Houzel1994EJN_axonNR

Page 1

European Journal of Neuroscience, VoL 6, pp. 898-917, I994

©European Neuroscience Association

Morphology of Callosal Axons Interconnecting Areas 17 and 18 of the Cat Jean-Christophe Houzel, Chantal Milleret and Giorgio Innocenti1 Laboratoire de Physiologie de la Perception et de I’Action, CNRS UMR9950, Collège de France, 11 Place Marcelin Berthelot 75231, Paris Cedex 05, France 1Institut d’Anatomie, 9 rue du Bugnon, 1005 Lausanne, Switzerland

Key words: corpus callosum, biocytin, cortex, vision, anatomical techniques Abstract Seventeen callosally projecting axons originating near the border between areas 17 and 18 in adult cats were anterogradely labelled with biocytin and reconstructed in 3-D from serial sections. All axons terminated near the contralateral 17/18 border. However, they differed in their diameter, tangential and radial distributions, and overall geometry of terminal arbors. Diameters of reconstructed axons ranged between 0.45 and 2.25 µm. Most of the axons terminated in multiple terminal columns scattered over several square millimetres of cortex. Thus in general callosal connections are not organized according to simple, point-to-point spatial mapping rules. Usually terminal boutons were more numerous in supragranular layers; some were also found in infragranular layers, none in layer IV. However, a few axons were distributed only or mainly in layer IV, others included this layer in their termination. Thus, different callosal axons may selectively activate distinct cell populations. The geometry of terminal arbors defined two types of architecture, which were sometimes represented in the same axon: parallel architecture was characterized by branches of considerable length which supplied different columns or converged onto the same column; serial architecture was characterized by a tangentially running trunk or main branch with radial collaterals to the cortex. These architectures may relate to temporal aspects of inter-hemispheric interactions. In conclusion, communication between corresponding areas of the two hemispheres appears to use channels with different morphological and probably functional properties. Introduction In the CNS the geometry of axonal arbors implements the spatial mapping rules between interconnected neuronal populations. In addition, as suggested by experimental findings and theoretical considerations (for references see Innocenti et al., 1994), it may determine transformations, in the temporal domain, of impulse trains generated at the cell body. Axons therefore are not only essential morphological components of nerve cells but also fundamental elements of the computations performed by individual neurons or neuronal networks. Unfortunately, with a few exceptions, notably the analysis of thalamocortical axons by bulk-filling or intracellular injection of HRP (Ferster and Levay, 1978; Blasdel and Lund, 1983; Freund et al., 1985; Humphrey et al., 1985), limitations of current tract-tracing techniques have thus far prevented the morphological analysis of CNS axons, in particular the long ones. Recently, however, the use of Phaseolus vulgaris leucoagglutinin has allowed considerable progress in the analysis of axons interconnecting several visual areas in the monkey (Rockland, 1989, 1992; Rockland and Virga, 1989) and of geniculocortical axons in kittens (Antonini and Stryker, 1993). Biocytin has similarly been used to trace corticofugal and corticocortical axons in

monkeys (Lachica et al., 1991; Yoshioka et al., 1992) and kittens (Assal and Innocenti, 1993). Not surprisingly, therefore, in the case of callosal connections attention has focused thus far on their topographical organization rather than on the morphology of callosal axons. A fundamental discovery of those studies was that of a discontinuous ‘columnar’ pattern of callosal terminations in rat and monkey (Heimer et al., 1967; Jones et al., 1975; Künzle, 1976), later confirmed in several areas of several species. In addition, in primary sensory areas both the origin and the termination of callosal projections were found to be circumscribed and spared large acallosal regions (for references see Innocenti, 1986). These studies, and their electrophysiological correlates (Choudhury et al., 1965; Berlucchi et al., 1967; Hubel and Wiesel, 1967; Berlucchi and Rizzolatti, 1968; Lepore et al., 1986; Milleret and Buser, 1993), gave rise to the powerful hypotheses that (i) callosal connections re-establish the structural and functional continuity between the ‘split’ sensory representatiqns in the two hemispheres and (ii) the role of callosal connections may be substantiallyknilar to that of intracortical connections between more peripheral parts of the visual field

Correspondence to: G. M. Innocenti, as above Received I6 September 1993, revised IO November 1993, accepted 5 January 1994


