transmission.6 Moreover, triple knockouts of the Nogo receptor family resulted in abnormally high synaptogenesis in the hippocampus.7 The functional significance of NgR1 has been hypothesized to mediate circuitry in development – its expression is required to finetune synaptic networks, preventing excessive aberrant synaptic connections. In fact, NgR1 loss-of-function mutations have been associated with schizophrenia disease phenotypes.8 NgR1 has been further implicated in reducing subependymal NSC proliferation9, thereby affecting multiple aspects of plasticity after injury and during aging. In Akbik et al, the authors hypothesized that NgR1 and its ligand Nogo-66 are molecular determinants of age-dependent anatomical synaptic dynamics; their function is to down-regulate novel synapse formation and stabilization in the aged cortex. They investigated their hypothesis using time-lapse imaging and behavioural tests of motor learning and conditioning, ultimately demonstrating that conditional gene deletion and complete knockout of ngr1 restored adolescent levels of synaptic plasticity, contributing to accelerated motor learning, enhanced fear memory extinction, and a reduced sensory threshold required for experience-dependent anatomical

Neuroscience Matters plasticity. Overall, NgR1 is a plasticity inhibitor and a convergent therapeutic target for aging, anxiety, motor skill learning, and neurodegenerative disorders such as Alzheimer’s in which overexpression of NgR1 is implicated.10 The significance in this research lies in bridging basic plasticity research with translational neurology.

concluded that NgR1 acts in a constitutive manner to stabilize spines to an ‘adult-level’ of stability, a function NgR1 that is reversible upon its deletion.

II. REVIEWED RESEARCH MATERIALS AND METHODS Mice Transgenic Thy1-YFP-H and Thy-1-eGFP-M mice were bred with conditional mutant ngr1 null and ngr1 flx/flx mice for timelapse imaging. Conditional gene knockout experiments utilized ngr1 flx/flx mice with or without an Actin-Cre-ERT2 transgene. Tamoxifen was then injected i.p for rearrangement of the ngr1 gene and loss of NgR1 mRNA in Cre mice. Time-Lapse Imaging Synaptic turnover and persistence were imaged using repeated transcranial two-photon confocal microscopy. L5 pyramidal neuronal dendrites in the S1 barrel field cortex were imaged in three-dimensions. Persistent spines were defined as spines that were observed on 2 separate imaging occasions 2 days apart. In vitro Dissociated cortical neurons (either WT or ngr1 null) cultured for 19 to 22 days were treated with 100nM Nogo-22 protein to study spine gains and losses. Behavioural studies Ngr1 null and heterozygous mice were housed in standard versus enriched environments to study effects of NgR1 expression on sensory experience-dependent spine plasticity. Bilateral whiskers were trimmed in both groups to confirm necessity of sensory input. The Rotarod test and fear extinction paradigms were used to compare motor and extinction learning rates of ngr1 KO versus WT mice. Fear conditioning training (tone-shock pairings) occurred on day 1, while days 2 and 3 were extinction trials. RESULTS NgR1 Limits Pre- and Postsynaptic Dynamics in Adult Cortex The first set of experiments were performed in vivo using two-photon imaging of YFP-H transgenic mice with the goal of observing pre- and post-synaptic dynamics in NgR1 null mice(ngr-/-) as compared to controls. The authors noted that their use of time-lapse imaging was necessary since spine density and morphology did not change while spine dynamics did.

Figure 1. Adapted from Akbik et al.11, 2013. Nogo ligand restricts dendritic spine and axonal varicosity dynamics in adult rodent brain.

Spine morphology was normal in NgR1 null and wildtype mice however, the authors noticed double the spine dynamics in NgR1 null mice, measured in the form of ‘gains’ and ‘losses’ (Figure 1A, 1B) over a two-week period. They also noticed that the number of stable or ‘persistent’ spines (i.e. lasting 2 days or longer) decreased in the NgR1 null mice and that over a two week period there was greater persistent spine loss in NgR1 null mice (Figure 1C). The authors wanted to examine whether the restriction of spine dynamics observed in mice expressing NgR1 was age-dependent. To do so, they observed the longitudinal dynamics in both NgR1 null and wildtype mice over the course of 360 days. They observed that wildtype mice and NgR1 null mice were similar from P26-P40 (days postnatal) followed by a slowing of spine dynamics to adultlevel in wildtype mice over a time course that corresponded to myelination of the cortex (Figure 1E). Conversely, NgR1 null mice maintained the same juvenile-like spine dynamics from P26-P180. Finally, the authors examined whether the restrictive/ stabilizing effects of NgR1 are reversible by creating a conditional knockout model using a CRE-ERT2 system that deleted the NgR1 gene when Tamoxifen (Tmx) was added. By adding Tmx at a point well into the onset of stable adult-like spine dynamics (P330), the authors observed a reverse in stability as spine turnover increased to levels similar to those of adolescent mice (Figure 1E). This result suggests that the effects of NgR1 are reversible and that its action in vivo is constitutive. Similar patterns were seen for presynaptic axonal varicosities (not shown). The authors concluded from these in vivo results that NgR1 normally acts to inhibit initial protrusions and retractions prior to synapse formation as well as to promote the establishment of persistent spines similar to those found in synapses. As normal mice develop from adolescence to adulthood, their brains experience a rapid increase in synaptic stability coinciding with myelination (i.e. Nogo ligand expression). Akbik et al. observed a release on the adult plasticity ‘gate’ in NgR1 null mice, resulting in juvenile-type spine and varicosity dynamics. The authors also

Nogo Ligand controls dendritic spine and axonal varicosity turnover in the adult cortex Next, the authors wanted to confirm the role of Nogo ligand in regulating dendritic and axonal turnover in baby mice using both in vitro and in vivo methods. For in vitro examination of juvenile neurons, GFP-labelled cells were imaged with confocal imaging from P19-P22. The authors observed that without the addition of the NgR1 ligand ‘Nogo 22’ juvenile wildtype and NgR1 null neurons showed similar spine and dendrite dynamics. Conversely, with the addition of Nogo22, wildtype mice with intact NgR1 receptors exhibited an 80% decrease in new spine protrusions, spine gains and losses over time in both acute (2 hour, not shown) and chronic (6 days, Figure 2C) conditions. NgR1 null neurons showed no change when Nogo22 was added (Figure 2C). Next, the authors studied mice null for Nogo A/B ligand in vivo using twophoton imaging. Ng A/B null mouse brains showed a doubling in spine gains and losses at P180 compared to control, similar to the NgR1 null mice dynamics (Figure 1A-E). Varicosities exhibited similar patterns for all parts of the experiment (data not shown). Finally, the authors wanted to observe whether there was any genetic interaction between NgR1 and Ng A/B by creating a series of heterozygous mutants. They observed that compound heterozygotes (ngr1+/- and nogo A/B +/-) exhibited the same spine dynamics as NgR1 and Ng A/B null mice i.e. juvenile level gains and losses.

Figure 2. Adapted from Akbik et al.11, 2013. Nogo ligand restricts dendritic spine and axonal varicosity dynamics in adult rodent brain.

The authors concluded that deletion of the Nogo A/B ligand phenocopies NgR1 null spinal turnover both in vitro and in vivo and that there is an in vivo interaction between NgR1 and NogoA/B. Reduced sensory threshold in ngr1-/- mice drives high experiencedependent spine turnover in chronic standard caging Two-day spine plasticity was observed in heterozygous control and

Neuroscience Matters ngr1-/- mice in both Standard (SE) and Enriched environments (EE). As expected, in control mice, caging in EE lead to an 8.5% increase in 2-day spine turnover when compared to standard caging. However in ngr1-/- mice, percent spine turnover was insignificantly different between SE and EE, demonstrating that reduced sensory stimulation is adequate in eliciting high levels of spine turnover in ngr1 null mice. The authors questioned whether vibrissal sensory input was necessary for this effect. Thus they deprived control and ngr1-/- mice of sensory stimulation by whisker trimming, housed the mice in SE, and found little spine turnover in control mice but control levels of turnover in ngr1/- mice. From these results, they concluded that loss of NgR1 enhances anatomical sensitivity to sensory experience, even when receiving little to no vibrissal input. Furthermore, spine stabilization was observed and it was found that spine persistence in control mice chronically caged in EE mirrored that of ngr1-/- mice in only SE. There was again no significant difference in stabilization between ngr1-/- mice in SE and EE, and control mice housed in SE exhibited poor spine stabilization. Therefore, NgR1 expression was shown to restrict both transient spine turnover and synaptic tenacity. Knockout of NgR1 accelerates motor learning and enhances extinction of fear memory Previous studies have shown that motor learning and extinction of fear memories are associated with high spine turnover rate.1 Since knockout of NgR1 leads to enhanced spine dynamics and sensitivity to environmental experience, the authors assessed whether conditional ngr1 KO mice learned quicker and performed better at the Rotarod task than controls. In fact, ngr1 KO mice outperformed control mice in latency to fall and produced a steeper learning curve, requiring almost 4 times less training (1.1 vs. 4.1 trials) (Figure 3).

Figure 3. Accelerated Rotarod motor skill learning and enhanced fear extinction in ngr1-/- mice. (A) The learning curve for ngr null mice (red) shows greater latency to fall in fewer trials than control mice (black). (B) Fear conditioning and extinction protocol for both knockout and WT mice. (C) Test of freezing response to tone on day 3 showed greater fear extinction in ngr1-/- mice. Adapted from Akbik et al, 2013.11

Neuroscience Matters Subsequently, a 3-day extinction experiment of conditioned fear memory revealed enhanced extinction of freezing behaviour to the tone in ngr1-/- mice. The authors postulated that the accelerated extinction of the CS-US pairing was due to NgR1dependent spine gains. Thus the rapid spine turnover elicited by loss of NgR1 helped to accelerate both motor and fear extinction learning paradigms. DISCUSSION In summarizing their results, Akbik et al. propose a model whereby NgR1, in response to its ligand Nogo-A/B, gates the transition from adolescent dendritic spine dynamics (rapid turnover) to the more stable, slower dynamics of adult spines. This role of NgR1 operates in a constitutive manner to suppress turnover of synapses and is cell autonomous in nature. The authors suggest that postinjury recovery may be delimited by this NgR1-dependent gating system, a conclusion supported by studies that inhibit Nogo/NgR1 action in order to improve rehabilitation of rodent models of CNS lesions12 and stroke recovery13. Since the conversion from rapid juvenile spine turnover to stable adult synapses corresponds to cortical myelination (i.e. when NgR1 ligand presents at ~P30) the authors propose this gating model is dependent on myelination. The authors also showed that NgR1 stabilized fear memories and hastened learning of motor tasks by decreasing the threshold for experience-dependent memory. With all of this in mind, the authors conclude that the ‘low set point’ for anatomical plasticity in adult rodents is effectively determined by NgR1 expression, a receptor that ultimately serves to restrict the impact of experience on changes in cortical anatomy. It is important to note that this study and its clinical applications, as proposed by the authors, is not without shortcomings. For starters, the lack of pre-synaptic data for compound heterozygotes suggests that either the authors did not carry out these measurements on axonal varicosities or that the interaction of NgR1 and Ng-A/B is limited to post-synaptic neurons. Another shortcoming was the failure of the authors to examine the role of other NgR1 ligands such as MAG and chondroitin sulfate proteoglycans. Their choice of using ‘prototypical’ Ng22 and Ng-A/B targeted models leaves out a large variety of complex NgR1-ligand interactions that occur in vivo. In suggesting the use of NgR1 antagonists for rehabilitation in a future clinical setting, the authors overlooked findings that show reduced NgR expression during development may cause disease phenotypes such as schizophrenia14. Finally, we propose that a follow-up experiment examining electrophysiological recordings of the mutants modeled here should be carried out in order to confirm the functional properties of the cells examined; without this data the conclusions drawn remain incomplete.

