Report andrew hemmings

Summary and Acknowledgements This report investigates the cause and function of stereotypic behaviours exhibited by farm animals and horses. It contains 1) a thorough literature review on stereotypic behaviour from a neurochemical perspective, 2) documentation of the stages of information retrieval and sample collection leading to a laboratory based study of equine stereotypic behaviour, 3) discussion of the results of the study in the context of animal welfare.

Sincere thanks are extended towards the Farmers Club Charitable Trust for their generous funding which enabled sample collection and retrieval of information regarding experimental methodologies and the brain in relation to animal welfare.

Visits were made to the Universities of Edinburgh, Cambridge Rome for information retrieval. An equine abattoir in the south of England provided the necessary samples.

I am also massively indebted to the Royal Agricultural College for funding the laboratory work mentioned in this study and allowing me the time away from work to enable its completion. All laboratory work was conducted at the Institute of Rural Studies, Aberystwyth.

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1.0 Introduction To Stereotypic Behaviour Captive animals have performed stereotypic behaviour since various domestication events some ten thousand years ago (Bahn 1980).

Stereotypies are repetitive invariant sequences, which have no obvious goal or function (for review see Mason1991). Behaviour patterns fitting this description are numerous including crib-biting, weaving and box walking in horses (McBride 1999), pacing in big cats, (Mason 1991) and chain chewing in pigs and mice (Cabib 1993).

Management practices associated with higher stereotypy incidence relate directly to restricted eating and lack of visual contact with conspecifics (Mason 1991; McGreevy et al.,1995b; Redbo et al., 1998). However, not all animals develop stereotypy under these same conditions suggesting genotypic predisposition. This is supported by results that indicate heritability of these behaviours (Cabib and Bonaventura 1997 ; Cabib et al., 1998). Furthermore, research using rodents has also identified genotype to be an important factor in stereotypy development (Nolte 1999). The physiological basis for this pre-disposition appears to be neurochemical differences in a region of the brain stem known as the basal ganglia (Cabib et al., 1998). As cross species homology of neurochemical mechanisms controlling stereotypy exists between farm animals and the rodent (Frohner 1896; Shuster et al., 1984, Cabib1993) the existence of similar neurochemical differences would be anticipated. This document follows the stages involved in a laboratory based project aimed at the investigation of the afore mentioned neurochemical differences in the horse. It contains 3 main sections: 1) a review of the available literature concerning brain pathways underlying stereotypy performance, 2) generation of suitable methodology to allow neural investigation and 3) a discussion of the results in relation to the welfare of companion and farm animals.

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1.1 Neuro-circuitry underlying stereotypy performance In order to understand the neurochemical mechanisms involved with the onset of stereotypy, the underlying neurobiology of these behaviours will first be considered.

The neural circuit responsible for mediation of a wide range of motor functions [including stereotypic behaviour] is known as the basal ganglia – thalamocortical loop (De Long et al., 1992; Cabib 1993) see (figure 1A). Brain areas connected in this loop include 1) The neo and pre-frontal cortical areas, both of which are aspects of the cerebral cortex. 2) The striatum, a structure belonging to the basal ganglion, which is divided into dorsal and ventral components. The dorsal region contains the caudatus and putamen (CP), which continue ventrally and rostrally to form the nucleus accumbens (NA). 3) The pallidum, also part of the basal ganglion, and 4) the thalamus which feeds back into the cerebral cortex to complete the circuit. Input into the loop initiates from the substantia nigra, the hippocampus and amygdala of the limbic system, and an area of the mesencephalon known as the ventral tegmental area (VTA) (Nolte 1999) (see figure 1 B).

The neurotransmitters contained within the basal-ganglia thalamocortical circuitry are also shown in (figure 1). Drugs agonistic to these neurotransmitters have the ability to cause stereotypy in individuals previously free of these behaviours (Cabib et al., 1984). This phenomenon has previously been termed pharmacologically induced (P.I) stereotypy (McBride 1999). Strategic manipulations of basal ganglia-thalamocortical circuitry in the stereotypy free animal, provide crucial information with regards to the neurochemistry of the stereotypic motor response.

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BEHAVIOUR

(A)

Pre-frontal cortex

Neocortex

Glu.

Glu.

NA

CP

G.A.B.A

Thalamus G.A.B.A

Pallidum

BEHAVIOUR

(B)

Pre-frontal cortex

Neocortex

Glu.

Glu.

Glu.

S.N P.C

Hippocampus

P.R

D.A Glu.

Amygdala

NA

CP G.A.B.A

D.A

G.A.B.A

Thalamus

V.T.A Pallidum

G.A.B.A

Figure 1. Basal ganglia-thalamocortical circuit. Adapted from Wang and McGinty 1999; Nolte 1999; Frackowiak 1997; De Long et al., 1992. (A) shown without inputs, (B) with inputs inclusive.

(Neurotransmitters) Dopamine (D.A) Glutamate (Glu) Gama Amino Butyric Acid (G.A.B.A).

Inhibitory connections shown in green, excitatory connections shown in red. (Brain structures) Ventral Tegmental Area (V.T.A) Nucleus Accumbens (N.A) Caudatus and Putamen (CP) Substantia Nigra (S.N) (Pars Reticula (.P.R) Pars Compacta (P.C).

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Dopaminergic systems Administration of the DA agonist apomorphine, caused crib-biting behaviour in the horse (Frohner 1896). Subsequent trials have induced stereotypy using amphetamine and apomorphine in laboratory rodents, cats, sheep, pigeons and humans (see Terlouw et al; 1992 for review). Investigations such as Goodman (1981) which induced stereotypy in pigeons via intra peritoneal (i.p.) apomorphine administration, lend useful information with regards to neurotransmitters mediating P.I stereotypy, but give little indication of specific brain regions involved. To these ends, Creese and Iverson (1975) used the D.A and noradrenaline toxin 6-hydroxydopamine (6-OHDA) to selectively lesion brain regions during DA agonist induced behaviour, and discovered that local injection into the caudatus blocked stereotypy, whilst intraccumbens administration blocked locomotion. As specific depletion of noradrenaline did not effect either locomotion or stereotypy, it was concluded that dopaminergic systems within the caudatus and accumbens control stereotypy and locomotion respectively.

However, other trials have given contrasting results; direct injection of a DA agonist into the accumbens of rats has resulted in stereotypic behaviour, whilst concurrent stimulation of the caudatus and accumbens produced stereotypy of higher intensity (see McBride 1999 for review). The latter phenomena may be explained by the neuroanatomical relationships between the dorsal and ventral striatum (Cabib 1993).