Visual callosal axons 899

BA16

BA 20

BA 22

F IG. 1. Camera lucida drawings and photomicrographs of injection sites. Code numbers of the experiments and the stereotaxic levels (in mm) of the centres of injection anterior (A) or posterior (P) to the interaural line are indicated. Low and high power views of each site are shown in coronal sections of the left hemisphere (seen from the front, to facilitate comparison with following figures). The region containing densely packed, labelled cell bodies and neuropil (the core of the injection) is drawn in solid black; axons radiating from the injection site are drawn over variable distances and the few labelled neurons clearly separated from the injection site are marked by dots. The drawings represent the maximal extent of the injection sites in superpositions of 5 - 15 adjacent sections. The core of the injection extended anteroposteriorly over 1200 µm in BA16, 600 µm in BA19, 1050 µm in BA20, 1200 µm in BA21 and 2250 µm in BA22. In the higher power views continuous lines denote the bottoms of layers I and III-VI. Arrowheads are centred on the border between areas 17 and 18. Notice that in BA21 and BA22 the part of the injection site extending below layer VI consists exclusively of densely packed axons labelled from the overlying cortex. The photomicrograph shows an enlarged view of a section through the centre of injection of BA20. Calibration bars are 500 µm for the drawings and 100 µm for the photomicrograph.

representations in areas 17 and 18 (see Discussion). Consistent with these views, it was recently found that callosal connections, like other corticocortical connections, are involved in the synchronous, stimulusdependent activation of spatially separate cortical sites, which may be relevant for visual perception (Engel et al., 1991). At the cellular level, the organization of callosal comiections (and corticocortical connections in general) is unclear. Although the dendritic patterns of callosally projecting neurons have been described in some detail (for data and references see Vercelli and Innocenti, 1993), current knowledge of the morphology of callosal axons (Hartenstein and Innocenti, 1981; Hendry and Jones, 1983; Fish et al., 1991; see Discussion) amounts to little more than a drawing from Ramon y Cajal (1894), a chalk-on-blackboard reproduction of which until recently existed at the Instituto Cajal in Madrid (DeFelipe and Jones, 1992). The results of this study based on computer reconstructions of biocytin-filled individual callosal axons reveal that visual areas in the

two hemispheres are interconnected by a heterogeneous population of axons. The geometries and patterns of termination of these axons suggest that they may perform specific operations in the spatial and temporal domains. Preliminary results of this study were presented at the XVth annual meeting of the European Neuroscience Association (Houzel et al., 1992).

Materials and methods Animals, surgery, injections and perfusions

Five adult cats (of unknown age) were inially anaesthetized with 1.2 ml/kg (i.m.) Saffan (10.8 mg/kg Alfaloxon and 3.5 mg/kg Alfadolone acetate; Pittman-Moore, UK). Rectal temperature was maintained at 38°C and the electrocardiogram was continuously monitored. Additional anaesthetic (1:1 Saffan in saline) was delivered


900

Visual callosal axons

TABLE 1. Reconstructed axons 9

column

number of tufts

a

2 2

b f; e

For each reconstructed axon (Identified by number of the ammal and a trunk diameter (m pm, median value of the class m brackets); total length architecture (see text for definitions; ‘simple’ refers to axons termmatmg pretermmal branches (pt) per column; column locatlon (termmatlon area), for ussue shrinkage (see Mater& and methods)

>3 3 2

fennlnatlor area

number of boutons m

10 10 17110 17110

: Ii

2 1 2 3

10 18 17110 10

:

21

17118 17110

C E

1 1

17110 17110 10

i iY

1pt 2 21

17110 18 17110

:

lpt 1

17110 17118 17110

a

1

l7fl0

:

73 146

94

290

15’:::::.!0, 20 55 ‘.:.::.: 47, ~:;,./:::/ ‘:/,,/ 55 2, ::l:i;.i: jljj$jjj 45 0 292’.;i,;iI;67. 7

50 59 55 526 66 394

0 41

Q

4

25’:‘. lO3’~.i....:‘::I 100 j:.j; ‘:j‘;:j;;;: Q ‘.,;,,:;:j:;, ::::j

66:,:i”::,:’

7:

7

2 1

17110

iz

21

17110 17

C

1

17110

E

21

17110 l7ll0

2

70

a

2

17110

32

56

a

1

17110

3

b

2

17110

i?