SIGNIFICANCE OF THE WORK/FUTURE DIRECTIONS Despite the need for further validation of their results, Akbik et al. present novel findings about the role of NgR1 in gating the restriction of neuroanatomical plasticity in the adult cortex. The implications of these findings are not only far reaching but crucially relevant to an ageing society that continues to struggle with the personal and economic burdens associated with neurodegenerative disease. It is feasible to suggest that within the next 10 to 20 years, Akbik et al. and their colleagues will make considerable headway in efforts to transfer current NgR1 related discoveries from bench to bedside. Experience dependent plasticity and its functional outcomes, learning and memory, are the primary substrates of disease-related rehabilitation. In light of this, NgR1 serves as a putative target for pathologies where learning and memory are critical to recovery. Some of the potential pathologies where NgR1-focused therapies may be useful include ischemic stroke, spinal cord damage, post traumatic stress disorder (PTSD), psychomotor disorders such as Parkinson’s disease, and a host of neurodegenerative diseases that plague young and elderly alike. Therapies for these conditions may one day include the use of viral vectors expressing NgR1 antagonists, NgR1 silencing with RNAi, and pharmaceutical NgR antagonists (i.e. Cethrin, Alpha Tocopherol). By lowering the anatomical threshold for learning, the targeting of NgR1 gated plasticity creates a rehabilitation model that is more focused on the design of effective training programs and less concerned with overcoming insurmountable physiological boundaries. We foresee a future where NgR1 serves as a convergent target for reactivating dynamic adolescent levels of learning and plasticity, not only to combat disease but perhaps one day to improve the capabilities of society as a whole. Whether it be to extinguish fear memories in individuals plagued by PTSD, or to learn several languages in a short time, as our understanding of the factors that regulate experience-dependent plasticity improves, we are that much closer to unlocking the full potential of an ageless, and even limitless, human brain. REFERENCES 1. Yang, G., Pan, F., & Gan, W.B. Stably maintained dendritic spines are associated with lifelong memories. Nature, 462(7275), 920–4 (2009). 2. Holtmaat, A. J. G. D., Trachtenberg, J. T., Wilbrecht, L., Shepherd,

G. M., Zhang, X., Knott, G. W., & Svoboda, K. Transient and persistent dendritic spines in the neocortex in vivo. Neuron, 45(2), 279–91 (2005). 3. Grillo, F. W., Song, S., Teles-Grilo Ruivo, L. M., Huang, L., Gao, G., Knott, G. W., Maco, B., et al. Increased axonal bouton dynamics in the aging mouse cortex. PNAS, 1–10 (2013). 4. Fournier, A. E., Grandpre, T., & Strittmatter, S. M. Identification of a receptor mediating Nogo-66 inhibition of axonal regeneration, 409, 341– 346 (2001). 5. Park, J. B., Yiu, G., Kaneko, S., Wang, J., Chang, J., He, X. L., Garcia, K. C., et al. A TNF receptor family member, TROY, is a coreceptor with Nogo receptor in mediating the inhibitory activity of myelin inhibitors. Neuron, 45(3), 345–51 (2005). 6. Petrinovic, M. M., Hourez, R., Aloy, E. M., Dewarrat, G., Gall, D., Weinmann, O., Gaudias, J., et al. Neuronal Nogo-A negatively regulates dendritic morphology and synaptic transmission in the cerebellum. PNAS, 110(3), 1083–8 (2013). 7. Wills, Z. P., Mandel-Brehm, C., Mardinly, A. R., McCord, A. E., Giger, R. J., & Greenberg, M. E. The nogo receptor family restricts synapse number in the developing hippocampus. Neuron, 73(3), 466–81 (2012). 8. Willi, R., & Schwab, M. E. Nogo and Nogo receptor: Relevance to schizophrenia? Neurobiology of disease, [Epub ahead of print] (2013). 9. Rolando, C., Parolisi, R., Boda, E., Schwab, M. E., Rossi, F., & Buffo, A. Distinct roles of Nogo-a and Nogo receptor 1 in the homeostatic regulation of adult neural stem cell function and neuroblast migration. The Journal of Neuroscience, 32(49), 17788–99 (2012). 10. Karlsson, T. E., Karlén, A., Olson, L., & Josephson, A. Neuronal overexpression of Nogo receptor 1 in APPswe/PSEN1(ΔE9) mice impairs spatial cognition tasks without influencing plaque formation. Journal of Alzheimer’s disease, 33(1), 145–55 (2013). 11. Akbik, F. V., Bhagat, S. M., Patel, P. R., Cafferty, W. B. J., & Strittmatter, S. M. Anatomical Plasticity of Adult Brain Is Titrated by Nogo Receptor 1. Neuron, 77(5), 859–866 (2013). 12. Buchli, A. D. & Schwab, M. E. Inhibition of Nogo: A key strategy to increase regeneration, plasticity and functional recovery of the lesioned central nervous system. Ann. Med. 37, 556-567 (2005). 13. Lee, J. & Lee, J. Nogo Receptor Antagonism Promotes Stroke Recovery by Enhancing Axonal Plasticity. The Journal of Neuroscience 24, 6209; 6209-6217; 6217 (2004). 14. Willi, R. et al. Constitutive genetic deletion of the growth regulator Nogo-A induces schizophrenia-related endophenotypes. J. Neurosci. 30, 556-567 (2010).

Neuroscience Matters

mTORC2 Plays Key Role in Improving Long-term Memory Consolidation Helen (Bomin) Kim1, Hunaid Husain1 and Marzia Niamah-Hussain1 Human Biology Department, The University of Toronto. Toronto, Ontario CA.

The role of mTORC2 – mammalian target of rapamycin (mTOR) complex 2 in long term memory consolidation has been studied by Huang et al in postnatal murine forebrain using electrophysiological manipulation and behavioral conditioning. Rictor (rapamycin-insensitive companion of mTOR) deletion was found to have a direct correlation with mTORC2 deficiency and a selective impairment of long term memory processes; conversion of E-LTP to L-LTP and the formation of LTM were found to be impaired in the hippocampus of Rictor knockout mice. TORC2 deficiency in Drosophila showed a similar effect on LTM, indicating an evolutionarily conserved pathway. Regarding

the underlying mechanism, mTORC2 was found to mediate actin polymerization at the synaptic level, indicated by L-LTP and LTM impairment as a consequence of diminished actin polymerization in hippocampal neurons of mTORC2-deificient mice. However, restoration of memory was seen upon revival of actin dynamics. Pharmacological stimulation of mTORC2 activity enabled conversion of E-LTP to L-LTP and boosted LTM. It can be concluded that the role of mTORC2 in memory consolidation can serve as a therapeutic target and possible treatment of cognitive dysfunctions seen in patients suffering from neurological disorders.

mTORC2 selectively nurtures long term memory processes It was found that L-LTP inducing stimulation activated mTORC2 in CA1 neuronal slices of wild type mice, as opposed to Rictor fb-KO slices In contrast, E-LTP inducing stimulation failed to activate mTORC2 in either group (Figure 1: a,b). In addition, single train, E-LTP producing stimulation resulted in similar E-LTP in both control and Rictor fb-KO mice whereas a four train L-LTP inducing stimulation resulted in normal L-LTP in control mice but impaired L-LTP in Rictor fb-KO mice (Figure 1: c,d); indicating that reduced mTORC2 activity in forebrain neurons arrests the conversion of E-LTP to L-LTP, a crucial component of the memory consolidation process.

unaffected (Figure 2: a, b). Spatial LTM was also impaired in mTORC2 deficient mice, as shown by the Morris Water maze test and a probe test on day 7 (Figure 2: c, d).

Keywords: mTORC2 (mammalian target of rapamyacin 2);Rictor; memory consolidation; actin polymerization; long-term potentiation (LTP), dementia; Alzheimer’s Disease INTRODUCTION Memory formation and storage has been studied extensively by neuroscientists to identify important brain regions, pathways and molecules involved in both processes. More recently, it has been shown that post-transitional synaptic protein modifications address the transient changes in synaptic efficacy, such as shortterm memory (STM) and the early phase of long-term potentiation (E-LTP, lasting 1-3hrs). However, new proteins are synthesized for long-term memory (LTM) and L-LTP (lasting several hours)1-4, both of which also require changes in actin dynamics5-7. MTOR has two components, mTORC1 and mTORC2, both of which are functionally distinct complexes8,9 and evolutionarily conserved. Both components integrate input from several upstream pathways including insulin, growth factors (IGF-1 and IGF-2) as well as amino acids10. Rapamycin targets this pathways, specifically, mTORC1, to suppress the immune system by blocking the G1 to S phase transition in T-lymphocytes of rats11. The first component, mTORC1, is comprised of Raptor and mLST8 (GβL) and is the sensitive to rapamycin and has been suggested to regulate mRNA translation rates3. However, mTORC2, containing Rictor and mSIN1, is notably insensitive to rapamycin and the function of the component is less understood12. Rictor has been suggested to be linked to membranes and is associated with the regulation of the actin cytoskeleton.

Rictor has been shown to be critical for brain development and function, as mice lacking Rictor die in early embryogenesis12,13. The importance of Rictor has also been indicated by genetic deletion in developing neurons, which leads to adnormal brain development including smaller brain and neurons, as well as cognitive disruptions 14-15. Actin dynamics, including stability of the cytoskeleton and polymerization, underlie the mechanism of memory consolidation5-7 and are regulated by mTORC28,9. The connection between the role of mTORC2 and the memory consolidation regulated by actin polymerization was looked at specifically by Huang et al.14 to observe the sustain changes in synaptic efficacy (LTP) in hippocampal slices and memory tasks. The group focused on both upstream as well as downstream molecular mechanisms by which mTORC2 regulate L-LTP and LTM. It was found that mTORC2 is an important component of memory consolidation, through the regulation of actin dynamics14. It was also shown that a small molecular activator of mTORC2 and actin polymerization was important in sustaining L-LTP and LTM. RESULTS Rictor is crucial for mTORC2 activity in the forebrain Camk2a-Cre dependent Rictor deletion in mice caused reduction of mTORC2 function, localized to the forebrain (CA1 and amygdala); evident from reduced phosphorylation of Akt at Ser473, a known marker of mTORC2 activity 8,9.

Figure 2: (a) – Contextual STM and LTM measured as percent freezing at 2h and 24h post training respectively. (b) – Auditory STM and LTM and STM measured at 2h and 24h respectively, upon tone and foot shock pairing. (c) – Morris water maze test using a hidden platform. (d) – Probe test performed on day 7 in the absence of platform. Taken from Huang et al, 2013

Figure 1: mTORC2 activity measured as a function of Akt (Ser473) phosphorylation in WT (a) and Rictor fb-KO mice (b). (c) – single train (1 x 100 Hz) tetanic stimulation produces normal E-LTP in control and Rictor fb-KO mice. (d) – impaired L-LTP in mTORC2 deficient mice upon four train (4 x 100Hz) tetanic stimulation. Horizontal axes for c & d: Time(min) Taken from Huang et al, 2013.

With regards to memory storage, freezing responses in WT vs. Rictor fb-KO mice after Pavlovian fear conditioning (Contextual and auditory) training revealed that mTORC2 deficiency resulted in contextual and auditory LTM impairment but STM remained

Mechanism of mTORC2 regulation It was speculated that mTORC2 regulates actin dynamics at the synaptic level through Rac1-GTPase signaling, based on a reduced F-actin to G-actin ration and diminished Rho-GTPase activity in CA1 neurons of Rictor fb-KO mice. Jasplakinolide (JPK), an actin polymerization stimulant, restored normal L-LTP in mTORC2 deficient mice, confirming that actin polymerization plays a crucial role in mTORC2 regulated L-LTP formation. Similar results were obtained for LTM; post-training JPK infusion into CA1 neurons of Rictor fb-KO mice enhanced contextual LTM, whereas amygdaladependent auditory LTM remained unaffected. Furthermore, it was found that actin-mediated formation of L-LTP was dependent on new protein synthesis, evident from blocked JPK-facilitated L-LTP in the presence of Anisomycin, an inhibitor of protein synthesis. Lastly, application of A-443654, a small molecule drug that enhances mTORC2 activity, was sound to enhance mTORC2 activity, actin polymerization and PAK phosphorylation in WT slices but not Rictor fb-KO slices, which was evident from a corresponding enhancement in L-LTP and LTM in WT slices.