The accumbens receives input from the A10 dopaminergic neurones of the ventral tegmental area (Gobert etal., 1995), whereas D.A. afferents from the substantia nigra pars compacta innervate the caudatus (Frackowiak 1997) (see figure 1). Neuronal relationships between the dorsal and ventral striatia are such, that the accumbens can 5

influence DA activity in the caudatus via a glutamatergic feedback pathway to the substantia nigra pars reticula, whereas the reciprocal possibility is absent (McGinty 1996) (see figure 1 B). And so, by injecting a DA agonist into the accumbens, indirect activation of the caudatus may be achieved. Therefore, increased activation of the caudatus is likely to occur during concurrent stimulation of both structures, which explains the higher intensity of stereotypy within this scenario. In conclusion, it would appear that the accumbens and caudatus work in synergy to produce stereotypy in response to a DA agonist.

Glutamate induced stereotypy The dorsal and ventral striatia receive massive glutamatergic inputs from the cerebral cortex, and limbic system respectively, (See figure 1). This neurotransmitter has mainly excitatory effects in the CNS (De Long et al., 1992). Intrastriatal administration of the glutamate agonist NMDA to rats resulted in stereotypic circling (Thanos et al; 1992) and hyperactivity (Arnt 1981). However, the relationship between glutamate and stereotypy is not clear, as rats injected with a DA agonist within a novel environment showed no stereotypy. If lesions were then made in the same rats to the hippocampus and neocortex thereby greatly reducing glutamate transmission to the striatum, then stereotypic behaviour was observed (see Robins et al., 1990 for review). This would suggest that glutamate has an inhibitory influence upon stereotypy performance. If this were the case, then the increase of local striatal glutamate tone with NMDA by Thanos et al; 1992, would not have elicited a stereotypic response. This apparent contradiction may be attributable to the subclass of glutamate receptor being activated. So far, 26 genetically distinct subtypes have been cloned, all of which are present in the striatum (see Wang & McGinty 1999 for 6

review). These can be divided into two main types; 1) Excitatory ionotropic receptors, which through intrinsic or trans-membrane ion channels increase Ca2+ influx into the neurone, thereby activating a cellular response i.e. neurotransmitter release (Blaha et al., 1997), and 2) metabotropic receptors (mGlurs) which directly influence second messenger cascades, and sometimes cause cellular inhibition (Calabresi et al., 1997).

For example, mGlur types 2&3 reduce cyclic cAMP formation, the presence of which is important for signal amplification following receptor binding (Voet and Voet 1995). Thus, opposite behavioural effects could result depending on the type of glutamate receptor stimulated (Willins et al 1992); a bias towards the inhibitory mGlur 2&3 receptors at synaptic targets of hippocampal and neocortical projections, would lead to suppression of striatal activity [and thus stereotypy] following glutamate binding (see below). In this scenario, lesions to glutamatergic projections as previously discussed, may constitute removal of the inhibitory influence. This may explain why a DA agonist caused stereotypy in this context.

Neurotransmitter flow culminating in stereotypy In studies attempting to define the sequence of interactions between dopamine glutamate and GABA systems during P.I. stereotypy, it has been observed that intrastriatal administration of the glutamate antagonist MK-801 and the GABA antagonist picrotoxin block stereotypy induced by a DA agonist administered by the same route.

However, a DA receptor antagonist (haloperiodol) had no effect, whereas a GABA antagonist blocked stereotypy induced by the glutamate agonist NMDA. With stereotypic behaviour induced by the GABA agonist muscimol, both dopamine and glutamate antagonists were ineffective (Karler et al; 1995). Thus it appears that the

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primary sequence of neurotransmitter flow in the striatum during P.I. stereotypy is as follows: dopamine, glutamate, and lastly GABA (see figure 2).

Cerebral cortex (2) Glutamate

S.N & V.T.A

(1) Dopamine

STRIATUM

(3) G.A.B.A

Pallidum

Figure 2. Flow of neurotransmitter through the striatum during stereotypy.

Considering that the only dopaminergic input to the motor circuit arrives from the SN and VTA, it would seem that stereotypic motor sequences originate here. The exact nature of motor circuit activation during stereotypy can be deduced with regard to the excitatory properties of glutamate and dopamine, and the inhibitory nature of GABA. For example, intra VTA dopamine (Blaha et al., 1997) and intra-striatal glutamate (Thanos etal.,1992) agonist administration is correlated with increased striatal dopaminergic tone and stereotypy. As the latter pharmacological manipulations increase ‘burst firing’ of striatal neurones (see Lejeune and Millan 1995 for review) a state of general excitement has been induced. The behavioural response to striatal excitement is found to be dose dependent. Low doses of the DA agonist apomorphine promote sequences of ‘normal’ behaviours which gradually decrease in complexity, becoming more stereotypic as dosage levels increase (Robbins and Sahakian 1980).

Thus, it seems that a pre-requisite for stereotypy performance, is striatal activation beyond that required for normal behaviour.

Considering the neurochemistry of the motor circuit, subsequent events following increased striatal activation will be increased GABAergic transmission from the striatum to the pallidum, bringing to it heightened inhibition and reduced efferential 8

output (Nolte 1999). And so, reduced GABA to the thalamus means disinhibition of its excitatory projections towards the cerebral cortex (De long et al., 1992) Thus, the neurochemical culmination of DA agonist administration into the striatum, would increased cortical output, and the manifestation of stereotypic behaviour (see figure 3 (A)).

(A) More activation

Cerebral cortex More output

Striatum

(B) Less activation

Thalamus

Cerebral cortex Less output

Striatum

Thalamus

Figure 3. Motor circuit response to differential striatal activation (Adapted from Nolte (1999).

Inhibitory connections are shown in green (GABA), excitatory connections are shown in red (Glutamate). Arrow size indicates magnitude of neurotransmitter flow.

Endogenous opioids and stereotypy The response to exogenous opioid administration is largely behavioural sedation ranging from somnolence to catatonia (Yamada and Nabeshima 1995). However, exceptions to this behavioural response do exist, whereby opioid agonists bring excitement and sometimes stereotypy (Katz et al., 1978). Opioid peptides are not among the main neurotransmitters in brain regions controlling stereotypic behaviours (McGinty 1996), so propagation of opioid-induced stereotypy is believed to occur via 9

stimulation of receptors residing upon neurones containing DA (Spanagel et al., 1991). Agonists specific to three subtypes of opioid receptor (mu delta and kappa) are considered applicable to the study of P.I stereotypy (see Cabib 1993 for review).

These are discussed below.

Mu receptor agonists Mama et al., 1992, induced crib-biting and increased locomotion in horses via systemic administration of the opioid agonist fentanyl, which binds preferentially to the mu subgroup of opioid receptor. Additionally, in experiments designed to study sensitisation to the motor stimulant effects of morphine (a mu agonist) in the horse, Shuster et al., 1984 reported cross sensitisation of the locomotor response to apomorphine (a DA agonist). This suggests that morphine induced behavioural sensitisation of the D.A system (i.e. increased behavioural response to the same dose), an adaptation normally associated with dopamine release and stereotypy (Cabib and Bonaventura 1997) (to be expanded in later sections). Together, these results implicate dopamine release during opioid-induced equine stereotypy, although brain regions involved await identification.