2 1

17110 10

5:

89

461:'::':; *: 56...'.3 170 66 26) Ill 1301

Fi

7(

46 63 269 241

X

00 4

92

9

1 81

29 22

5

1

65 222 72

50

100 1; ;; i:i.:j41 Ii 72 j::: 15

115

20 ::;,.;' 12

2

65

:

>2 1

17110

QQ :j:,;$;:/3::

e

1

f

,5 :;:i:/i: ..' ..: :. : 4:j:,j

Q

lpi

17110 17

z

>2 2

17110

c+d 9

4 3

a

1

17110 17110 17110

17

letter) the followmg features are reported: area of mjectlon (orlgm area, 17, 18, 17118 transmon), class of to their we of entry m the grey matter (m pm; asterisks refer to axons fully reconstructed from the mldlme), wth smgle tufts), termmal columns (Identliied by small letter5 a~ m the figures); number of tufts or isolated number of boutons per layer, column and arbor Nottce that values of axon length and diameter are uncorrected

when needed through an i.v. catheter. Animals were positioned in a stereotaxic frame and 5 % biocytin (Sigma) in 0.05 M Tris buffer (pH 8.0) was injected in the lateral gyrus through a 20 pm (tip diameter) glass micropipette cemented to a stainless steel needle and connected to a Hamilton syringe via an oil-filled polyethelyne catheter. Two or three 0.2-0.5 ~1 pressure injections were delivered at 500-2000 pm depth (usually 800 pm apart) aimed at the border between areas 17 and 18 near the representation of the central (stereotaxic coordinates about P3 - L4) or paracentral portion (between P 1 - L3 and A3 - L2) of the central vertical meridian of the visual field. Post-operatively

antibiotics were supplied (Extencilline; 200 000 U/kg i.m.; Specia, France). Fifty to 60 h later sodium pentobarbital was injected (Pentobarbital, 60 mg/kg i.p.; Sanofi, France). The animals were then perfused transcardially with 0.1 M PBS, pH 7.4, followed by 4% paraformaldehyde in PBS (in some case&: 0.3% glutaraldehyde was added). In two cases, in order to evaluate tissue shrinkage, several electrolytic lesions were placed in the injected hemisphere before the perfusion at various depths and A/P coordinates (tungsten microelectrcde; 15 PA DC for 10 s; tip negative).


Visual callosal axons 901 TERMINAL

I

COLUMNS

ITUF,,\

COLLATERAL SECOND-ORDER

FIRST-ORDER

t TRUNK

\ FIG. 2. Schematic drawing of a terminal arbor ending in the cerebral cortex (WM = white matter, GM = grey matter) and illustrating the nomenclature employed. ‘Terminal columns’ refer to the volumes delineated by interrupted lines; one column is supplied by an isolated preterminal branch. For further details see text.

Experiments were carried out in accordance with the European Communities Council Directive regarding the care and use of animals for experimental procedures. Histological procedures Histological procedures were similar to those published elsewhere (Assal and Innocenti, 1993). Briefly, the brains were kept in cold (4°C) fixative for 4 h and stored in cold, 30% sucrose, PBS for 2 days; 75µm-thick frontal sections were cut frozen, collected in PBS and processed according to King et al. (1989). Sections were rinsed four times in PBS with 1% Triton X-100 and incubated in avidin-peroxidase complex for one night [ABC Standard or Elite kit (Vector) 1:200 in PBS -Triton] . Peroxidase was then revealed using the nickel-intensified diaminobenzidine reaction (Adams, 1981). Sections were mounted, dehydrated and coverslipped in DPX or Eukitt medium. Every second or fourth section was counterstained with cresyl-violet or toluidine blue. Cytoarchitectonic criteria (Otsuka and Hassler, 1962) were used for defining areas 17 and 18, the transition zone between them (17/18 border) and cortical layers. The Elite kit (Vector PK6000; cats BA21 and BA22) revealed biocytin more intensely than the ABC standard kit (Vector PK4000; cats BA 16, BA 19 and BA20) and also raised the background staining. This could be reduced by adding glutaraldehyde (see above) in the fixative. However, higher background staining did not obscure stained processes and allowed us to identify layers without Nissl counterstaining. 3-D reconstructions Axons were chosen for 3-D reconstruction according to the following criteria. In nine out of 17 cases, axons were selected at their site of termination in the cortex on the basis of their quality of filling, which was judged from the intensity of staining and the presence of stained boutons. This procedure could have biased the selection in favour of