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Neuroscience Matters

CONCLUSIONS

REFERENCES

In conclusion, this paper identified the necessity of mTORC2 pathway in formation of long term memory (LTM) and the late phase of long term potentiation (L-LTP). Observation of memory formation on the behavioural level was shown using Morris water maze test; this showed its function in the hippocampus specifically. It was identified that actin polymerization, as noted in increased F-actin: G-actin ratio (specifically in the CA1 of the hippocampus), the structural regulation of memory consolidation is under the control of this conserved pathway. This was proven in mice models deficient in mTORC2; whose neurons showed reduced spine density in conjunction with inability to form LTM and carrying out L-LTP. Hence, their results shown that mTORC2 is responsible for the actin-dynamic-dependent L-LTP and LTM. This paper looked further into the downstream effectors of mTORC2; whose action was seen to take effects via the activation of Rac114. However, other downstream effectors of mTORC2 such as Akt and PKCalpha were not studied14. In order to confirm that these effectors do not play a role in actin polymerization, a possible direction to take is by using gene silencing method such has microRNA or short hairpin RNA. Using this method, these downstream effectors can be selectively knocked down in a time specific manner15. This pathway can be applied to higher mammals, such as humans since drosophila models that are deficient in mTORC2 homologue, dTORC2 – showed normal STM and E-LTP but not LTM nor L-LTP. Hence, this proves that this is an evolutionarily conserved pathway and can be applied to homo sapiens14.

1. Kandel, E.R. The molecular biology of memory storage: a dialogue between genes and synapses. Science 294, 1030–1038 (2001). 2. McGaugh, J.L. Memory–a century of consolidation. Science 287, 248– 251 (2000). 3. Costa-Mattioli, M., Sossin, W.S., Klann, E. & Sonenberg, N. Translational control of long-lasting synaptic plasticity and memory. Neuron 61, 10–26 (2009). 4. Wang, S.H. & Morris, R.G. Hippocampal-neocortical interactions in memory formation, consolidation and reconsolidation. Annu. Rev. Psychol. 61, 49–79 C1–4 (2010). 5. Cingolani, L.A. & Goda, Y. Actin in action: the interplay between the actin cytoskeleton and synaptic efficacy. Nat. Rev. Neurosci. 9, 344–356 (2008). 6. Lamprecht, R. & LeDoux, J. Structural plasticity and memory. Nat. Rev. Neurosci. 5, 45–54 (2004). 7. Lynch, G., Rex, C.S. & Gall, C.M. LTP consolidation: substrates, explanatory power and functional significance. Neuropharmacology 52, 12–23 (2007). 8. Guertin, D.A. & Sabatini, D.M. Defining the role of mTOR in cancer. Cancer Cell 12, 9–22 (2007). 9. Wullschleger, S., Loewith, R. & Hall, M.N. TOR signaling in growth and metabolism. Cell 124, 471-484 (2006). 10. Hay N, Sonenberg N. Upstream and downstream of mTOR. Genes Dev 18 (16): 1926–45 (2004). 11. Magnuson B, Ekim B, Fingar DC. Regulation and function of ribosomal protein S6 kinase (S6K) within mTOR signalling networks. Biochem. J. 441 (1): 1–21(2012). 12. Guertin, D.A. et al. Ablation in mice of the mTORC components raptor, rictor or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCalpha, but not S6K1. Dev. Cell 11, 859–871 (2006). 13. Shiota, C., Woo, J.T., Lindner, J., Shelton, K.D. & Magnuson, M.A. Multiallelic disruption of the rictor gene in mice reveals that mTOR complex 2 is essential for fetal growth and viability. Dev. Cell 11, 583–589 (2006). 14. Huang, W. et al. mTORC2 controls actin polymerization required for consolidation of long-term memory. Nature Neurosci. 10 (2013). 15. Xiang, S., Fruehauf, J., Li, C. J. Short hairpin RNA–expressing bacteria elicit RNA interference in mammals. Nature Biotechnology 24 (6): 697– 702 (2006). 16. Tsai, V., Parker, W. E., Orlova, K. A., Baybis, M., Chi, A. W. S., Berg, B. D., Birnbaum, J. F., Estevez, J., Okochi, K., Sarnat, H. B., Flores-Sarnat, L., Aronica, E., and Crino, P. B. Fetal brain mTOR signalling activation in tuberous sclerosis complex. Cereb cortex published online, doi: 10.1093/ cercor/bhs310 (18 Oct 2012). 17. Zhou, J., Blundell, J., Ogawa, S., Kwon, C. H., Zhang, W., Sinton, C., Powell, C. M. and Parada, L. F. Pharmacological inhibition of mTORC1 suppresses anatomical, cellular and behavioural abnormalities in neuralspecific Pten knock-out mice. J. Neurosci. 29, 1773-1783 (2009).

SIGNIFICANCE AND FUTURE DIRECTIONS This finding is highly valuable in that it is both applicable to humans and can be used to further study debilitating symptoms seen in neurological disorders such as Alzheimer’s, Parkinson’s, Huntingtons and Autism Spectrum Disorder. These disorders commonly share dementia and memory deficits14. As proven in this paper, mTORC2 pathway is essential to memory consolidation and storage, hence closer look into this underlying pathway can give rise to a potential treatment method. A study in 2012 showed that mutations in PTEN and/or TSC1 and TSC2 which are negative regulators of mTORC1 were shared among patients suffering from autism spectrum disorder (ASD)16. Another group used in vivo mice models of ASD. These mice models exhibited ASD-like phenotypes, and upon injection of rapamycin, mTORC2 activity was seen to improve in these mice. Furthermore, ASD-like symptoms were also shown to be improved17. Hence, their findings as well as other groups’ observations show that a closer look into the role of mTORC2 in memory consolidation can lead towards finding a method for the treatment of cognitive dysfunctions seen in patients suffering from neurological disorders.

Redefining the Distinct Mechanisms of Lasting Memories: an example with Caenorhbaditis elegans Alexa Quach1,2 Human Biology Department, Neuroscience, The University of Toronto. Toronto, Ontario CA. Cell and Systems Biology, The University of Toronto. Toronto, Ontario CA.

1 2

Memory can be dissected into subtypes based on retention times, such as short-term, middle-term, and long-term memory. The nematode Caenorhabditis elegans is an excellent model system to study learning and memory. Relatively little is known about the mechanisms underlying massed trained memories, although it has been suggested that they are regulated by different cellular and molecular mechanisms based on the differences in temporal retention of the memory. Here it shown that the different in the temporal training protocol of spaced or massed training using tap habituation has a distinct mechanism that allows a lasting alteration in neurotransmission. Spaced training consists of postsynaptic changes, including AMPA receptor trafficking, de novo protein synthesis and the transcription factor CREB, whereas

massed training was shown to result in presynaptic changes in the mechanosensory neurons and the release of FMRFamide-related peptide (Phe-Met-Arg-Phe-NH2) FLP-20 at the interneurons of the neural circuit for a 12-hour massed trained memory. As subtypes of memory were defined earlier, this review explores the possibility of an independent pathway for this 12-h MTM, or even possibly anesthesia-resistant memory (ARM), and the significance this has on our knowledge of LTM storage. This reductionist approach can be used to decipher LTM mechanisms that may be conserved amongst simple invertebrates to complex vertebrates. Therefore the temporal protocol of training is able to cause specific types of lasting memory mediated by distinct mechanisms.

Key words: mechanisms of memory; short-term memory (STM); long-term memory (LTM); habituation; massed training; spaced training; neuropeptide neurotransmission; FMRFamide peptide INTRODUCTION Consolidated memory can be dissected into subtypes induced by different forms of training. First described by Ebbinghaus in 19131, temporally spaced training, where “rest” periods are provided in between blocks of training, induces longer lasting memory versus massed training, where the training is given in a single massed block of time. The dissection of memory subtypes was demonstrated in the model organism Drosophila by Tully2 where conditioned odor avoidance during spaced training after 1 day training was able to form two genetically distinct, independent, but parallel forms of memory, anesthesia-resistant memory (ARM) and long-term memory (LTM), where the memory mechanisms branch off from the progression of short-term memory and middle-term memory2 (Fig.1). The disruption of one form of memory using single gene mutations allows the other memory to remain intact2. It was also shown to be independent of new protein synthesis, shown using cyclohexamide, a protein synthesis inhibitor, to disrupt LTM, but not ARM, which is cycloheximide insensitive2. Spaced training has been shown to produce longer lasting retention of memories than an equivalent time of training during a single massed training block in other organisms, including mice3, humans4, and nematode Caenorhabditis elegans5. The nematode C. elegans is an excellent model to study learning and memory due to

their simple nervous system that consists of a mere 302 neurons, easily able manipulate genetically for the study of learning and memory. It was shown previously that massed training created memory that lasted only 12 hours, and not 24 hours created by spaced training using the tap habituation paradigm in C. elegans5. The tap habituation paradigm consists of an automated mechanical tapper that exerts 1-2 N of force to the side of the petri pate on which worms sit on agar5. This creates a reversal response, or the reversal movement by the worms, which after training results in habituation – the reduction of reversal responses.

Fig. 1 The genetic pathway that underlies the progression of memory formation after Pavlovian olfactory conditioning in Drosophila. Figure adapted from Tully et al. (1994).

It was determined that LTM 24-hour memory was dependent on glutamate training, post-synaptic AMPA receptors trafficking and the transcription factor CREB5. The common consensus for long-term potentiation involves glutamate release, binding

Neuroscience Matters to NMDA receptors for downstream signaling, that allows the recruitment of AMPA receptors to the post-synaptic membrane to strengthen the connection between the pre- and post-synaptic terminals, allowing easier excitation and depolarization of the cell. A simple learned behaviour, seen in the invertebrate Aplysia, the giant marine snail, with a defensive reflex of gill-withdrawal upon tactile stimulation of the siphon, and showed the distinction between a transient memory, or a short term memory (STM) that lasted several minutes to an enduring memory that lasts days, or LTM, using spaced repetition of training6 (Fig. 2)7.

Fig. 2 The temporal distribution of memory retention for different subtypes of memory, for STM, MTM, ARM and LTM. Figure adapted from Margulies et al. (2004).

It was then determined that LTM required new protein synthesis, distinctly different from STM, and that there was a conservation of biochemical mechanisms between Aplysia and vertebrates6. This reductionist approach focuses on using the simplest instance of memory storage to study learning and memory mechanisms on the basis of conservation and the cell and molecular level between these higher vertebrates and simple invertebrate models6. In this review of the Li et al. (2013) paper, the FMRFamide-related peptides (FaRPs) expressed in the mechanosensory neurons of the tap withdrawal circuit were explored because they were hypothesized to be involved in the plasticity of the tap response8. This shows new evidence for a distinct memory mechanism for this 12-h massed habituation memory (which can be referred to as middle-term memory, or MTM) that differs from LTM. MATERIALS AND METHODS Strains and transgenic animals In the Li et al. (2013) study7, C. elegans strains from the Caenorhabditis Genetics Center (University of Minnsesota, Minneapolis, MN) including wild-type N2 Bristol, KP4 glr1(n2461), MT6308 eat-4(ky5), and YT17 crh-(tz2). VG126 Pmec7::SNB- 1::GFP gifted from Dr. Michael Nonet (Washington University, St. Louis). Deletion allele strains flp-4(yn35) and flp-8(pk360), and flp-20(pk1596) were isolated from EMSmutagenesis, and double and triple mutants were generated8. Lines using the Pmec-7 or the flp-20 promoter were used to create transgenic animals, by amplification of the flp-20 region, or the

Neuroscience Matters cDNA flp-20 region, and microinjection with coinjection markers into plasmid constructs with the specific promoters. Heat shock treatment and training protocols Habituation assays were performed as described in Rose et al (2002). 4 day old worms were tested for 24-hour experiments, and 4.5 day old worms were tested for the 12-hour experiments. Spaced training consisted of 4 blocks of training with 20 taps at 60-sec interstimulus intervals (ISI), with 1 hour rest between each training block. Massed training consisted of 80 taps in a single training block with the 60 ISI. Habituation memory was measured at 22-28 hours after training for the 24-hour memory and 10-16 hours after the training for the 12-hour memory. 15-20 worms were tested on each plate for trained and untrained control worms. Heat shock at 32 degrees Celsius for 40 minutes was administered. Confocal microscopy and fluorescence quantification GFP transgenic worms were mounted onto slides, and GFP was measured where the processes of the PLM mechanosensory neurons form chemical synapses onto the interneurons within the tap withdrawal circuit. A fluorescent reporter was used to monitor vesicle proteins. Researchers were blind to the treatment groups analyzing the imaging files.