Systemic administration of a mu agonist causes locomotory activity and DA release in the nucleus accumbens and caudatus regions of the rat brain (Dichiara and Imperato, 1988). However, direct stimulation of mu receptors in the same regions induces motor inhibition and no increase in DA release (Longoni et al., 1991). The latter result may not be surprising, as mu opioid receptors have been found not to reside on striatal dopaminergic neurones (Wang et al., 1997) Moreover, the primary neurotransmitter contained in neurones of the striatum is believed to be acetylcholine (McGinty 1996), therefore direct stimulation of mu receptors in the caudatus, putamen or accumbens is 10

not likely to result in DA efflux. To these ends, local injection of mu agonist into the dopaminergic afferents of the dorsal and ventral striatum, the SN and VTA respectively, caused stereotypic circling in rats, which was attenuated with a DA antagonist (Leone et al., 1991, Matsumoto et al., 1988). Thus, the stimulant effects of systemic mu agonist administration would seem to depend upon stimulation of mu opioid receptors on dopaminergic neurones of the SN and VTA (Cabib 1993).

Moreover, the attenuation of stereotypy with a DA antagonist implicates opioid induced DA release, highlighting the importance of this neurotransmitter as an underlying mediator of stereotypy within the striatum.

Delta receptor agonists Intra-accumbens injections of Deltorphin II, a delta receptor agonist, induced stereotypic behaviour and dopamine efflux in rats (see Cabib 1993 for review). Thus, unlike the mu sub-group, delta receptors appear to reside upon dopaminergic neurones of the nucleus accumbens. However, both delta and mu agonists bring stereotypy when injected into the SN, suggesting that both sub types are expressed here (Matsumoto et al., 1988). In a similar manner to mu agonist induced behaviours, stereotypy induced by a delta agonist may be attenuated by a DA antagonist, once again implicating DA as intrinsic to P.I stereotypy.

Kappa receptor agonists Opioid receptors of the kappa subtype are generally considered to form part of a presynaptic negative feedback system, functioning synergistically with dopamine receptors on the SN and VTA to limit DA transmission towards the striatum (Jackisch et al., 1994), thereby inhibiting stereotypy performance. Indeed, systemic kappa 11

agonist administration attenuates metamphetamine induced stereotypy in rats (Ohno et al.,1989), whilst disablement of pre-synaptic Kappa receptors, leads to increased DA levels in the nucleus accumbens and caudatus of guinea pigs (Jackisch 1991).

However, several studies give contradictory results, in that stereotypic circling was induced via intra-nigral kappa agonist administration (Friederich et al., 1987; Matsumoto et al., 1988). This discrepancy could relate to preferential receptor activation in one of two functionally distinct but anatomically homologous nigral regions; the pars compacta opposed to the pars reticula (Delong etal., 1992). The former projects dopaminergic neurones to the striatum, and the latter directly innervates the thalamus via a GABAergic synapse (McGinty 1996) (see figure 1).

Kappa receptor activation in the pars compacta would mean decreased DA transmission to the striatum, culminating in thalamul inhibition, decreased cortical output thus behavioural depression (Nolte 1999). Conversely, inhibition of GABA flow to the thalamus, via activation of kappa receptors in the pars reticula, would result in thalamul disinhibition and increased cortical output (Pedro et al., 1994), thereby replicating motor circuit activation previously associated with increased cortical output and stereotypy (see figure 2 (A)). Thus, the behavioural response to kappa agonists may depend upon the brain region stimulated, with regards to the neurotransmitter contained in subsequent projections to the thalamus and cerebral cortex.

Serotonin (5-HT) and stereotypy There are numerous behavioural syndromes resulting from pharmacological manipulation of serotonin systems (see Yeghiayan et al., 1997 for review). Among

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these are three stereotyped movements; lateral head weaving, forepaw treading, and orofacial stereotypy (Green et al., 1981).

Serotonergic projections from mid brain raphe nuclei terminate in the striatum, where they are received by three subgroups of receptor (5-HT 1, 2 & 3) (Soghomonian et al., 1987). Infusion of serotonin into the NA induces intense orofacial stereotypy, although local administration of receptor agonists specific to 5-HT subtypes have no such effect (Yeghiayan et al., 1995). Moreover, DA antagonists attenuate, whilst serotonin antagonists are ineffective against 5-HT induced behaviours (Benloucif and Galloway 1991). Therefore, it seems that a non-receptor mediated, dopaminergic system underlies 5-HT stereotypy. It was suggested by Yeghiayan et al., 1995, that serotonin causes prolonged DA activity in dopaminergic synapses, [thus stereotypy] by reversing the DA transporter, which would normally function to reabsorb neurotransmitter.

Conclusion The stereotyped motor response has been shown to originate within dopaminergic terminals of the VTA and SN, progressing thereafter around structures of the basal ganglia- thalamocortical circuit. Predictably, drugs agonistic to the main neurotransmitters in this loop (glutamate GABA dopamine) cause stereotypy.

However, this may depend on the type of receptor / receptor sub-group being stimulated.

Opioid peptides and serotonin can be considered peripheral in the context of P.I stereotypy, as they seem to cause these behaviours indirectly by increasing synaptic transmission of dopamine. Finally, evidence suggests that cross species homology of underlying neurochemistry exists between rodents and horses, as DA agonists induce 13

crib-biting, whilst administration of mu specific opioid agonist appears to cause DA release in the horse.

Neurobiology of Environmentally Induced Stereotypy Stereotypies arising from environmental influence alone (E.I stereotypy), like their pharmacologically induced counterparts require motor co-ordination. It can therefore be envisaged that similar neuro-circuitry will be involved. However, only one study has directly linked brain region with E.I stereotypy; Antelman and Szechtman (1975) attenuated E.I stereotypy in rats with an injection of the DA neurotoxin into the dorsal striatum. The majority of work has focussed on attenuation of these behaviours with antagonists of neurotransmitters identified in P.I stereotypy.

Mele et al., (1995) discovered that administration of the glutamate antagonists MK801 or GM1 fully abolished stereotypic climbing in mice. Whilst D.A. antagonists acepromazine reduced weaving behaviour by 30% in one horse (Nurnberg et al., 1997). The use of haloperiodol, another DA antagonist has significantly attenuated stereotypy in pigs (P<0.05) (Von Borell and Hurnik 1991) and cats (P<0.01) (Willemse et al., 1994) No studies to date, have reported the effects of a GABA antagonist on E.I behaviour. However, the neurotransmitters dopamine and glutamate, and the striatal brain region, both of which were identified as intrinsic to P.I stereotypy, also appear important to E.I behaviours.

Neurotransmitters previously considered peripheral to motor circuitry are also linked to E.I stereotypy. The opioid antagonists naloxone naltrexone and nalmefeme caused complete cessation of crib-biting in horses (Dodman et al., 1987) whilst a selective serotonin transport inhibitor reduced weaving behaviour by 95% (Nurnberg et al.

1997).