richly branched axons and/or axons terminating at particular locations. Therefore, the other eight axons were chosen at the corpus callosum, paying attention not to bias the selection in favour of axons with a given diameter. Camera lucida sketches were used to prepare the 3-D reconstruction. The stained axons were individually reconstructed using a computercoupled microscope (Glaser and Glaser, 1990) and the Neurolucida software package (Microbrightfield Inc.). Each slide was placed on the motorized stage of the microscope and the operator could see a compound image of the preparation and of the computer-generated overlay by looking into the oculars. The contours of each section (pial surface), boundaries of cortical layers and blood vessels could be drawn at low magnification. The stained axonal processes were observed through X63 or X100 immersion objectives (final magnification of X1000 or more) and traced as sets of connected 5- to 100-µm-long segments delimited by two points. Each point was assigned x, y and z coordinates, a diameter and one of the following structural tags: node; passing or terminal bouton; and origin or termination. The terminations were of two kinds: real, or interruptions caused by sectioning. The latter were carried into the following section after the appropriate alignment of the computer overlay had been achieved. Reconstructions extended over a range of 3-62 (mean = 32) consecutive sections. Final corrections for shrinkage could be applied separately for the x, y and z dimensions. The whole set of sections enclosing an axon could then be analysed, rotated, re-scaled and plotted. Axon diameters were estimated at X1000 magnification or more using a graded ocular and were ascribed to classes of 0.46 or, in the second phase of the study (axons 21B, 21C, 22B, 22D and 22E), 0.26 µm width. In the following descriptions axon diameter will be given as the median (with two decimals) ± the half-width value of the class. As a rule, diameters were measured near the surface of the section where the staining is often most intense. The amount of tissue shrinkage resulting from ftxation and histological processing was determined by measuring the distances between the same sets of electrolytic lesions at different stages of processing. Measurements were performed after cutting, biocytin visualization and after dehydration and coverslipping. Total shrinkage was 35-40%) approximately isotropic in the x, y and z axes and occurred mainly during futation. A more severe tissue deformation (compression), perpendicular to the cut surface of sections, was caused by dehydration-coverslipping and reduced section thickness to ~ l/3 of its value at cutting. Shrinkage from fixation affects measurements of axon length and thickness. Since it is difficult to determine whether shrinkage is identical for every axon in grey and white matter, and for all axon dimensions, we did not compensate for it in our measurements. Compression causes axons travelling along the z axis to take a characteristic wavy trajectory which was followed faithfully during the reconstruction and measured as curvilinear length with appropriate software (Maxsim; Tettoni et al., 1993, 1994). This eliminated the need for further corrections except in the 3-D visualizations for which the z axis was expanded threefold. The definition of a ‘terminal column’ (see Results) required the identification of one or more distinct clusters of boutons in a plane tangential to the cortical surface. This plane was seldom perpendicular to the original plane of cutting; therefore the set of sections containing the reconstruction was rotated to obtain first ‘standard front views’ and then views perpendicular to the pial surface on which separation of clusters was judged (see Appendix). The separation was further evaluated in a medial view. Numbers of boutons per layer and/or column were counted in the standard views described above. Using immersion objectives, details of axonal terminations were drawn with a camera lucida and/or photographed.


902 Visual callosal axons

F IG. 3. Photomicrographs (collages) of details of biocytin-labelled callosal axons. (A) A bifurcation in layer II. (B) A preterminal branch with numerous boutons ‘en passant’ (small arrows) and two terminal boutons (arrowheads). (C-E) Details of terminal arbors with numerous ‘en passant’ and terminal boutons. (F) Axons of different diameter in the corpus callosum. In all panels, dorsal is upwards. Same magnification in B-E.