Increased synaptic vesicles in the presynaptic terminals of PLM follows massed training and is dependent on flp-20 activity The authors tagged synaptic vesicles synaptobrevin/SNB-1 with GFP in presynaptic terminals of the PLM mechanosensory neurons, and assayed GFP-tagged synaptobrevin. They found that only after massed training after 12-h there were significantly larger areas of fluorescence in presynaptic terminals, but these areas decreased in fluorescence after 24 hours to levels similar to control untrained worms, correlating to the length of the 12-h massed habituation memory, which similarly disappeared after 24 hours. flp-20 is required in mechanosensory neurons for memory following massed training flp-20 mutants were used to express flp-20 cDNA under the mec7 mechanosensory cell specific promoter, which showed rescue of the memory. flp-20 cDNA was also expressed under the flp-13 promoter, driving expression in many different neurons but not in mechanosensory neurons, did not show any memory. Therefore it was determined that flp-20 was required in mechanosensory neurons specifically for the 12-h habituation memory DISCUSSION

Data analyses ANOVA was used to compare the reversal distances in response to tap from each trained group to the untrained control group. An α significance value of 0.05 was used. RESULTS Flp-20 neuropeptide is required for 12-h memory retention during massed training The authors tested three FaRP neuropeptide genes flp-4, flp-8, and flp-20, expressed in mechanosensory neurons of the tap withdrawal circuit, using deletions in each of these genes and testing using massed or spaced training in 12 and 24 hours. It was determined that there was no evidence of massed trained memory in the flp-4; flp-8; and flp-20 triple mutant. It was then determined that the flp-20, expressed in all mechanosensory neurons, was critical for the massed 12-h memory (Fig. 3) Memory of massed training and spaced training are mediated by distinct mechanisms Disruption of protein synthesis by heat shock, worms with genetic mutations for glutamate receptors or mutations for transcription factor CREB pathway showed that these worms could still form the 12-h massed memory, and thus concluded that these processes required for memory tap habituation 24-h after training (LTM) were not required. Therefore this different training protocol memory is mediated by a distinct mechanism.

Fig. 3 flp-20 is required for massed training 12-h memory. (A) Wildtype worms showed 12-h massed training memory (denoted by * as significant decrease in mean reversal distance) (B) No memory formed in flp-4; flp-8; and flp-20 triple mutants. (C) Spaced training showed memory formation in flp-4; flp-8; and flp-20 triple mutants. (D) Massed training showed no 12-h memory in flp-20 mutants, unlike spaced training. (E) Genomic flp20 under endogenous promoter in flp-20 mutants rescued 12-h massed memory.

This study determined that temporal training protocols have distinct mechanisms to cause different lasting memories. Massed training resulted in a shorter memory that is not dependent on de novo protein synthesis or CREB, caused and maintained by presynaptic vesicle recruitment in mechanosensory neurons containing neuropeptide FLP-207. In contrast, the spaced training memory is longer and mediated by post-synaptic changes in interneurons7. Therefore these memories are mediated by different mechanisms. However how FLP-20 neuropeptides create a lasting alteration in neurotransmission for this 12-h memory is unknown. It is suggested that there is an increase in the area of vesicles of the PLM mechanosensory neuron terminals, that allow these neuropeptides to be release7. Neurotransmitter glutamate release was not proposed as a part of the mechanism as they determined the eat-4 mutant, which has insufficient glutamate release for neurotransmission, was still able to form the 12-h massed trained memory7. However the authors do not address where these distinct mechanisms branch from a common progression from STM for the different temporal training protocols. There is an overlap between the protocols for spaced and massed training, where the first training block of 20 taps in spaced training overlaps with the beginning of the first 20 taps in massed training. However if the resultant memory is mediated by different mechanisms, where exactly do these memory pathways diverge? According to Tully et al. (1994) it was shown that MTM progresses into either LTM or ARM (Fig. 1). However the results of this paper suggest there may be an even earlier branching of the mechanisms from STM to LTM, which may occur during memory acquisition shortly after the first 20 taps in the first training block, acting as a molecular switch determines the specific memory pathway. It is also possible that the ARM found in Drosophila2 is similar to the 12-h memory found in this study, as ARM also does not depend on de novo protein synthesis or CREB9. It is important however to remember that the Drosophila was an associative memory, whereas this study explored a non-associative memory in C. elegans. SIGNIFICANCE OF THE WORK This study is significant because it explores the distinct mechanism for this massed 12-h habituation memory from spaced trained memory. Using a reductionist approach to decipher molecular mechanisms of memory, it is possible that the authors have identified a new mechanism for lasting memories that may be conserved amongst more complex vertebrate organisms as well. While the human homologs of flp-20 have not been well established, these FaRPs are found within the animal kingdom and shown in many behaviours including pain modulation10, feeding11 and reproduction12. While a specific evolutionary advantage for having distinct mechanisms may not be immediately apparent, it is nevertheless advantageous to leave one memory pathway intact

Neuroscience Matters if the other was damaged, for survival reasons such as memories of food scavenging or a conditioned fear response. Therefore it should be kept in mind that previous studies using a simpler model of the nervous system have shown the conservation of cellular mechanisms of learning and memory storage in more complex vertebrates6, so even the smallest and simplest of organisms can have the greatest contribution for our knowledge of learning and memory. FUTURE DIRECTIONS It is not known how flp-20 neuropeptides in the recruitment of high trafficking core vesicles establish this 12-h habituation memory. Therefore further research with FaRPs in higher complex vertebrate organisms is needed to further explore this distinct memory mechanism. Also since there are similarities between ARM and the 12-h memory induced in this study, perhaps more experiments need to be performed, such as an anesthesia resistant memory in C. elegans, as described by Tully et al. (1994) in Drosophila. Genetic screens can also be used to discover important genes involved in LTM formation using manipulations between the spaced and massed training protocols, and possibly a identify a molecular switch that allows for specific LTM formation. REFERENCES 1. Ebbinghaus, H. Memory: A contribution to experimental psychology. (Teachers College, Columbia University, 1913). 2. Tully, T., Preat, T., Boynton, S. C. & Del Vecchio, M. Genetic dissection

Neuroscience Matters of consolidated memory in Drosophila. Cell 79, 35–47 (1994). 3. Scharf, M. T. et al. Protein synthesis is required for the enhancement of long-term potentiation and long-term memory by spaced training. J. Neurophysiol. 87, 2770–2777 (2002). 4. Kornmeier, J. & Sosic-Vasic, Z. Parallels between spacing effects during behavioral and cellular learning. Front Hum Neurosci 6, 203 (2012). 5. Rose, J. K. A New Group-Training Procedure for Habituation Demonstrates That Presynaptic Glutamate Release Contributes to LongTerm Memory in Caenorhabditis elegans. Learning & Memory 9, 130– 137 (2002). 6. Kandel, E. R. The Molecular Biology of Memory Storage: A Dialogue Between Genes and Synapses. Science 294, 1030–1038 (2001). 7. Margulies, C., Tully, T. & Dubnau, J. Deconstructing Memory in Drosophila. Current Biology 15, R700–R713 (2005). 8. Li, C. et al. The FMRFamide-related neuropeptide FLP-20 is required in the mechanosensory neurons during memory for massed training in C. elegans. Learning & Memory 20, 103–108 (2013). 9. Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71–94 (1974). 10. Li, C. & Kim, K. Neuropeptides. WormBook 1–36 (2008). doi:10.1895/ wormbook.1.142.1 11. Bechtold, D. A. & Luckman, S. M. The role of RFamide peptides in feeding. J. Endocrinol. 192, 3–15 (2007). 12. Kriegsfeld, L. J. Driving reproduction: RFamide peptides behind the wheel. Horm Behav 50, 655–666 (2006).

Received April 2, 2013; revised Month, ##, 200#; accepted Month, ##, 2013. Address correspondence to: Alexa Quach Email: alexa.quach@mail.utoronto.ca

Gene Therapy: The Future of Treatment for Neurodegenerative Disorders Kalachandran, Sallini1 and Raval, Jelum1 Human Biology Department, Neuroscience, The University of Toronto. Toronto, Ontario CA.

Parkinson’s Disease is a debilitating neurodegenerative disorder most prevalent in old age. Additionally, as more individuals enter this demographic due to surges in population as well as increased life expectancy the need for better treatment options are urgent. At this stage in the research and treatment of neurodegenerative disorders, there is still no cure to efficiently reduce and reverse the neuronal damage prominent in these disorders. Treatments that are currently available are limited in their therapeutic advantages as they act to ameliorate disease state and alleviate some of the symptoms, making for a somewhat better quality of life. However, these types of symptom-aimed treatments only prolong the inevitable demise of the central nervous system. With the expanding population of seniors, the need to develop treatments

that are neuroprotective and aimed at reversing neuronal loss are emphasized, especially so with the goal of preserving cognitive functioning. This review focuses on the efficacy of gene therapy with CDNF in comparison to GDNF, shown to have significant effects in neuronal rescue. The hopes of the study that this review focuses on is that CDNF gene therapy may be a potential neuroprotective treatment for Parkinson’s disease in which there are substantial losses of dopaminergic neurons in the pars compacta of the substantia nigra. Research as such has major implications in the future for possible prevention therapies for Parkinson’s disease and possibly other neurodegenerative disorders, if research is taken in the right direction.

Key words: Parkinson’s disease; neuroprotective treatment; gene therapy; cerebral dopamine neurotrophic factor (CDNF); neurodegeneration; dopaminergic neuronal loss; novel therapies for Parkinson’s disease; 6-OHDA PD mice I. BACKGROUND Parkinson’s disease (PD) is a common neurodegenerative disorder primarily affecting individuals over the age of 601,2. Although research in the past decade has tremendously benefitted our understanding of PD and its pathogenesis, there is still no definitive test for its diagnosis until post-mortem15. Clinicians therefore make assessments based on four cardinal features which include tremors at rest, rigidity of the body, postural instability, as well as akinesia or bradykinesia1,2,15. The pathology in the brain responsible for this clinical presentation is the hallmark loss of dopaminergic neurons in the pars compacta of the substantia nigra. PD is, however, a progressive disease with presymptomatic neurodegeneration beginning in areas such as the olfactory bulb and advancing to the nigrostriatal regions and eventually the neocortex3. The staging of the pathology is based on the spread of lewy bodies comprised of α-synuclein immunoreactive inclusions2,15. Although the idiopathic cases of PD are numerous, there are some familial incidences that suggest a genetic component, such as mutation in the α-synuclein gene found in some familial forms of PD2. Many of the treatments currently available for Parkinson’s disease alleviate the symptoms rather than targeting the neurodegeneration that is responsible for the symptoms to begin with. The first line of treatment for patients with PD and the most widely used is levopdopa, often in addition to a peripheral decarboxylase inhibitor2. Levadopa or L-dopa is a precursor to dopamine that acts to replenish the dopamine neurotransmitter lost in dopaminergic degeneration of PD. L-dopa is administrated in the place of dopamine as it has the ability to cross the blood brain barrier unlike dopamine1. The peripheral decarboxylase inhibitor is combined in the treatment to allow L-dopa to reach the neurons without degradation in peripheral tissue1. Unfortunately L-dopa does not target the continual loss of neurons and side effects include gastrointestinal trouble, hypotension, dyskinesias such as twitching and nodding with long term use1,4, and perhaps even a neurotoxic effect5. Inhibitors against catechol O-methyl transferase (COMT) and monoamine oxidase (MOA), both of which are involved in metabolism of dopamine, have also been used as treatment1,2. Surgery and Deep Brain Stimulation (DBS) are also potential treatment options2. Approaching therapeutic options from a neuroprotective approach is promising for targeting neurodegeneration and therefore subsequent motor and non-motor disabilities at its core. Cerebral dopamine neurotrophic factor (CDNF), as a neurotrophic factor, is important for regulating plasticity, promoting survival, differentiation, and maintenance of dopaminergic neurons6. It has

been implicated in playing a neuroprotective role in PD by reversing and preventing neurodegeneration. In a study by Lindholm et al. (2007), PD-like neurodegeneration of the nigrostriatal dopaminergic system was induced in rats, via administration of 6-hydroxydopamine (6-OHDA). A single administration of the CDNF protein before lesioning with 6-OHDA showed a rescue of almost all dopaminergic tyrosine hydroxylase-postive cells in the substantia nigra7. Similar recues were seen when CDNF was injected into the striatum up to 4 weeks post 6-OHDA treatment7. This review focuses on paper in which a novel CDNF gene therapy was used in opposition to CDNF protein injection. The rationale for such a study was that protein injections must be administered continuously over time, whereas gene therapy has been shown to be effective and safe over a longer period time8 after just the initial injection. II. REVIEWED RESEARCH MATERIALS AND METHODS

Figure 1 Viral vector (A) and behavioural test (B) set up.