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It has been suggested however, that information obtained from systemic antagonist administration may be of limited value, as the effects may not be stereotypy or brain region specific (Rushen et al., 1992). As well as a reduction in cribbing, Dodman et al., 1987 noted an overall decrease in behavioural activity, whilst McBride (1999) recorded significantly increased resting behaviour (p=0.02) following naloxone administration. Therefore, reduced stereotypy may be attributable to a general sedative effect within the CNS. However, as opioids are not among the main neurotransmitters in the motor circuit (see figure 1) their involvement in stereotypy reduction is potentially complex. On the other hand, antagonism of dopamine results in stereotypy inhibition without the presence of a sedative effect (Willemse et al., 1994). As dopamine is an integral part of the motor circuit, the effect of haloperiodol could be attributed to the reduction of striatal excitement, seemingly to the level that stereotypy no longer impinges upon the normal behaviour repertoire.

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1.2 Neurochemistry underlying stereotypy development In the following sections, stereotypy development is considered in the context of motor circuit response to repeated drug administration, stress and genotype.

Behavioural Sensitisation Behavioural responses to psychostimulant administration or certain environmental stressors are heightened following repeated drug use or stressful experience (see Stewart and Badiani 1993 for review ). This phenomenon is referred to as â&#x20AC;&#x2DC;behavioural sensitisationâ&#x20AC;&#x2122;, and has been associated with the neurochemistry of stereotypic behaviour development (Cabib 1993). Behavioural sensitisation arising pharmacologically (P.I.BS), or in drug free animals subjected to environmental stress (E.I.B.S), is considered separately.

Pharmacologically induced behavioural sensitisation (P.I.B.S) Many species (cats, guinea pigs, rats, and humans), when exposed to repeated doses of DA agonists become sensitised to the stereotypy inducing effects of these compounds (see Robinson and Becker 1986 for review), i.e. display a reduced time to the onset of stereotypy, increased stereotypy frequency, and new behaviours previously unrecorded with a single test dose (Cabib and Bonaventura 1997). This phenomenon indicates altered DA systems and is a typical example of behavioural sensitisation (Cabib et al., 1984). Additionally, cross sensitisation to the locomotor effects of morphine and DA agonists has been recorded in rodents (Vezina and Stewart 1989), and horses (Shuster et al., 1984), suggesting that opioids also sensitise DA systems indirectly by causing DA release. In the latter study, Shuster et al., 1984 noted that horses are particularly susceptible to behavioural sensitisation compared 16

with rodents, as much lower drug doses are required to increase locomotion (per kg body mass).

Experiments designed to determine brain areas responsible, reveal that injection of DA antagonist into the VTA selectively blocks, whilst selective lesion of the dorsal striatum has no effect on amphetamine induced behavioural sensitisation (Deroche et al., 1993), thus implicating the mesoaccumbens DA system as mediator. Additionally, blockade of DA agonist induced sensitisation can be achieved through administration of a glutamate antagonist (Karler et al., 1989), or by selective lesion of the fimbria fornix, the major glutamatergic hippocampal â&#x20AC;&#x201C; accumbens pathway (Yoshikawa et al., 1991). Thus, both glutamate and dopamine are required for the propagation of P.I.B.S, whilst the nucleus accumbens, and its respective glutamatergic and dopaminergic afferents are the brain areas responsible.

Environmentally induced behavioural sensitisation (E.I.B.S) Cabib and Bonaventura (1997) discovered that mice, when subjected to a nine-day regime of food restriction display a robust sensitisation to the locomotor effects of amphetamine. The same effect has been noted in response to stressors such as isolation rearing (Robbins et al., 1996) and intermittent immobilisation (Cabib et al., 1989).

All of the latter stressors are shown to reliably cause DA activity in the nucleus accumbens, (see McBride 1999 for review). In addition, endogenous opioids are massively released during stress (Anisman and Zacharko 1991). Thus it may be possible that sustained opioid release associated with stress, sensitises the mesoaccumbens D.A system via repeated D.A release. (for review see Cabib 1993).

In support of this hypothesis, opioid antagonists attenuate stress induced sensitisation 17

of the DA system (Cabib et al., 1984). A homology is therefore implicated, between P.I.BS and E.I.B.S. Moreover, Rivet et al., 1989 reported that corticosterone depletion via adrenalectomy abolished food deprivation induced behavioural sensitisation. This indirectly implicates glutamatergic involvement in E.I.P.S. Since evidence suggests that corticosterone release has the potential to cause glutamate efflux in the nucleus accumbens; Blaha et al., (1997) reported that corticosterone receptors are present in large numbers within the hippocampus. Additionally, their occupation induces extracellular accumulation of glutamate, which has the potential to elicit synaptic transmission of glutamate along the hippocampal-accumbens pathway (Moghaddam et al., 1994). Thus, by preventing corticosteroid receptor activation by adrenalectomy, glutamate release in the accumbens would potentially be reduced. Therefore P.I.B.S like E.I.B.S appears to require a level of glutamatergic input to the N.A.

behavioural sensitisation and stereotypy development E.I stereotypy, and E.I.B.S seem to be highly related, as rodents subjected to food restriction exhibit stereotypy development, which is temporally paired with the onset of behavioural sensitisation to amphetamine (Cabib and Bonaventura 1997). When considered alongside the findings that 1) Similar stressors bringing E.I.B.S, also cause E.I stereotypy (isolation rearing, food restriction) (Robins et al., 1996), and 2) both E.I stereotypy, and E.I.B.S are believed to share common brain regions (striatum) (Antelman and Szechtman 1975, Mittleman et al., 1991 ), E.I stereotypy could be regarded as an overt manifestation of neural pathways sensitised by stress.

Stressors that bring behavioural sensitisation and stereotypy in rodents, have also been strongly implicated as causal factors underlying equine stereotypy (Social isolation, 18

restricted eating) (McGreevy 1995b). This tentatively suggests stress-induced sensitisation as a factor behind equine stereotypy development. Lending credence to this theory is an observed of pairing of P.I.B.S and stereotypic head bobbing in the horse (anecdotal information, Shuster et al., 1984). Moreover, Cabib 1993 suggested that mesoaccumbens D.A pathways sensitised by stress â&#x20AC;&#x153;would be hyper-responsive to a range of different stimuliâ&#x20AC;?. Therefore, any stimulus that activates this region following sensitisation could potentially cause stereotypic behaviour. Indeed, in horses this appears to be the case: The highest intensity of crib-biting is observed post ingestion of a palatable ration (Gillham et al., 1994). Research indicates that endogenous opioids are released following ingestion of a palatable food (Dum et al., 1983) whilst the synaptic target for opioids produced in this context is thought to be the nucleus accumbens (Martel and Fantino 1996). Following sensitisation of this region or its afferents, it is thus perceivable how opioids derived from a palatable meal could bring a stereotypic response.

Additionally, stereotypic behaviour in horses is triggered by stimuli pre-empting meal delivery or exercise (Houpt 1995). This is also suggestive of behavioural sensitisation, as the nucleus accumbens and neostriatum appear central to the mechanism involved in the pairing of a conditioned stimulus with a response (Berridge 1996).