Injection sites As in a previous study in kittens (Assal and Innocenti, 1993), injection sites had sharp boundaries and consisted of intensely stained cell bodies and neuropil. The location and extent of five injection sites are shown in Figure 1. All sites, when compared with the electrophysiological maps of the visual areas of Rosenquist (1985) appeared to be located clearly within or near the transition zone between areas 17 and 18. This was confirmed by cytoarchitectonic criteria (Otsuka and Hassler, 1962). All injections

included layers II-V and in three cases (BA20, BA21 and BA22) layers I and VI. Their diameter ranged between 500 and 1500 µm mediolaterally and 370 and 2250 µm anteroposteriorly. In two cases (BA20 and BA22) they were wider in supragranular than in infragranular layers. All injections which reached layer VI also labelled axons terminating medially in the lateral geniculate body, as expected from the estimated retinotopic locations of the injections. Although biocytin was transported mainly anterogradely, it also labelled retrogradely a few neurons near the injection site and,


Visual callosal axons 903

.,, __....

,.*’

...

.‘ _. “ ..’.‘. ‘..

‘. .

‘.-. . . . . .._ _,___

‘1

\

\ \ .. . ..__.___ _\_.

FIG. 4. Computer-aided 3-D reconstructions of individual labelled axons. In this and the following figures code numbers refer to the experiments whose injection sites are shown in Figure 1. Each panel shows an axon within the outlines of a few of the serial sections (coronal sections of the right hemisphere seen from the back) used for the reconstruction;pial surface and bottom of grey matter are indicated by interrupted lines. Dorsal is upwards, medial to the left. Calibration


904 Visual callosal axons

bars are 500 Âľm. Arrowheads are centred on the transition between cytoarchitectonic areas 17 and 18. For quantitative data on the axons shown in this and the following figures see Table 1. Different views and/or enlargements of axons 21B, 22B and 22D are shown in Figures 7 and 9.


Visual callosal axons 905 8 18

18 5

l 7

3

1

2

nLm

0.46-0.90

0.91-1.35

1.3-1.90

1.91-2.25

DIAMETER (µm) FIG 5. Frequency of diameters in the sample of reconstructed axons (full black columns) and of all callosal axons labelled in BA22 from the injection shown in Figure 1 (shaded columns). Number of axons is marked above each column.

occasionally, in the lateral geniculate body. No labelled neurons were found in the hemisphere contralateral to the injection.

Results The following description is based on the analysis of 17 axons (Table 1) serially reconstructed either from the callosal midline (seven cases) or for various distances from their termination (10 cases). Three axons originated clearly in area 17, nine in area 18 and the other five at the unsharp transition between areas 17 and 18. The analysis was completed by inspection of axons not serially reconstructed. Individual axons differed greatly in their morphology. The following descriptions will deal with general aspects of axonal structure which may be of taxonomic or functional relevance (see Discussion). Terminology In the description of axonal morphology the following terms will be used (Fig. 2). The trunk is the part of the axon proximal to the first branching point (node). The part of the axon located distal to the first node is called the terminal arbor. Axonal branches will be identified by their topological order. Thus, first-order (or primary) branches originate from the first node and give rise to second-order (or secondary) branches; the latter give rise to third-order (or tertiary) branches, etc. Branches which are particularly conspicuous by their length and/or thickness will be designated as main branches. Preterminal branches carry boutons (presumably synaptic boutons). The latter can be terminal boutons, characterized by the presence of a connecting stalk or ‘en passant’boutons, i.e. swellings along the preterminal branch (Fig. 3). A tuft is a part of an arbor characterized by densely ramified and tightly distributed high-order and preterminal branches originating from a common stem. Collateral branches (collaterals) are first- or higher-order branches clearly distinct from the rest of the arbor and usually shorter and thinner than their parent branch. They can ramify modestly and/or carry boutons. Trajectories and diameters of callosal axons directed to areas 17 and 18 Labelled axons (Figs 4, 10 and 11) were routed unbundled through the dorsal half of the splenium of the corpus callosum. After entering the medial wall of the hemisphere they coursed below the bottom of the splenial cortex and then laterally and dorsally in the optic radiation.