AAV-CDNF and AAV-GDNF plasmids were made by cloning human CDNF and GDNF ORFs into the appropriate restriction enzyme sites of AAV-MCS. The plasmids were then co-transfected with helper plasmids. The rats were given isoflurane to anaesthetize them before the rats’ left striatum was injected with the viral vector using a stereotaxic operation; the right striatum wasn’t injected so that protein expression could be compared between the two sides later. The rats were divided randomly into six groups such that they received different titers of AAV2-CDNF, AAV2-GDNF, or

Neuroscience Matters a PBS negative control. All the rats were injected with 6-ODHA two weeks after the viral injection to lesion the Dopaminergic system in the rat midbrain in a similar stereotaxic operation with anesthesia. Behavioral tests were performed bi-weekly two weeks after injection for up to 10 weeks. 12 weeks post-injection, the rat brains were fixed and immunohistochemical staining and immunofluorescence was performed on brain sections6. III. RESULTS Viral Vector Mediated Expression Human CDNF expression was followed using a CDNF-ELISA assay post injection of AAV2-CDNF into the rat striatum. The expression patterns suggest dependence on both titer and time. Pictured in Figure 1A, the results showed that CDNF protein levels, measured as pg/mg of total protein in the striatum, increased as the virus vector titer increased. A five time increase between the second and third titers (2 x 108 vg to 1 x 109 vg) lead to a significantly substantial increase in CDNF expression from 160pg to 530pg of CDNF/mg of total protein. The results also showed time-dependent expression. The CDNF levels were measured as a function of time from the start of the study and for 12 weeks onward, for the highest titer.

Figure 2 Rat hCNDF expression levels measured in striatum and substantia nigra post AAV2-CDNF injection, using CDNF-ELISA assay. A Quantified hCDNF expression 4 weeks post injection for different titers. B hCDNF protein expression levels as a function of time, measured from the beginning to the end of study. C hCDNF expression measured in substantia nigra of rats with highest titer injection.

Neuroscience Matters 180pg of CDNF/mg of total protein was detected at just 1-week after the viral vector titer injection. This was followed by an accelerated increase in expression to 490pg by the second week. The Expression continued to increase until the 8th week, and remained till the 12th week, when the study ended. When the highest titer was injected into the rat striatum, hCDNF appeared to also be expressed in the substantia nigra starting at 2 weeks post injection.

IV. DISCUSSION To summarize, the main results of this paper were that intrastriatal expression of the CDNF gene therapy leads to the expression of hCDNF in the brain, as well as functional recovery of neuronal circuits involved movements of the 6-OHDA. The results of this study are promising for CNDF gene therapy as a potential treatment for Parkinson’s disease. However, there are certain caveats that must be further considered. One major concern when delivering therapeutic agents with viral vectors is that the level of the expression is difﬁcult to control, and sustained expression of the transgene could cause negative effects. This problem could be solved in the future by developing more efficient means of controlling inducible vectors. Furthermore, this study did not directly asses for the death of dopaminergic neurons, since lack of staining for TH-immunoreactivity may not directly infer to death of these neurons. They may not have stained for multiple reasons, one being that although they were alive, they perhaps had not been producing enough TH. A better understanding of the molecular mechanisms by which CDNF and GDNF act to protect dopaminergic neurons in this context should be further studied to better interpret the results showing a reduced THimmunoreactivity.

Detection of Protein Expression Figure 3 on the right shows immunohistochemical staining of the rat brain twelve weeks post injection of the viral vectors. The CDNF in the striatum was found in the areas into which it was injected and stayed within the neurons, whereas the GDNF injected in the striatum diffused into the surrounding tissue. CDNF was expressed in the Global Pallid us (GP) and Substantia Nigra (SN) in addition to the striatum. Both CDNF and GDNF expression was significant when compared to endogenous levels already expressed in the GP and SN. GDNF was also found in the ipsilateral GP, SNpc, and SNpr. Furthermore, the CDNF colocalized with NeuN in the striatum and with TH-immunoreactive cells in the SNpc upon immunoflourescent staining6. NeuN is a nuclear protein that is specific for neurons and it is expressed in mostly all types of neurons in adult mice making it a good neuronal marker9. Co-localization with TH means it was found within dopaminergic neurons. TH-Immunohistochemistry Further staining was done using antibodies against Tyrosine Hydroxylase. Measuring the amount of TH-hydroxylase immunereactive fibers allowed the researchers to determine the amount of dopaminergic neurons in the striatum of the rats. Ten weeks after the 6-ODHA lesions, the control rats, when compared to the intact non-lesioned side, had lost approximately 78% of the TH-immunoreactive fibers. Whereas rats treated with GDNF showed only a 58% loss and CDNF treated rats showed 63% loss. This indicated that both CDNF and GDNF served some protective function for dopaminergic neurons. Furthermore, there was no significant protective function found in the SNpc, but with the highest titer of vector used (1 x 109 vg) CDNF showed significant protective function in just the central SNpc. Similarly, GDNF showed no significant protection but there was increased protection with increased titer of viral vector in the SNpc. Not only does GDNF serve a protective function, TH-fiber sprouting was also seen in the striatum, lateral GP, and SNpr of rats who were injected with GDNF prior to the lesioning. AAV2-CDNF injections, conversely, showed no tendency to cause TH-reactive fiber sprouting. However, rats that were treated with CDNF protein showed sprouting in the striatum. This suggests that there is a difference in mechanism of how intracellular versus extracellular CDNF might be working to effect dopaminergic neurons6. Further studies need to be done to study the mechanisms of CDNF and GDNF action.

Figure 3 (above) CDNF and GDNF expression in the rat brain. CDNF was found to be expressed in the striatum (A), global pallidus (B) and substantia nigra (D) and it was significant expression when compared to endogenous levels in the GP (C) and SN (E). GDNF was also expressed in the striatum, but diffused into surrounding tissue as opposed to CDNF which stayed intracellular (F). GDNF was also found in the ipsilateral global pallidus (G) and the substantia nigra pars compacta (I). Similar to CDNF, GDNF expression was significant and more than endogenous levels in the GP and SN (H, J). CDNF co-localized with NeuN in the striatum (K) and with TH in the SNpc (L).

Amphetamine-induced Rotational Behaviour Lesioned rats were injected with amphetamine and rotational behaviour was monitored for a period of 120 minutes. Amphetamine-induced turning behaviour was measured 2,4,6,8 and 10 weeks post 6-OHDA lesion as net ipsilateral rotations. At 2 and 4 weeks, the group administered the second highest viral vector titer showed significant attenuation of the ipsilateral rotation tendency of the rats. At 6 and 10 weeks, the group with the highest titer administration showed significant improvements in rotation symmetry.

Another aspect of the study that must be considered is the animal model. The neurodegeneration in these rats were chemically induced with 6-OHDA, and was limited to the nigrostriatal pathway. These mice do not replicate the progressive nature of Parkinson’s disease. In addition, these mice do not produce the characteristic lewy bodies comprised of pathogenic α-synuclein inclusions present in PD. This model is therefore not true model of Parkinson’s disease. Furthermore, this study bases the efficacy of CDNF treatment against the effects of glial-derived neurotrophic factor (GDNF), which has been shown to have protective function in many models of PD except the α-synuclein models of PD. This posits the question whether the results of this experiment can then reliably extrapolate to human Parkinson’s disease in which synucleinopathy is characteristic10. As a treatment for humans, this type of therapy is still questionable at this stage. A viral injection of CDNF does prove more promising than injecting the CDNF protein into the striatum itself, in that there is generally longer lasting expression. The exact length of the expression is however unknown. The paper demonstrates that with the highest titer expression continually rises till about 8 weeks and remains stabilized till the 12th week when the study was concluded. The method of administration in the rat models was extremely invasive, with an injection directly into the striatum. Therefore, the benefit of such a therapy is shadowed by its invasiveness nature. Furthermore, if there is not sustained expression for a reasonable length of time, then the requirement for repeated treatments within short intervals of time further shadow the benefits.

Neuroscience Matters Aside from these concerns, alterations and improvements of this gene therapy is viable as a possible future prophylactic treatment of Parkinson’s disease, slowing down the progression of the disease by targeting and repairing the neuronal cell death responsible for the clinical manifestations of PD. V. SIGNIFICANCE AND FUTURE DIRECTIONS According to the U.S. Census Bureau, age distribution patterns projecting from 2010 to 2030 show large growth individuals entering the age range when PD is prevalent11. As of now, the baby boomer generation occupy the age range of 45-60 years, and it is predicted that within a decade or so, the largest population group will be between 65 and 85 years old11. This surge in the elderly population is furthered by increasing life-expectancy11. With more individuals entering this demographic, the urgency behind understanding and treating neurodegenerative diseases in general, is extremely important. Advances in genetics and technology have reified gene therapy as a potential therapeutic opportunity for neurodegenerative diseases. The ability to deliver genes as a means of neuroprotection in the brain, or even replacement and replenishment of deficient proteins implicated in the disease pathology is becoming more and more a potential option today. The field of gene transfer technology has developed quite a diverse array of genetic tools and current experiments with animal models have been investigating the versatility and efficacy of them14. However, it is important to consider the gap between animal models and human subjects in the context of gene therapy outcomes. Within the next decade or so, with an increase pressure for more promising treatment options, the safety and efficacy of gene transfer methods are expected to improve so that gene therapy becomes a viable option for treatment. In fact, Phase I trials for certain gene therapies in humans are already underway. Phase I trials have already been completed for AADC gene therapy for Parkinson’s disease and it was found that the viral vectors were stable, without any adverse effects, for up to four years after they were administered to the patients but further long term follow-ups were not done8. It has also been used in adults with congenital blindness12 and AAV2NGF (nerve growth factor) gene therapy has been shown to prevent cardiomyopathy in Type 1 Diabetic Mice13. Understanding the mechanisms by which growth factors and other molecules work in the brain to serve a protective function against neuronal damage or death can provide significant insights into neurodegeneration as well as pave the way for developing new, innovative, and effective therapies for treatment and maybe even prevention of neurodegenerative diseases.