Geneology of stereotypy development E.I.B.S alone, may be an overly economic explanation of underlying factors behind stereotypy development, as an element of heritability has been implicated; in a population of 1035 Thoroughbreds, the prevalence of crib-biting, weaving and box walking increased from 2.4, 2.5 and 2.5%, to 30, 26 and 13% respectively, in families 19

originating from stereotypy sires (Vecchiotti and Galantini 1986). However, limited data from this study prevented firm conclusions from being drawn. More recently, results obtained from two highly inbred mouse strains (DBA and C57) substantiates the evidence relating to geneology and stereotypy development. For example, in response to chronic stress, mice of the DBA strain exhibit behavioural sensitisation to amphetamine and develop stereotypy, whilst individuals of the C57 strain show no such sensitisation, and exhibit behavioural depression (Cabib and Bonaventura 1997).

If cross species homology exists between rodents and horses, the latter results offer a model that can be used to explain why stress induced sensitisation cannot be taken exclusively as the cause of equine stereotypy, and instead implicates a stress / genotype interaction (Cabib et al., 1994). This concept will be expanded below.

1.3 Neurochemical alterations associated with stereotypy development Stereotypy development has been directly linked to stress induced sensitisation of striatal brain regions (see above). Therefore, certain neurochemical changes may be envisaged, leading to sensitisation / stereotypy development.

The biological response elicited by a neurotransmitter will be governed primarily by ligand / receptor interactions, and the nature of subsequent second messenger events (Nolte 1999; Frackowiak 1997). It is therefore perceivable, that stress induced alterations to receptor density, or to second messengers culminating in sensitisation, could result in stereotypy (Puglisi-Allegra et al., 1994). So far, few experiments have investigated stress-induced changes in receptor mechanisms. Work already conducted, has focussed upon sub-groups of dopamine and opioid receptors.

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Dopamine receptors Sharman et al.,1982 discovered a significant increase in the binding (P<0.01)of a D2 DA receptor radioligand (3H Spiperone) within the caudatum of piglets subjected to early weaning stress. This indicated a stress-induced increase in D2 numbers compared with sow reared counterparts, in a brain area previously implicated in stereotypy. Additionally, increased 3H Spiperone binding in this study was positively correlated with the performance of stereotypic snout rubbing. The functional significance of this finding (from a neurochemical perspective) is difficult to establish, as a correlation with receptor sensitivity was not attempted. To these ends, rodent work again bridges some of the gaps remaining in farm animal studies. Cabib et al; (1998) used mice of the DBA and C57 strains to investigate DA receptor plasticity. Following ten days of restraint stress, D1 and D2 receptor binding at both the afferent terminal regions and projecting areas of the mesoaccumbens, and nigrostriatal DA systems was assessed. Marked strain differences were noted with regards to densitometrical analysis of receptor numbers, reflecting opposite adaptations to the imposed stressor. Within the VTA and SN (projecting areas), the DBA strain had significantly reduced binding (P<0.05) of 3H sulpiride to D2 receptors compared to an unstressed control population of the same strain, whilst in the terminal fields of the latter regions, (the NA and CP respectively) D2 binding increased (P<0.05). Binding of the D1 ligand 3H SCH23390 was unchanged in all regions apart from the NA, which displayed proliferation of this subtype. The significance of these changes relates to altered function of the D2 DA receptor depending on its location on the synapse (Kostrzewa 1993, Cabib 1998). Pre synaptic D2 receptors on DA neurones in projecting regions are known as â&#x20AC;&#x2DC;autoreceptorsâ&#x20AC;&#x2122; (Jackisch 1994). They 21

operate a retrograde negative feedback mechanism, as the binding of dopamine released from local synapses to autoreceptors inhibits further production and release of neurotransmitter, and electrical impulse flow (Gobert et al; 1995). Conversely, D2 Receptor populations located in terminal areas are made up of both inhibitory autoreceptors, and postsynaptic receptors, the latter of which propagate the release of neurotransmitter. (Nolte 1999) Therefore, the decrease in D2 receptor density in the VTA and SN as seen in the DBA strain, constitutes decreased inhibitory influence upon neurones along the mesoaccumbens and nigrostriatal projections, and so neurotransmitter flow is facilitated (Cabib et al; 1998). Moreover, proliferation of excitatory D2 receptors in the CP and NA would provide additional binding sites to receive the dopamine influx, leading to heightened striatal excitation thus stereotypy.

This stereotypy induction hypothesis, is also supported by the adaptive increase in D1 receptors seen in the NA, as costimulation of D1 and D2 subgroups is required to elicit stereotypic behaviour (La Hoste & Marshall 1996).

Conversely, the C57 strain of mice used in the Cabib et al., 1998 study, showed trends towards inhibition of neurotransmission within the striatum; D2 autoreceptor numbers in the VTA were increased (P<0.05) in comparison with a control group of the same strain, whilst the post synaptic D1 subgroup significantly decreased (P<0.05).

Cabib et al., 1998 did not conduct observations with regards to behavioural sensitisation to DA agonists or stereotypy performance. However, extremely low genetic variation within the DBA and C57 strains means that populations of either strain can be treated as a single â&#x20AC;&#x2DC;individualâ&#x20AC;&#x2122;. This allows accurate correlation of behavioural and physiological data obtained from two different studies, so long as the strain type remains consistent. Therefore, the resistance and susceptibility to E.I 22

stereotypy and behavioural sensitisation displayed respectively by C57 and DBA mouse strains (Badiani et al., 1992; Cabib and Bonaventura 1997) can now be reliably connected to stress induced alterations to DA receptors in the same strains, within nigro-striatal and mesoaccumbens D.A systems. For instance, in the DBA strain, stereotypy development and behavioural sensitisation is correlated with enhanced D.A transmission to the striatum.

Opioid receptors It was mentioned above, that opioids may participate in stress induced sensitisation of D.A. pathways leading to stereotypy development. Additionally, evidence suggests stress-induced change in opioid systems themselves.

Zeman et al., (1988) showed that rats subjected to acute immobilisation stress demonstrated increased binding of tritiated ligands to mu and delta opioid receptors in the striatum compared to non stressed counterparts, whilst increased kappa receptor binding in the same region following stress was reported by Wolinsky et al., (1994) and Tsujii et al (1986). As no behavioural observations were undertaken in the latter experiments, the increases in receptor density cannot be related to stereotypy performance. However, sham chewing in tethered sows has been correlated with reduced kappa receptor binding in the frontal cortex (Zanella et al., 1996). Clues relating to the functional significance of this finding are scarce, although pre-synaptic kappa receptors normally exert an inhibitory effect upon neurotransmitter release (Jackisch et al., 1994). Decreased kappa receptors in the frontal cortex could therefore mean disinhibition of glutamate transmission to the striatum, a scenario connected with stereotypy propagation (Wang et al., 1999).