Although a few axons remained unbranched until they entered the grey matter, most of them branched in the white matter, invariably between the fundus of the lateral sulcus and the convexity of the lateral and postlateral gyri, often beneath area 18. The average length of callosal axons measured between the midline of the corpus callosum and the bottom of layer VI in seven axons was 12 376 µm (range: 11 70013 490 µm; see Table 1). The trunks of the reconstructed axons ranged between 0.65 ± 0.13 and 2.02 ± 0.23 µm (Table 1) in diameter. In order to determine whether the values for reconstructed axons were representative of the full range of axons originating near the 17/18 border, 48 labelled axons were measured at the callosal midline of BA22. The majority of the axons ranged between 0.69 ± 0.23 and 1.14 ± 0.23 µm in diameter (Fig. 5) while most of the reconstructed axons ranged between 0.69 + 0.23 and 1.59 ± 0.23 µm, but the two distributions were very similar. In general the diameter remained constant right up to a node, although occasionally the axons appeared to taper before reaching a node. Both first-order branches maintained the size of their trunk of origin in four out of 13 cases. More frequently the diameter of one (five cases) or both (four cases) decreased to 10% of the trunk diameter. Conservation or decrease of diameter could occur at any other level of the arbor but the diameter of preterminal branches invariably ranged near the limit of optical resolution at ~0.26 µm. Variations in axon diameter in an arbor are interesting because they contribute to its computational properties (Innocenti et al., 1994).

Terminal structure of callosal axons Although many axons started branching in the white matter at variable distances from their site of entry into the cortex, all of them formed a more or less complex arborization in the grey matter (Figs 4, 6, 7 and 10-14). Axons were found to end with one or several tufts and in addition could have more modestly ramified collateral branches. Tufts usually had an approximately conical shape, were radially oriented and extended over one or more layers (Fig. 6). Tufts and/or collaterals of one axon often terminated in tangentially segregated (and radially oriented) volumes of cortex (Figs 6, 7, 9 and 11 - 14) including one or, more frequently, several layers. In the following descriptions these volumes will be called terminal columns. The characterization of a terminal column required the identification of distinct clusters of preterminal branches and boutons in a view perpendicular to the cortical surface (see Materials and methods and Appendix). Columns were usually 300-600 µm in diameter and separated by spaces of 120-2770 µm. Thirty-six terminal columns were unequivocally identified. However, ambiguities in the separation of clusters prevented the recognition of at least four more columns; in addition, five clusters consisted of only 2 - 9 boutons and therefore their identification as ‘columns’ was tentative. Columns could be supplied by separate branches originating in the white or grey matter. Often however, two or more branches converged into the same column (Fig. 7, axon 21B) or else collaterals or preterminal branches extended from one column into another (Fig. 7, axons 16C and 22D). The number of terminal columns established by one axon varied between one and 7 (Table 1). Between two and 526 boutons were counted per column, giving a total of 33-864 boutons per axon (Table 1). Terminal and ‘en passant’ boutons were charted separately. However, because their numbers and topographic distributions were similar, the distinction will not be maintained in the present description.


906 Visual callosal axons

19 A

medial view 90ยบ FIG. 6. Computer-aided 3-D reconstruction of the tuft of axon 19A. (Top-left panel) Location of the axon as in Figure 4; two incompletely reconstructed branches are directed towards the convexity of the gyrus. (Bottom-left panel) Enlarged front view of the terminal structure of the axon in the supragranular layers; dots along the branches are boutons. (Top-right and bottom-right panels) Top and medial views respectively (90ยบ rotations from front view). D = dorsal, M = medial, L = lateral and A = anterior. In this and in the following figures, arrowheads point to the transition between areas 17 (medially) and 18 (laterally) in front and/or top views. Notice that the axon distributes preterminal branches and boutons in two separate terminal columns, the smaller of which is encircled by dots.

Laminar distribution of boutons The laminar distribution of boutons varied across columns and axons (Fig. 8 and Table 1). In the first and most common pattern (19 in 41

columns), boutons were restricted to supragranular layers II and III and often included layer I (and in two cases layer IV). This pattern of distribution was described as supragranular. In the second pattern


Visual callosal axons 907

16 Cv

22 D

/

c

-

i

ioopm

loopm

FIG. 7. Computer-aided 3-D reconstructions of axons 21B, 16C and 22D showing terminal columns fed by converging branches. For 21B (front view) two firstorder branches are shown which distribute in the same column but at different depths. In 16C (top view) three branches distribute within column c; one of them also feeds column d. In 22D three branches terminate in column a; each of them also supplies other columns. Axons 21B and 16C are shown at two different magnifications; arbors and boutons are separately represented for axons 16C and 22D. In each panel, distinct branches or their boutons are graphically differentiated by full-black or shaded traits. Bars are 100 Âľm. P = posterior. M, L and D as in Figure 6. Tapering of orientation bar points away from the viewer. An enlargment of axon 16C is shown in Figure 13.