Neuroscience Matters REFERENCES 1. Tandon, A. (2012) Parkinson Disease: Clinic to Biochemistry [PDF document]. Retrieved Lecture Notes Online 2. Davie, C. A. A review of Parkinson’s disease. Br Med Bull 86, 109-127 (2008). 3. Braak H., Ghebremedhin, E., Rüb, U., Bratzke, H., & Del Tredici, K. Stages in the development of Parkinson disease-related pathology. Cell and Tissue Research 318, 121-134. (2003). 4. Schrag, A., & Quinn, N. Dyskinesias and motor fluctuations in Parkinson’s disease: A community-based study. Brain 123, 2297-2305 (2000). 5. Fahn, S. Parkinson Disease, the Effect of Levodopa, and the ELLDOPA trial. Arch Neurol 56, 529-535 (1999). 6. Bäck, S. et al. Gene therapy with AAV2-CDNF provides functional benefits in a rat model of Parkinson’s disease. Brain and behavior 3, 75-88 (2013). 7. Lindholm, P., Voutilainen, M. H., Laurén, J., Peränen, J., Leppänen, V. M., Andressoo, J. O., et al. Novel neurotrophic factor CDNF protects and rescues midbrain dopamine neurons in vivo. Nature 448, 73-77 (2007). 8. Mittermeyer, G. et al. Long-Term Evaluation of a Phase 1 Study of AADC Gene Therapy for Parkinson’s Disease. Hum. Gene Ther. 23, 377381 (2012). 9. Mullen, R. J., Buck, C. R. & Smith, A. M. Neun, a Neuronal Specific Nuclear-Protein in Vertebrates. Development 116, 201-211 (1992). 10. Stefanis, L.. α-Synuclein in Parkinson’s Disease. Cold Spring Harb Perspect Med 2, a009399 (2012). 11. OpenStax College. (2012, July 16). Who Are the Elderly? Aging in Society. Retrieved from the Connexions Web site: http://cnx.org/content/ m42874/1.5/. 12. Bennett, J. et al. AAV2 Gene Therapy Readministration in Three Adults with Congenital Blindness. Science Translational Medicine 4, 120ra15 (2012). 13. Meloni, M. et al. Nerve Growth Factor Gene Therapy Using AdenoAssociated Viral Vectors Prevents Cardiomyopathy in Type 1 Diabetic Mice. Diabetes 61, 229-40 (2012). 14. Carter, J. E., & Schuchman, E. H. Gene therapy for neurodegenerative diseases: fact or fiction? The British Journal of Psychiatry 178, 392-394 (2001). 15. Jankovic, J. Parkinson’s disease: clinical features and diagnosis. J Neurol Neurosurg Psychiatry 79, 368-376 (2008).

Received April 5, 2010; revised Month, ##, 200#; accepted Month, ##, 2013. Address correspondence to: Sallini Kalachandran s.kalachandran@mail.utoronto.ca Jelum Raval jelum.raval@mail.utoronto.ca Copyright © 2013 Dr. Bill JU, Human Biology Program

Retinal Prosthetic Strategy with the Capacity of Restoring Normal Vision Vision for the Blind: A Revolution in Neuroscience Research Joyce Tang1 and Leora Sazant1 Human Biology Department, Neuroscience, The University of Toronto. Toronto, Ontario CA.

Conventional treatments for retinal degeneration are unable to halt the disease progression and are ineffective at restoring vision. In an attempt to resolve this situation, a group of researchers altered the current retinal prosthetic device to allow for the user’s brain to form more accurate mental representations of the presented visual stimuli. Modern retinal prosthetics are ineffective because they directly stimulate the ganglion cells without accounting for the visual processing that should have occurred in higher circuitry cells (photoreceptors, bipolar, amacrine, horizontal cells). As such, a revolutionary mathematical logarithm was incorporated into the device which mimics the visual processing that should have occurred in the higher retinal cells; visual stimuli would be processed by the logarithm which transforms the information into a pattern of action potentials that the ganglion cells in a normal retina would produce after all the visual processing. In

this manner, the brain would receive information that has been appropriately processed and would accurately decode the series of action potentials as the presented stimuli. Tests were completed on rats to evaluate the accuracy of this prosthetic in relation to the standard device. These tests included the usage of ganglion cell response tasks, confusion matrices, image reconstructions, and optomotor tracking tasks. Results from these experiments demonstrated that this prosthetic, with the new logarithm, was very efficient. In fact, results indicated that through the usage of this prosthetic, the retinal processing in a blind retina was nearly as accurate as that of a normal retina. Given the promising results obtained in this animal study, human clinical trials should be conducted in the future. A more exciting prospect is that this revolutionary technique may be applicable for restoring other sensory losses as well.

Key words: retinal prosthetic devices; retinal degeneration (RD); retinitis pigentosa (RP); age related macular degeneration (AMD); retinal processing; optogenetics; confusion matrices; image reconstruction tasks; optomotor tracking task 1. BACKGROUND Blindness is one of the most feared disability around the world.1 Some causes of blindness, like cataract, can be effectively treated through laser surgery but other conditions such as retinal degeneration (RD) still lack an adequate treatment strategy. Unfortunately, the 25 million individuals worldwide who have RD not only suffer a debilitating loss in their quality of life, they also become a costly burden to the health care system.2 Retinal degeneration is an inherited disease that primarily affects the photoreceptors.1 It can be divided into two classes: retinitis pigmentosa (RP) and age related macular degeneration (AMD). Retinitis pigmentosa is characterized by the degeneration of rods which initially causes night blindness, then progressive loss of peripheral vision in daylight and finally, complete blindness may develop in severe cases.3 The only treatment option for RP patients is the administration of vitamin A supplements which only delays the disease progression.1 While RP has an early disease onset, AMD mainly emerges in individuals over 55 years of age.1 It involves the degeneration of cones and thus results in the loss of central vision.1, 4 AMD can be classified based on the presence or absence of vascular irregularities that invade the retina; they are

labeled as the wet and dry form of AMD respectively.5 Similarly, patients with dry AMD have the option of nutritional therapy where a combination of antioxidants has been shown to slow the disease progression. Those with wet AMD have the option of antineovascular drugs but they only constitute 10% of total AMD patients.1 Currently, patients with retinal degeneration do not have an effective treatment strategy to restore their vision. It is in the interest of the patients and their government to develop more effective treatments so these otherwise healthy individuals may restore their quality of life and contribute to society. Since the conventional therapies are unsuccessful for RD patients, an alternative route was developed in attempt to restore vision - the use of electronic prosthetic devices. However, the current retinal prosthetic is not without fault. Visual information is captured by a camera located behind the user’s glasses. It is subsequently transmitted to an encoder which transforms the information into electronic signals. Those signals are then sent to a microelectrode array that is tacked onto the layer of ganglion cells in the user’s eye. The electrodes directly stimulate the ganglion cells which fire action potentials through the optic nerve. This allows visual information to be transmitted to the brain without depending on the degenerated photoreceptor cells which normally sense the

Neuroscience Matters

light.1 Unfortunately, the current prosthetic directly stimulates the ganglion cells but does not account for the visual processing that should occur in the photoreceptors, bipolar, amacrine and horizontal cells before it is relayed to the ganglion cells.2 Therefore, the information that is transmitted through the optic nerve would be underprocessed and the brain would not be able to construct the appropriate mental image. Hence, current retinal prosthetics only allow individuals to see spots of light or high contrast edges.2

Impact on Prosthetic Capability The researchers then measured the accuracy and capabilities of the encoder-ChR2 prosthetic through three methods: confusion matrices, image reconstructions, and optomotor tracking task. Once again, these tests were conducted on 3 experimental groups - normal mice, Thy1-ChR2 rd1/rd1 mice using the standard prosthetic, Thy1-ChR2 rd1/rd1 mice using the encoder-ChR2 prosthetic2.

The article that will be reviewed in this paper features a solution which dramatically increases the efficacy of current retinal prosthetics. The authors of the article developed a mathematical logarithm where the input is the visual stimulus, the equation mimics the visual transformations that occur within the higher circuitry sensory cells (ie. photoreceptors, bipolar, amacrine and horizontal cells) and the output is the appropriate ganglion cell firing pattern. This solves the problem of bypassing the photoreceptors since the visual processing is replicated by the logarithm. The computation is carried out by an encoder, which then sends electronic pulses in the pattern that is computed by the logarithm (Figure 1). This is sensed by a minidigital light projector (MDLP) which subsequently sends light pulses that replicate the electronic pulse pattern. The light pulses activate a sheet of ganglion cells, which are made to express channel rhodopsin 2 (ChR2). Thus, the original ganglion cell firing pattern that is computed by the logarithm is relayed from the encoder to the MDLP and finally to the ganglion cells which would send the appropriately processed firing patterns to the brain. The authors of this study refer to this device as the “encoder-ChR2 prosthetic”. However, prior retinal prosthetics have used optogenetics to activate the ganglion cells but this is the first to develop a logarithm to mimic retinal processing.2 The following sections will discuss how the incorporation of this logarithm may allow retinal prosthetics to restore the vision of RD patients to near normal capacity.

From the confusion matrices task, the authors calculated the probability that the brain would accurately decoded the series of action potentials as the presented stimulus2. The probability was 88% for the blind retinas that used the encoder-ChR2 prosthetic2. This was in comparison to the normal mouse retina, which resulted in a fraction-correct percentage of 96%2. On the other hand, a blind retina that used the standard prosthetic only resulted in a fraction-correct percentage of 25%2. Thus, this indicates that with the new prosthetic, the blind mouse retina was capable of possessing the same quality of information as that of the normal mouse retina2.

Encoder (logarithm) RESULTS Production of Normal Output The authors had compared the ganglion cell firing patterns that were recorded in normal mice and Thy1-ChR2 rd1/rd1 mice using either the standard prosthetic and or the new encoderChR2 prosthetic in response to viewing the same movie. From Figure 2, it is clear that the normal retina and the blind retina that used the new prosthetic had produced similar patterns of action potentials. However, the blind retina that used the standard device did not produce a similar firing pattern. Thus, the encoder-ChR2 prosthetic could enable the blind retina to produce normal retinal output while the standard prosthetic was unable to produce such a result.

II. REVIEWED RESEARCH MATERIALS AND METHODS Subjects The subjects in this study were mice. Two types of mice were utilized – those with normal retinas and those with retinal degeneration accompanied with the expression of Channel Rhodopsin 2 in their retinal cells (Thy1-ChR2 rd1/rd1 mouse line). The Thy1-ChR2 rd1/rd1 mice with retinal degeneration were completely blind. In addition, it is important to note that the standard prosthetic that was used for comparison had also utilized optogenetics to activate the ganglion cells. However, it lacks the logarithm that is incorporated into the new encoder-ChR2 prosthetic.

Figure 2. Ganglion cell firing patterns in response to a visual stimulus as shown by S.Nirenberg and C.Pandarinath (2012). Response patterns resulting from normal retinas and blind retinas through the encoderChR2 prosthetic are similar in their action potential firing patterns. The action potential firing patterns resulting from the blind retina through the standard prosthetic are completely random2.

Next, for the stimulus reconstructions, an image of a baby’s face was presented to each condition and the ganglion cell responses were recorded and decoded to determine the mental image that the animal would perceive2. To do this, the researchers used the resulting ganglion cell recordings and converted them into the reconstructed images2. When the firing patterns that were driven by the encoder-ChR2 prosthetic was reconstructed into an image, it highly resembled the presented stimulus2 (Figure 3C). Additionally, this new prosthetic would enable subjects to distinguish between different faces, an otherwise challenging task for those using the standard prosthetic2. In comparison, the reconstructed image from the standard prosthetic had much poorer resolution and recognition of the object would be difficult2 (Figure 3D).

Figure 3. Stimulus reconstruction images as shown by S. Nirenberg and C. Pandarinath (2012). The image shown in A is reconstructed from the firing patterns of a normal retina; the image shown in B is reconstructed from the electronic pulse patterns produced by the encoder-ChR2 prosthetic; the image shown in C is reconstructed from the ganglion cell firing patterns induced the encoder-ChR2 prosthetic; the image shown in D is reconstructed by the ganglion cell firing patterns induced by the standard prosthetic2.