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Additionally, in chickens displaying oral stereotypies, when compared with nonstereotypy counterparts, binding of the mu specific ligand [3H] DAGO increased within the paleostriatum, a homologue of the mammalian globus pallidus (Stewart et al 1996). As mu agonists have previously caused stereotypic circling when injected into this area (Hoffman et al., 1991) it is possible that this apparent upregulation results in facilitated efferential neurotransmitter flow, thus disinhibition of the thalamus (Frackowiak 1997) culminating in stereotypy.

Evidence connecting changes in opioid receptor function to stereotypy is presently inconclusive. Moreover, all studies so far have employed between individual comparisons of animals from outbred populations. They make no attempt at genetic analysis, or temporal correlations between stereotypy development and onset of stress.

Because of this, differences in receptor density arising potentially from genotype, stress, and stereotypy performance, or from combinations of these factors are hard to dissect (McBride 1999). However, even without functional adaptation of opioid receptors, the binding of endogenous ligand to opioid receptors of a pre-sensitised mesoaccumbens D.A system is likely to result in a stereotyped response (Cabib 1993).

Conclusion Current neurochemical research in rodents has identified changes in neurotransmitter density which may be linked to the performance of stereotypy. Dopamine receptors in striatal regions have provided the most reliable results in this sense; Strain dependant opposite adaptations to DA receptor number, reflect facilitation or inhibition of DA release in DBA and C57 mice respectively. Facilitation in the case of DBA individuals results in behavioural sensitisation, a symptom of which is stereotypy.

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In the horse, observations of behavioural sensitisation suggest that similar neurochemical changes may occur, causing the development of stereotypic behaviour.

However, only limited effort has been applied to receptor based mechanisms: McBride and Hemmings (2001) discovered a significant upregulation of D1 receptors in the accumbens of stereotypy versus non-stereotypy controls.

1.4 Functional Significance of Neural Sensitisation The complex nature of stereotypies means they are difficult to describe solely in terms of behavioural sensitisation. Therefore, to fully examine the occurrence of these behaviours, the motivation behind stereotypy performance will be considered.

Stereotypies are not â&#x20AC;&#x2DC;hard wiredâ&#x20AC;&#x2122; innate behaviours, and will therefore be performed when the animal is motivated to do so. Hughes and Duncan (1988) proposed a model to explain motivational processes underlying all goal directed behaviours (see figure 4). This will be used to explain the motivation behind stereotypy performance.

Organism variables

- ve

Motivation +ve

Perception of external stimuli

Modulates

Appetitive behaviour +ve followed by -ve Consummatory behaviour

Functional consequences

Figure 4. The model proposed by Hughes and Duncan (1988) showing appetitive and consummatory phases of behaviour in relation to motivation and organism variables.

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The model is based on the assumption that goal directed behaviours have an appetitive (anticipatory) and a consummatory (terminal) phase (Lawrence and Terlouw 1995). In response to organism variables (e.g decrease in blood bourne metabolites), motivation to perform consummatory behaviour (e.g eating) may result.

As a consequence, appetitive behaviour, (e.g. foraging) takes place. Appetitive behaviours have a positive feedback on motivation, and so they continue until the consummatory act begins. Consummation initially has positive, then negative feedback on motivation with the onset of satiation. Additional effects include 1)‘functional consequences’ (e.g elevated metabolite levels) which have negative feedback on organism variables, and 2) a change in the animals perception of the environment. Ultimately, both these factors influence the underlying motivation of the behaviour. If the animal is placed within an environment that prevents consummatory aspects of behaviour (e.g a stable), then negative feedback on motivation may not take place (Hughes and Duncan 1988). Appetitive behaviours therefore continue in a positive feedback loop, and due to the restrictive nature of the environment, become channelled into a limited number of acts performed repeatedly. Lawrence and Terlouw (1993) suggested this to be the basis of stereotypic behaviour.

When the above model is considered in a neuroanatomical context, a small portion of the diencephalon known as the hypothalamus is responsible for monitoring many organism variables. Hypothalamic efferents then feed into limbic areas such as the hippocampus and amygdala, which process ‘drive’ related feelings or motivation (Nolte 1999). In turn, the amygdala and hippocampus project neurones to the nucleus accumbens, which forms part of the motor circuit. This constitutes a pathway through which motivation may influence a range of behaviours (Frackowiak 1997) including stereotypy. Additionally, the nucleus accumbens is among areas responsible for the 26

mediation of reward (Chen 1993), and becomes activated when appetitive behaviours are performed (Robbins and Evritt 1996). It is therefore possible, that this structure provides the positive feedback that appetitive behaviours are proposed to have on motivation (see figure 3). When the accumbens becomes sensitised as a result of stress, the reward threshold is reduced (Jones et al., 1989; Jones et al., 1990) (i.e. the same amount of stimulation produces enhanced reward). In this context, stereotypic repetition of appetitive behaviour is likely to have increasingly rewarding consequences for the animal as sensitisation progresses. Eventually, motivation to perform such behaviour may no longer stem from organism variables such as metabolite deficit, and could instead arise from the pleasurable sensation of stereotypy performance alone. This would explain why facultative stereotypies are commonly observed.

A motivational model can also be used to further describe stereotypy performance in response to meal ingestion . For instance, it was mentioned earlier that endogenous opioids are released following a palatable meal. When this effected is mimicked with the administration of exogenous opioids like morphine, motivation to perform rewarding behaviour (i.e sexual) increases (Wise 1996). The reward value of these behaviours is also enhanced (Wise et al., 1992). In the horse, post meal opioid release would increase the motivation to perform rewarding behaviour (i.e. stereotypy) which according to the above model would have enhanced reward qualities. McBride (1999) suggested that this is the reason why stereotypies are performed at the highest intensity post consumption of a palatable meal.

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2.0 Scheme of Work The previous equine receptor based study was flawed in that receptor affinity for the radioligand was not considered. Neural sensitisation could manifest as stress induced alterations in receptor affinity as well as overall receptor density (Nolte 1999). The peers which reviewed this work recommended that future studies should focus on the measurement of affinity as well as overall density. This study aims to 1) obtain expert advice regarding receptor quantification and neuroscience in relation to animal welfare 2) obtain brain material for further study and 3) perform the laboratory work associated with receptor affinity quantification.

1) Obtaining expert advice Expert advice was obtained from two institutions; The institute of neuroscience, Edinburgh, and the institute of psychobiology, Rome. A full oral presentation was presented to relevant workers in Rome to convey my experimental hypotheses.

Overall, an experimental process known as the saturation assay was recommended.

In a saturation-binding assay, binding at various concentrations of radioligand is investigated. The highest concentration should saturate all target binding sites, giving a measure of receptor density (Bmax). Decreasing ligand concentrations from this point allows the formation of a binding curve (see figure 11). The ligand concentration (nM) at which 50% of binding sites are occupied is the radioligand dissociation constant (Kd). This is taken as a measure of receptor affinity for the ligand. Maximum binding (Bmax, expressed as fmols of ligand bound per mg tissue) is identified on the curve as a plateau, after which no further increase is evident (Keen and MacDermot 1993).