16 YV

22 B r

16 E

I

F IG. 8. Front views of terminal columns showing the four patterns of laminar distribution of boutons found in this study: respectively granular (16Y), supragranular and bilaminar (22B), and transgranular (16E). Layers I-VI are indicated in each panel. Calibration bars are 100 Âľm. Area 17 is on the left and area 18 on the right of the arrowheads. For the arbors of the same axons see Figure 4.


908 Visual callosal axons

22 B

22 D

22 E

v

M

P

FIG. 9. Top views of a composite 3-D reconstruction of axons (22B, 22D and 22E) are shown on the left, their synaptic boutons on the right. In each panel one individual axon and its boutons are graphically differentiated by full-black traits. M = medial, P = posterior. For different views of the same axons see Figures 4 and 10.

(12 in 41 columns) the highest density of boutons was still in the supragranular layers; there were few or no boutons in layer IV, but there was a second, often substantial fraction of boutons in infragranular

layers V and VI. This pattern of distribution was described as bilamimar. A third pattern, complementary to the latter, was found in two columns from different axons: boutons were concentrated in layer IV (granular


Visual callosal axons 909

22 E

FIG 10. Computer-aided 3-D reconstruction of axon 22E, using the same conventions as in Figure 6. Notice that this arbor distributes IV (denoted by interrupted lines) and at the bottom of layer III. distribution). The fourth pattern (eight in 41 columns) had boutons distributed across both supra- and infragranular layers, including layer IV (transgranular distribution). An axon could provide boutons bilaminarly or transgranularly to one column and supragranularly to another; only three axons (16C, 16D and 20B) distributed boutons both bilaminarly and transgranularly (Table 1). No relation could be found between the laminar pattern of termination of an axon and the areal location of either its cell body or terminal columns (Table 1). Areal distribution of terminal arbors All axons had at least one site of termination at the transition between areas 17 and 18 within 2 mm from the region homotopic to the site

only in layer

of injection (compare Fig. 1 with Figs 4,6,7 and 10-14). However, the tangential span of individual axons varied. Some (six out of 17) terminated within a roughly cylindrical or conical volume 100-500 Âľm in diameter and including one or two terminal columns. Others (10 out of 17) spanned a broader, usually elongated, terminal territory extending up to 3500 Âľm and including up to seven terminal columns. The seventeenth axon was incomplete. Axons with broad terminal territory still distributed more heavily near the 17/18 border and in addition projected to either area 17 or 18, or to both. Axons with the widest tangential spread were found in the posterior part of the visual cortex near the representation of area centralis. None of the axons was found to give additional branches to area 19, to more lateral visual


910 Visual callosal axons

500pm

.’ .. . . . . . ..:

V

FIG. 11. Computer-aided 3-D reconstruction of axon 20C, using the same conventions as in Figure 6 but in the enlarged views arbors and boutons (dots) are shown separately. Notice that the axon terminates in two anteropostcriorly separate columns which become visible in medial or top views; the two columns are supplied by long branches running close to each other, typical of parallel architecture

areas or elsewhere. However, independent axons running from the midline to area 19 were identified. Figure 9 shows three axons labelled from the same injection site. They terminated in distinct, partially overlapping terminal columns within a territory tangentially spanning ~2 mm. They also differed in their radial distribution since one of them (22E) ended mainly in layer IV (Fig. 10) while the others terminated supra- and infragranularly (see axon 22B in Fig. 8 and Table 1).