Lastly, the researchers measured the mice’s optomotor tracking abilities to determine the efficacy of the ChR2-encoder prosthetic

at the level of behavior. The mice were placed in front of an LCD monitor, which displayed an oscillating sinusoidal wave. Results showed that the optomotor tracking in blind mice was random, because they displayed eye movements even when no stimulus was present2. Similarly, the blind mice that used the standard prosthetic was unable to track the moving stimulus2. However, with the usage of the encoder-ChR2 prosthetic, the blind mice exhibited an increase in eye movement that accurately followed the oscillating sinusoidal wave2. DISCUSSION In the article that was reviewed, the effectiveness of the retinal prosthetic was demonstrated through four assays. The first test compared the firing patterns produced by a blind retina with this new prosthetic to that which was produced by a normal retina when viewing a movie of natural scenes. It was found that the firing patterns were comparable. This suggests that the logarithm can reliably replicate the retinal processing of visual information in a normal eye and compute a ganglion cell firing pattern that is highly similar to that produced by a healthy retina. Next, through the confusion matrix, the authors answered the question that, if the brain receives a stimulation that is induced by this artificial source, would it be able to decode the spike trains accordingly? Results had shown that almost 90% of the time, the brain would decode the firing patterns computed by the logarithm as the stimulus that was presented to the blind retina. This indicated that the brain is able to interpret the spike trains that are induced by the encoder-ChR2 prosthetic. In addition to this confusion matrix, the fidelity of the computed firing pattern was demonstrated in a reconstruction task, where a blind retina that used the new prosthetic was shown an image of a baby and a reconstruction of what the brain would perceive was generated from the resultant ganglion cell firing pattern. Once again, the spike trains from the ganglion cells would produce an accurate representation of the image in the brain. Also, an interesting finding was observed in a variation of this task where the pattern of electronic pulses produced by the encoder was treated as the ganglion cell firing pattern and was subsequently used to reconstruct the mental image (Figure 3B). The results showed that the image formed directly from the electronic pulse pattern is an even more accurate representation of the baby’s face than that which was produced from the ganglion cell firing pattern (Figure 3C). This suggests that that is a slight loss of information as the electronic pulse pattern from the encoder is relayed to the ganglion cells through the minidigital light projector (MDLP) (refer to Figure 1). Ideally, the MDLP should project pulses of light in a pattern that directly replicates that of the electronic pulses but perhaps, the replication was not as accurate. As a result, the ganglion cells would not be activated in the exact firing pattern that was originally computed by the encoder. Another possible scenario is that the light pulses are in the pattern that precisely replicates that of the electronic pulses but the ganglion cells are

Neuroscience Matters not always activated by the pulses of light. Hence, the firing pattern that is computed by the logarithm and that which is transduced by the electronic pulses is not precisely relayed to the ganglion cells, thus explaining the loss of information. Lastly, the efficacy of the prosthetic was demonstrated through behavioural changes in blind rats. Results had shown that while the rats that had used the standard prosthetic could not track a drifting stimulus in the optomotor tracking task, those that had utilized the prosthetic with the new logarithm were able to do so. This shows that the encoder-ChR2 prosthetic allows for functional improvements in an in vivo experiment. In sum, the results presented by the authors suggest that the incorporation of a logarithm which mimics normal retinal processing may significantly improve the efficacy of current retinal prosthetic devices. SIGNIFICANCE OF THE WORK The implications of this work is vast; if the incorporation of the logarithm into retinal prosthetics can indeed restore the vision of RD patients to near normal capacity, then 25 million people worldwide can hope to restore their quality of life.1 It would permit these individuals to lead independent and productive lifestyles, ultimately allowing them to contribute to their societies. They would no longer require the long term social and financial support which are provided by the government but at a cost to the economy. This is especially true for individuals who have retinitis pigmentosa since their blindness has an early onset so they would become dependent on government assistance at an early age.2 Even though the prosthetic that is proposed by the authors were only tested and shown effective in rats, it has a very high probability of success in humans. This is the reason why the work is significant - there is an immense potential for this retinal prosthetic to effectively restore vision in human patients. The logarithm is already shown to replicate the retinal processing in normal eyes and restore tracking ability in in vivo experiments so there is reason to believe that its incorporation will improve the effectiveness of current retinal prosthetics. This argument is further strengthened when one recalls that current prosthetics only allow patients to see spots of light or stark edges. Even if vision is not completely restored through the proposed logarithm, it is still likely that a large improvement will be observed when compared to current prosthetics. Moreover, the mechanisms behind the encoder device and the optogenetics methodology have been developed and refined for a number of years; they do not require drastic modifications to be applied on human users. Thus, the authors of the article have developed the building blocks for an effective retinal prosthetic for humans. Finally, the concept of developing a logarithm to mimic the processing of degenerated sensory organs may be applied to restore other sensory losses. For example, if the cause of hearing loss is

Neuroscience Matters in the degeneration of hair cells, then perhaps a logarithm could be developed to mimic the processing of information in those sensory cells and the appropriate firing pattern of the auditory nerve could be computed for the given stimulus. Theoretically, the auditory nerve could be activated to fire in that computed pattern and the brain would decode the series of action potentials as the presented auditory stimulus. Through this method, the perception of sound could be induced in the brain. Hence, in addition to the 25 million RD patients worldwide, many more may benefit from the implications of this article. FUTURE DIRECTIONS A few critiques about this study should be noted as well. Firstly, this procedure is invasive because the chip that contains the logarithm for the encoder-ChR2 prosthetic would have to be inserted directly into the retina.6 This procedure may be uncomfortable for some individuals. Additionally, this study was limited due to the fact that mice were the primary subjects. Thus, it is uncertain whether this method would be effective on humans; the possible side effects are also unknown. Also, even though this new prosthetic is not able to restore vision completely because the logarithm still does not account for all the visual processing that occurs in the retina; there is still room for improvement. As well, this prosthetic may not be effective for other retinal problems like glaucoma, which involves damage to the optic nerve7. Similarly, it may not be efficient for those who are blind from birth since their brains have not yet learnt to decode action potential firing patterns in relation to visual stimuli8. Thus, in the future, researchers have several issues to address. First, the current logarithm needs to be improved to ensure 100% accuracy - more specifically, it should more accurately mimic the visual processing in the retinal cells to restore vision to full capacity. In addition, human clinical trials should be conducted with the encoder-ChR2 prosthetic to identify its potential side effects and its overall efficacy in the human retina. If this prosthetic will be as effective as these researchers claim, then it will have the ability to restore the quality of life for the young and old with retinal degeneration. With further research, it is possible that this type of prosthetic could cure other sensory losses (hearing impairments) as well. This is the reason why the technique employed in the encoder-ChR2 prosthetic is revolutionary – not only may it allow the blind to see, it may also provide hope for the deaf to hear! REFERENCES 1. Chader, G.J., Weiland, J. &Humayun, M.S. Artificial vision: needs, functioning, and testing of a retinal electronic prosthesis. Prog Brain Res 175, 317- 332 (2009). 2. Nirenberg, S. &Pandarinath, C. Retinal prosthetic strategy with the capacity to restore normal vision. Proc NatlAcadSci USA 107,

15012-7 (2012). 3. Hamel, C. Retinitis Pigmentosa. Orphanet J Rare Dis1, 40 (2006). 4. Voleti, V.B. &Hubschman, J. P. Age-related eye disease. Maturitas. Advance online publication. doi:10.1016/j.maturitas.2013.01.018. 5. Ambati, J. & Fowler, B.J. Mechanisms of age-related macular degeneration. Neuron75, 2-39 (2012). 6. Winter, J.O., Cogan, S.F. & Rizzo J.F. Retinal prostheses: current challenges and future outlook. Journal of Biomaterials Science 18, 1031-1055 (2007). 7. Chidlow, G., Ebneter, A., Wood, J.P. & Casson, R. The optic nerve

head is the site of axonal transport disruption, axonal cytoskeleton damage and putative axonal regeneration failure in a rat model of glaucoma. Acta Neuropathologica 121, 737-751 (2011). 8. Kupers, R., Chebat, D.R., Madsen, K.H., Paulson, O.B. & Ptito, M. Neural correlates of virtual route recognition in congenital blindness. PNAS 107, 12716-21 (2010). Received March 30, 2012; revised Month, ##, 200#; accepted Month, ##, 2013. Copyright © 2013 Dr. Bill JU, Human Biology Program

Implantable Neural Interfaces: Cutting the Cord Pavel Yarmak1 Human Biology Department, Neuroscience, The University of Toronto. Toronto, Ontario CA.

A brain-computer interface (BCI) allows the brain to communicate directly with a machine using an array of electrodes implanted in an area of the brain which detects neuronal activity. This neuronal activity is then digitized and transferred to a computer for processing. Currently the ultimate goal of BCI devices is to enhance the quality of life of individuals suffering from motor dysfunctions by allowing them to interact with their environment through various assistive devices such as prosthetic robotic arms. However BCI devices also have great potential research applications as well. This review paper will discuss various types of BCI devices currently available while highlighting their

advantages and shortcomings. This paper will then introduce a recently developed novel wireless long-term implantable neural interface device. This new device is capable of broadband wireless transmission of neuronal firing data. This advancement in the field of BCI devices could potentially pave the way for the next generation of neural prosthetic devices while simultaneously opening up new avenues of neuroscientific research. This device has the potential to provide a research method which does not require the subjects to be restricted to a certain location thus eliminating confounding variables and increasing the ecological validity of the experiment.

Key words: brain computer interface (BCI); neural interface; electroencephalogram (EEG); prosthetics; biological control systems; applied bioengineering I. BACKGROUND Simply put, brain-computer interfaces (BCI) are devices which allow the brain to communicate directly with digital devices. This is achieved by recording the electrical activity related to neuronal firing in the brain then analyzing this data and extrapolating from it the intended action behind the particular pattern of signals in question. After determining the intention behind the brain generating certain patterns of electrical activity, various mechanical systems may be used in order to execute actions in response to them. The use of BCI systems holds great potential for both compensating for different types of disabilities, such as motor dysfunctions, and even augmentation of existing abilities such as hands free control of digital devices. Currently, the ultimate application of BCI devices seems to be in computerized prosthetics used to improve the quality of life of individuals suffering from disabilities by allowing them to interact with their

environments through the help of devices such as robotic actuator arms or by simply allowing them to access a computer without a mouse and keyboard.1,2 However, BCI devices also see extensive use in research applications where they can be used to study things such as motor control and the somatosensory system.3 Generally speaking, there are two major types of BCI devices. The first is a non-invasive electroencephalogram (EEG) based system which entails attachment of electrodes onto the user’s scalp and recording gross electrical activity from the surface of their scalp. The recorded activity is a rough estimation of the neuronal firing in the brain seeing as the electrical activity measured by an EEG is caused by the opening and closing of neuronal ion channels which drive action potentials. However, since it requires a great population of neurons in order to produce usable EEG signals, an EEG based BCI system much processing has to be conducted on the recorded signals. One strategy which has been gaining

Neuroscience Matters in popularity as of late is using event-related potentials (ERPs); most commonly the P300 component observed during decision making tasks. Tests have shown that P300 based BCI systems can be quite accurate (>80%) and fast (7.8 characters per minute on a grid based typing task) which speaks to the practicality of such systems.4 Furthermore, a more recent study corroborated these findings while adding that 89% of subjects (n=81) were able to achieve 80-100% accuracy on a grid based typing task but only 19% of subjects (n=99) were able to achieve comparable accuracy in a similar experiment using a motor imagery task.5 While these findings bode well for EEG based BCI systems, it is important to note that both of the aforementioned papers utilized healthy subjects and there have only been a few studies testing these types of systems in clinical populations which have had a very limited participant pool.6 The other major type of BCI systems under current active development are more invasive than EEG based solutions but offer far greater precision thus offering more control options and a much wider array of potential applications. This type of BCI systems involves the implantation of a microelectrode array (MEA). The MEA is a chip with multiple electrodes which is placed in direct contact with the brain tissue of interest, e.g., the motor cortex. The MEA then begins collecting electrical signals from the area surrounding the electrodes thus providing great spatial and temporal precision. The collected electrical signals are then digitized and transmitted to another device such as a computer for processing. While this approach to BCI devices holds crucial benefits such as precision over EEG based devices, up until this point, it has seen little clinical application due to some significant risks. Most current BCI systems require the MEA to be percutaneously cabled to various electrical receiving instruments which then carry out signal processing and finally execute the appropriate actions. This poses a challenge because this approach requires a socket to be made in the skull where the wires are connected whenever the device is used. The problem here is then twofold: (1) the socket may become infected overtime, (2) requiring a cable to run to the subject at all times greatly restricts movement and effective use range of the device. Thus cabled implementations of MEA dependent BCI systems are not ideal for use in human and other mobile subjects. While recently there has been an increase in wireless BCI units, they have also required percutaneously mounted, external to the skin, modules which then transmitted information to external processing devices. Furthermore, these wireless units have also had a limited number of channels of information (electrodes on the MEA). Luckily, recent advancements in these types of devices has yielded the first fully implantable, wireless, neural interface perfectly suited for mobile subjects.7