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Bmax

100 Specific binding (fmol mg protein-1)

50

Kd 0 10-11

10-10

10-9

10-8

10-7

10-6

Radioligand concentrations Figure 11. Specific binding curve plotted on a semi-logarithmic scale (Keen and MacDermot 1993).

The upper asymptote of the curve is the total number of receptors (B max). The concentration at which half these receptors are occupied is the dissociation constant (K d).

Choice of radioligand Financial constraints dictated that only one ligand could be purchased for this initial study (D1).

Both agonist and antagonist ligands with D1 affinity were commercially available.

Generally, antagonists are preferred, as the characteristics of agonist binding are more prone to influence by factors such as local Guanine Tri Phosphate (GTP) or cation concentration. Additionally, agonist ligands are readily internalised in whole tissue preparations, thereby hindering transmission of the radioactive signal (Keen and MacDermot 1993). Therefore, a radiolabelled D1 antagonist ([3H] SCH23390) was purchased from New England Nuclear (NEN) to label D1 receptors in this study.

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Binding characteristics of SCH23390 Although SCH23390 has most affinity for the D1 receptor, it will also bind to D2 dopamine and 5-HT2 serotonin subtypes (Lidow 1993), both of which are present in the striatum (McGinty 1996). In order to reduce binding to these subtypes, they were ‘blocked’ with high specificity non-labelled ligands. After Cabib et al., (1998) 5-HT2 receptors were blocked with ketanserin tartrate, and D2 receptors were blocked with sulpiride, both of which are available from research biochemicals international (R.B.I). However, the radioligand may still bind to other yet uncharacterised receptor subtypes, or to non- receptor sites (Lidow 1993). To assess the level of this ‘nonspecific’ binding, two parallel incubations were performed. One contains just radioligand and blocking agents, within which the ligand is free to bind to any site other than those being blocked, and is thus referred to as ‘total binding’. The other incubation, also contains radioligand and blockers, but has added to it a saturating quantity of an unlabelled ligand with high affinity for the receptor being quantified.

This is known as the ‘non-specific’ incubation. It is presumed that all the target receptors will be occupied by unlabelled ligand, therefore, any binding of radioligand in this scenario is to sites other than those being quantified. Subtraction of nonspecific from the total values gave a more reliable measure of ‘specific’ binding to the receptor in question.

Tissue treatment and signal detection method Because relevant scintillation equipment was not available, the ligand was bound to whole tissue sections opposed to a striatal homogenate. Any signal emitted from the tissue sections was picked up on radiosensitive film. The images obtained were then densitometrically quantified with relevant computer equipment.

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2) Obtaining Material for Further Study When the research proposal was submitted to the Farmers Club, there was not an equine slaughterhouse with sufficient throughput in the England, therefore travel to Northern Ireland was agreed. However, a slaughter company originally situated outside Bristol, moved to Taunton and increased its throughput, effectively making it the largest in England. This made travel to Northern Ireland unnecessary.

Over a total of five trips, 10 brains of crib-biting horses were obtained.

Details of slaughterhouse processes In order for the brain to be quickly removed with minimal damage, bone had to be removed from the cranium. Using whole heads, cranial resection was attempted with three cutting tools; 1) Hacksaw, 2) orbital disk cutter 3) Electric band saw. Of these, the hacksaw allowed quickest resection with minimal damage to brain tissue and was thus employed:

1)

A transverse cut was made 4cm rostral from the nuchal crest, continuing downwards to the level of the occipital condyle (see figure 7);

2) A caudal-rostral cut was then made starting at the dorsal aspect of the occipital condyle, bisecting cut 1; 3) Two parallel cuts are then made, one to each temporal bone running from the lateral most aspects of cut 1, in a caudo-rostral direction ending at the level of respective zygomatic arches; 4) Finally, a downwards cut made between zygomatic arches allows removal of a cranial section, forming an opening of sufficient size to accommodate the brain.

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1) Nuchal crest Temporal bone

3)

Zygomatic arch

4)

1)

4) 3)

2)

Occipital condyle

32

Figure 7. Detail of cranial resection.

Brain removal Following cranial resection, four anatomical structures prevent brain removal: 1) ligamentous meninges, which firmly attach the brain to the cranium. 2) remains of the occipital protuberance, a bony elongation of the cranium dividing fore and hindbrain regions, 3) the brain stem, which remains firmly lodged in the occipital foramen and 4) the two optic tracts, that project from the optic chiasm on the underside of the forebrain, toward respective eyes.

These structures were removed to facilitate brain removal as follows: 33

1) Fragments of occipital protuberance were removed with a sharp scalpel, along with any meningal tissue covering the dorsal surface of the cerebral hemisphere.

2) A transverse cut is then made through the cerebellum and brain stem, thereby separating fore, and hindbrain regions.

3) The olfactory bulbs were deflected upwards, allowing the optic tract to be severed.

4) Light finger pressure was then applied around the underside of the cerebral hemisphere, paring away any final ligamentous attachments before the forebrain was lifted from the cranium.

Brain dissection and preservation Brains were sliced into 5mm coronal sections with a P.M knife before being frozen to â&#x20AC;&#x201C;40oC as previously described by Turchan et al., (1999) Given that the striatal region lies rostral to the optic chiasm, 3 coronal slices from this point were made (see figure 8).

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Optic Chiasm

5mm slices taken rostral of chiasm Figure 8. Coronal slicing detail (Adapted from Frandson and Spurgeon 1993) .

3) Laboratory work associated with receptor quantification 34

The following protocol was adapted from Cabib et al., (1998), who achieved saturation of D1 receptors with the following range of [3H] SCH 23390 concentrations (0.27-4.1 nM). Initially, two horse brains were used to test whether saturation of receptors could be achieved within a similar range (1.025-8.2 nM).

Method for coating glass slides The coating medium was 0.1gm chrome alum and 1.0gm gelatin (Sigma U.K) added to 400ml distilled water. A 500ml stainless steel tank and corresponding slide rack (Fisher U.K) were used for the dipping procedure.

The chrome alum and gelatin solution was dissolved at 55oC using a hot plate stirring device. Following a one-hour cooling period at 4oC, the medium was then transferred to the dipping tank. Conventional glass microscope slides were loaded into the rack and dipped momentarily. They were then baked at 40 oC for 2 hours to set the coating medium.

Solutions required for the binding experiment Ice cold 3% paraformaldehyde (Sigma U.K) was used to fix tissue prior to incubation.

Chilling took place in a polystyrene box filled with ice. Two tissue buffer solutions were required: 1) a rinse buffer containing Tris HCL pH 7.4 (50 mM) produced by adding 7.88 gms Tris HCl pH 7.4 (Sigma U.K) to a litre of distilled water. 2) An incubation buffer containing Tris HCl pH 7.4 (50mM), NaCl (120mM), KCl (5mM), CaCl2 (2mM) and mgCl2 (1mM) produced by adding 7.88 gms Tris HCl p.H 7.4, 7.01

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gms NaCl, 0.37 gms KCl, 0.29 gms CaCl2 and 0.095 gms MgCl2 to 1 litre of distilled water.

The total binding solution was incubation buffer containing sulpiride (10 mM) and ketanserin tartrate (1µM). The non-specific solution was identical, except for the addition of 10 µM butaclamol. To produce these dilutions, stock solutions of the three compounds were made up: 1) 500 µM ketanserin tartrate solution, made by adding 10mg ketanserin tartrate to 40ml buffer, 2) 1mM butaclamol solution; 8mg butaclamol was dissolved in 8ml ethanol, then diluted with 12ml buffer, 3) 1M sulpiride stock solution; 0.341gms sulpiride was added to 1ml ethanol.

The final concentration for total binding was achieved by adding 20 µl of ketanserin, and 100 µl sulpiride stock to 9.88 ml buffer. The non specific solution was produced by adding 20 µl of ketanserin, 100 µl sulpiride, and 100 µl butaclamol stock to 9.78 ml buffer.

‘Hot’ ligand was added to both the total and non-specific solutions. 1ml of each solution was aliquoted into 1.5 ml plastic ependorf containers held in a suitable rack.

1.2 microlitres of [3H] SCH23390 (specific activity 75.5 Ci/mmol) was pippeted into each, giving an 8.2 nM dilution of ligand. 5 double dilutions were then performed for each solution, giving a range of five ligand concentrations from 1.025 to 8.2 nM. Incubation method Eight slides from each brain (sufficient for all ligand dilutions, total and non specific) were taken from storage at –80oC and placed in glass coplin jars containing ice cold paraformaldehyde for 20 minutes. Following this period, jars containing the slides were removed from the ice for 10 minutes, before transferral to additional coplin jars containing incubation buffer at room temperature for a further 10 minutes. Slides were then loaded into Shandon sequenza cover slips and placed into a Shandon 36

sequenza staining rack. 100 µl of ligand was added per slide and allowed to bind at room temperature for 60 minutes. Each sequence of ten slides was subject to concurrent total and non-specific incubations at all five ligand dilutions.

Binding was terminated with a series of three five-minute washes in rinse buffer cooled to 4oC. Incubated slides were removed from the coverslips and dried in a fume hood for twelve hours, then loaded into X-Ray cassettes along with tritiated [3H] standards, and exposed to radiosensitive film (hyper-film 3H) for 3 weeks (all autoradiography materials were purchased from Amersham radiochemicals). Following this period, films were developed for 2.5 minutes in Kodak D 19 developer, and fixed in any commercial fixative solution.

Quantification of autoradiographs The images were captured with a conventional flat-bed scanner, and loaded into the Scion image analysis program for the P.C. Background coloration derived from autoradiograph ‘fogging’ was removed with the ‘subtract background’ command.

Images were also clarified by a factor of 1 with the ‘sharpen’ function. The software was then calibrated to measure nCi / mg of tissue, through densitometrical analysis of the image obtained from the tritiated standards. Measurements of nCi / mg of tissue were then taken from the caudatus, putamen and accumbens regions of the total, and non-specific images. After Woodruf et al., (1993) the accumbens was defined as any tissue residing below the ventral most portion of the internal capsule, (see figure 12 ) Caudatus Nucleus accumbens 37

Putamen Figure 12. Sampling detail.

For each region and ligand concentration, the non-specific value was subtracted from the total, to obtain a measure of â&#x20AC;&#x2DC;specificâ&#x20AC;&#x2122; radioactivity per mg of tissue. This value was then converted to fmol of ligand bound per mg of tissue with the following equation:

specific activity of ligand = fmol of ligand / mg tissue. nCi / mg tissue

Ligand binding curves Using a curve fitting programme (minitab for windows) scatter graphs of specific binding values were plotted against ligand concentration. To these, best-fit regression lines were added. The curves were then analysed for evidence of saturation binding.

After Keen and MacDermot 1993, this was identified by a plateau in the binding curve following linear evolution, after which no changes occur).

3.0 Results & Discussion In line with McBride and Hemmings (2001), saturation binding studies revealed that the nucleus accumbens region of stereotypy horses possessed significantly more D1 receptors than non-stereotypy controls (p<0.01) indicating regional sensitisation.

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There were no differences in receptor density in the caudatus or putamen.

Additionally, no significant differences were observed in terms of receptor affinity between the two groups in any brain region.

As reported previously, by considering the role of the nucleus accumbens, the function of stereotypy performance can be partly elucidated. For example, in section 1.4 stereotypies were considered as highly motivated appetitive behaviours. When the neurochemistry of appetitive behaviour is considered, the nucleus accumbens along with VTA and medial pre-frontal cortex are considered important (Jones 1980). As these regions are all intrinsic to the generation of reward, when the nucleus accumbens becomes sensitised as a result of repeated stress, the reward threshold would potentially be reduced. It is therefore likely, that the performance of stereotypy has highly rewarding consequences for the animal. Thus, the common practice of preventing equine stereotypic behaviour (McBride and Long 2001) may have a welfare reducing effect, especially if the behaviours function to counter the effects of an aversive environment. In humans, reward behaviours such as consumption of palatable foods or alcohol are often used to counter the effects of stress (Cabib 1993).

Conversely, stereotypic behaviours observed in farm species such as pigs and poultry are normally not prevented by their owners. In this sense, animals which are free to stereotype could be considered to be in a higher state of welfare than the nonstereotypic equivalents. However, when faced with a stressor, due to genotypic differences discussed earlier some individuals react differently to others. For example, when isolated, some mink perform cage stereotypies, whilst others exhibit behavioural depression (see Mason 1991 for review). In this sense, behavioural depression may act in a similar capacity to stereotypic behaviour. To consider stereotypy as the sole coping strategy is therefore flawed.

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Conversely, some authors have previously used stereotypies as indicators of poor welfare, as stress has been inextricably linked with stereotypy performance. When considered in the context of this study, stereotypy development depends upon an element of genetic pre-disposition. In this sense, these behaviours do not depend solely upon stress exposure and thus cannot be relied upon as an index of stress exposure. Moreover, the putative reward value associated with stereotypy is originally been instated as a result of stress induced neural sensitisation. Beyond this point, repetitive behaviours continue out of the context of stress because of their hedonic properties (facultative stereotypic behaviour). When assessing living conditions in relation to animal welfare, using stereotypy performance may again prove unreliable as any stereotypy exhibited may not be a reflection of the current accommodation.

To conclude, a thorough investigation of the neurochemical pathways underlying stereotypy has enabled a novel view of these behaviours from a welfare perspective.

The results of the laboratory investigation suggest these behaviours have rewarding qualities, a finding which is perhaps more relevant to horse owners, rather than farmers of the UK, mainly due to the common practice of physically preventing equine stereotypy and therefore blocking the activation of a potential coping mechanism. When this information is disseminated via the lay press to horse owners it has the potential to substantially benefit the welfare of the UK horse population.

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