Neuroscience Matters II. REVIEWED RESEARCH MATERIALS AND METHODS Subjects In addition to bench tests conducted on the new BCI devices a number of tests used animals as test subjects to test the system in the environment of its intended application. The subjects that this review paper focuses on which were used for testing of the newly engineered BCI units were two swine initially implanted with device which were simply allowed to roam in their home cages with the device in operation. This test was done as an initial test and verification of the system as well as to assess its longevity. An additional two units were implanted in two rhesus macaque monkeys in order to test the neuroscientific and clinical utility of the new BCI devices in highly mobile primates which more closely resemble human subjects than swine.7 The devices were operated for a duration of over twelve months in both swine and non-human primate macaque models. Design, Engineering and Verification The device used a silicone based 100 element MEA capable of recording the full spectrum of circuit information composed of action potentials, ﬁeld potentials and low frequency rhythms from each electrode.7 The MEA was wired to the module at the center of this review paper which houses all electronic components within a 56 mm × 42 mm × 9 mm two-piece grade 2 titanium alloy enclosure hermetically sealed via laser welding.7 The device included a brazed single-crystal sapphire window 29.2mm in diameter designed to (1) reduce wireless signal loss and (2) maximize transcutaneous wireless inductive charging potential. The device included an amplification board which integrated a preamplifier, two digital to analog converters and a 24 MHz crystal oscillator with the ultimate goal of increasing the signal to noise ratio. Connected to the amplification board was the transmission board tasked with wireless data transfer to the external processing units. At the heart of the transmission board was a commercial chip antenna (Fractus) capable of transmitting the 24 Mbps data stream from the amplification board tuned to the lesser common 3.2 and 3.8 GHz frequencies in order to minimize interference from other wireless devices. Also located on the transmission board was the wireless charging circuit consisting of an inductively coupled secondary (receiving) coil tuned to 2 MHz. The charging circuit was in turn connected to a 200 mAh Li-ion rechargeable battery which provided the power for the device to function during its normal operation. For a more detailed diagram of the device components see Figure 1. The final part of designing the implantable unit consisted of

modeling the heat dispersion properties of the implantable device which can become an issue with any inductively charged device. The team utilized both computerized modelling as well as bench top testing in order to make sure that the implantable device met the FDA standard requiring no outer surface of a device rise more than 2°C above body temperature (37°C).7 An external receiving unit was also designed in order to transfer data transmitted by the implanted unit to processing devices. However because the design of the external unit has far fewer constraints and is largely depended on the specifications of the implanted unit, it will not be discussed in great detail in the scope of the current review paper. Suffice it to say, the external unit was tuned to the same 3.2 and 3.8 GHz frequencies and amplified the received signal to further increase the signal to noise ratio. Surgical Implantation and in vivo Recording The two swine were induced using a combination of telazol (4 mg kg-1) and xylazine (2 mg kg-1) and then maintained using isoﬂurane (1.4% MAC) with continuous IV injection of Ringers solution.7 A custom stereotactic frame was then used to guide implantation of the BCI device subcutaneously and the MEA into the primary somatosensory cortex. The procedure for macaques was quite similar with the only difference being that they were induced using ketamine (15 mg kg1 ) and the MEA being implanted into the arm area of the primary motor cortex.7

In swine the devices were charged and recorded from once per week for the first month and variably throughout the next twelve months for a total of over 380 recording days.7 For charging purposes, the swine were sedated using dexmedetomidine and monitored. The macaques were sedated during charging of the device using ketamine (5 mg kg-1) then allowed to freely roam their home cages while the device was used to record data with time-locked video being recorded simultaneously. All data processing was done using METLAB. 7 RESULTS Device Performance Testing The team has managed to create a full spectrum implantable BCI device with a 100 element MEA capable of broadband (24Mbps) wireless transmission. The device is capable of recording the full spectrum of circuit information composed of action potentials, ﬁeld potentials and low frequency rhythms from each electrode. The device itself performed extremely well under both bench top and implanted conditions despite being only 64g in weight and 56 mm × 42 mm × 9 mm in size. The device’s miniscule power consumption of 90.6 mW allowed the device to be used continuously for 7 hours on a single charge with new iterations aiming for up to 16 hours of operation. The system allowed for a functional operation range of up to 1-3 meters without significant signal loss. Furthermore, the electronic degradation over the 13 month testing period has shown no electrical seepage into surrounding tissue and electrode survival comparable to current MEA literature.7 One of the major concerns with any implantable inductively charged device is heat dispersion as well as charging efficacy. As previously mentioned, the team utilized a COMSOL model in order to predict heat generation during charging (the device generated virtually no heat during normal operation) by the device which predicted temperatures similar to those gathered from bench top testing falling within a 70-80°C range. Now these types of temperatures would be extremely damaging to brain tissue however the model predicted temperatures around the 40°C mark for the tissue model which is far more acceptable. Having said that, the device did exhibit acceptable charging results with a maximal efficacy of 31% at a distance of 5mm between the coils.7

Figure 1 (Above). To scale diagram representing the components of the implantable BCI device. Taken from Borton et al., 2013.

Neuroscientific Pilot Data The team used recorded signal data acquired from the rhesus macaque monkeys and showed dynamic state changes of neural networks using neural trajectory analysis for activities including free movement, eye scratching, touching an apple and turning the head.7 These analyses were conducted in order to demonstrate the

Neuroscience Matters

Neuroscience Matters augmentative technologies which would grant people abilities beyond their wildest imaginations such as operating appliances in their homes using the power of their thoughts. Furthermore, these types of devices could be used as a more advanced diagnostic tool for disorders such as epilepsy thus eliminating patients’ prolonged hospital visits being tethered to an EEG machine thus improving patient satisfaction, test accuracy. However that would involve invasive procedures and the accompanying costs thus it would likely be restricted to only very severe cases. Outside of clinical practice it could prove to be an invaluable tool for neuroscientific research to better understand the dynamic aspects of the cortex through novel behavioral tasks conducted in an ecologically valid, natural, environment. In research results are commonly acquired from procedures requiring the subjects to be tethered to some sort of an apparatus whilst performing various tasks in order to allow recording of various neurological data. With this device the requirement for such bulky, constrictive, apparatuses would be eliminated thus allowing subjects to conduct themselves more naturally which would eliminate various confounding variables and effectively raise the ecological validity of the experiment.

Table 1 (Above). Comparison between current state of the art BCI systems including the one presently being discussed. Taken from Borton et al., 2013.

potential usefulness of this BCI device in research settings. No novel discoveries were made during the experiment in question. DISCUSSION It is baffling how far BCI technology has progressed in the past decade. Maximal transfer rates of BCI devices circa the year 2000 used to be in the bits per minute, with the absolute best transfer rate at the time being 5–25 bits/min.8 In contrast, the current device can transfer data wirelessly at a rate of 24 Mbps which is hundreds of times faster; essentially being just about half the theoretical transfer rate of wireless G (the most common WiFi technology today). Furthermore, compared to any other BCI device currently available, this novel implantable version surpasses even much larger, non-implantable versions. The longevity and performance of this novel BCI unit is up to par with current state of the art BCI devices. Many input channels survive past the 12 month mark and no electrical leakage or significant performance decline were detected after 13+ months suggesting that the device remained sealed. Furthermore, the signal to noise ratio as well as the bandwidth, power consumption, size, and number of channels of this new BCI unit show far better characteristics than any of the other BCI units currently available (see Table 1). As alluded to previously, heat generation during charging is currently one of the primary concerns when it comes to this type

of implantable BCI device. Seeing as the device is designed to be ultimately implanted into humans with various dysfunctions in order to improve their quality of life, it would be extremely important to gather extensive data regarding heat generation in the surrounding tissue. Current data from the aforementioned tissue model suggested that overall system temperature during charging would increase by about 7°C and observed skin temperature may only increase by 2°C. However this data does not provide any insight into what effect the temperature increase from the BCI device is having on surrounding brain tissue therefore it is impossible to know whether or not damage is being done to it. In current implementations of this device, the potential damage to surrounding tissue was mitigated using external cooling of the surface of the skin around the area between the two coils. This however is merely a temporary solution seeing as the current iteration of the device was not designed with heat minimization in mind. SIGNIFICANCE OF THE WORK This device could pave the way for the next generation of neural prostheses devices for people with severe motor dysfunctions. People who could not previously function independently could potentially gain some valuable independence through the use of these devices. These individuals could gain functions such as grabbing things for themselves, using a computer or even moving their wheelchair without the use of their limbs.1,2 Perhaps thinking more daringly, one these devices may become the ancestors of

FUTURE DIRECTIONS As mentioned a number of times before, while the BCI device performed admirably in terms of heat production during charging measured via surface skin temperature, it is important to note that the temperature of brain tissue surrounding the device was not measured. Increased temperature to the surrounding tissue puts it at risk of being damaged during the charging process. This potential risk should be investigated before any potential human applications. In addition to the heat dissipation questions, signal interference could prove to be an issue due to the wireless nature of the device. In its current iteration, when used in an area with many conducting materials, the device is susceptible to increase background electromagnetic (as well as RF) interference. Thus the transmitter and perhaps the receiver modules should be investigated as areas of potential improvement.

REFERENCES 1. Hochberg, L. R. et al. Reach and grasp by people with tetraplegia using a neurally controlled robotic arm. Nature 485, 372-375 (2012). 2. Sung-Phil Kim et al. Point-and-Click Cursor Control With an Intracortical Neural Interface System by Humans With Tetraplegia. Neural Systems and Rehabilitation Engineering, IEEE Transactions on 19, 193-203 (2011). 3. O’Doherty, J. E. et al. Active tactile exploration using a brainmachine-brain interface. Nature 479, 228-231 (2011). 4. Donchin, E., Spencer, K. M. & Wijesinghe, R. The mental prosthesis: assessing the speed of a P300-based brain-computer interface. Rehabilitation Engineering, IEEE Transactions on 8, 174179 (2000). 5. Guger, C. et al. How many people are able to control a P300based brain? computer interface (BCI)? Neurosci. Lett. 462, 94-98 (2009). 6. Sellers, E. W. & Donchin, E. A P300-based brain-computer interface: Initial tests by ALS patients. Clinical Neurophysiology 117, 538-548 (2006). 7. Borton, D. A., Yin, M., Aceros, J. & Nurmikko, A. An implantable wireless neural interface for recording cortical circuit dynamics in moving primates. J. Neural Eng. 10, 026010 (2013). 8. Wolpaw, J. R. et al. Brain-computer interface technology: a review of the first international meeting. IEEE transactions on rehabilitation engineering 8, 164-173 (2000). Received March 30, 2013; revised Month, 04, 2013; accepted Month, 04, 2013. Address correspondence to: Pavel Yarmak email: pyarmak@ gmail.com Copyright © 2013 Pavel Yarmak, Human Biology Program

Finally, though perfectly acceptable for animal research in their current state, operation time should be increased before any human applications. Luckily, work has already begun on trying to maximize operational time of the device.7 This is another critical part of what would make these types of devices prevalent in clinical practice. The longer the device can function on a single charge, the less time individuals would have to spend being tethered to a single location.

ÂŠ Jenise Chen | Neuroscience Matters | 2013