Architecture of individual terminal arbors Axons varied in the complexity and spats organization of their terminal arbors. Some examples are described and illustrated below. Only two axons, 16Y and 22E (Figs 4 and 10, and Table l), terminated with a single tuft wIthin an approximately conical volume 150 and 400 Âľm in diameter respectively, centred on layer IV, close to the cytoarchitectonic 17/18 border. Axon 22E is shown in detail in Figure 10; it originated in area 17;


Visual callosal axons 911

i oopm FIG. 12. Computer-aided 3-D reconstruction of axon 16F, using the same conventions as in Figure 6. Notice that the axon terminates in two mediolaterally spaced columns, one of which is supplied by a radial collateral to the cortex typical of serial architecture.

its trunk was 1.43 ± 0.13 µm in diameter; and it distributed 24 boutons to layer IV and 10 to the bottom of layer III. More complex arbors could display parallel and/or serial architecture. Parallel architecture (for typical examples see below, axon 20C and Fig. 11) was characterized by the formation, usually in the white matter, of first- or higher-order branches of comparable length which supplied different columns or converged onto the same column. These branches usually ran close to each other over several millimetres. Serial architecture (for a typical example see axon 16F below and in Fig. 12) was characterized by a tangentially running trunk (or else first- or higher-order main branch) with roughly radial collaterals to the cortex. Usually the tangentially running branch also maintained a larger calibre than the collaterals. Seven cases of axons with purely (or overwhelmingly) parallel arbors were found. Converging branches to the same column were found in 21B as well as in 16H (the latter also had a short collateral distributing four boutons to layer VI; Figs 4 and 7, and Table 1). 21B (Fig. 7) originated near the 17/18 border. Its trunk was 0.65 ± 0.13 µm in diameter but decreased to 0.39 ± 0.13 µm ~2 mm before its first node. The latter was in the white matter, 500 µm below the bottom of layer VI at the convexity of the lateral gyrus. The two primary branches maintained the diameter of the trunk and ascended into the grey matter where they formed two tufts, in layer III (with a few branches in IV) and in layers I-III respectively. In total this arbor distributed 92 boutons, predominantly supragranularly, but with a few in layer IV (Table 1). 20C (Fig. 11 and Table 1) was another example of purely parallel

architecture. This axon was labelled by an injection near the 17/18 border. Its trunk was 2.02 ± 0.23 µm in diameter and bifurcated in the white matter of the lateral gyms, giving rise to primaty branches, one of which kept the diameter of the trunk, the other of which was 1.12 ± 0.23 pm in diameter. Both branches ran tangentially for > 2 mm before reaching the 17/18 border; here they took a radial trajectory and terminated in two columns separated anteroposteriorly by ~200 µm. Both columns were elongated in the radial direction. The anterior column originated from the thinner primary branch and consisted of a single terminal tuft distributing 80 boutons to layers I-III over a volume extending 500 µm radially and 250 µm across. The caudal column originated in the white matter from the thicker primary branch and consisted of at least two tufts distributing 202 boutons to all layers, but mostly supragranularly, within a volume extending 1200 µm radially and ~400 µm across. Only two axons, 16F and 16T (Figs 4 and 12, and Table l), had purely serial architecture. 16F (Fig. 12) was labelled by an injection in area 18. Its trunk was 1.12 ± 0.23 µm in diameter and established a thin (~0.26 µm) collateral branch in area 18 which formeda sparsely ramified tuft and distributed 63 boutons to all layers. The other branch maintained the diameter of the trunk and ran tangentially for 1880 pm before entering the 17/18 transition, where it established a tuft distributing 269 boutons to layers III-VI, including layer IV. Other axons showed a mixed parallel-and-serial architecture (Figs 4, 13 and 14, Table 1). 16C (Fig. 13) originated in area 18. Its trunk was 1.12 ± 0.23 µm


912 Visual callosal axons

16C

J

F IG. 13. Computer-aided 3-D reconstruction of axon 16C. (Middle-top panel) Arbor within outlines of some sections. (Top-left panel) Front view of the arbor (the region in the box is shown enlarged in the middle-bottom panel). (Bottom-left panel) Corresponding view of boutons with the bottom of layer VI marked by a thick interrupted line. (Top-right and bottom-right panels) Top and medial views of the arbor respectively; terminal columns are encircled by dots and denoted by small letters. Tapering of orientation bars points away from the viewer. The way terminal columns are supplied is typical of mixed parallel and serial architecture (see text for description).

in diameter. In the white matter under the lateral part of area 18 the trunk gave rise to thin ( ~ 0.25 µm) collaterals entering the grey matter radially. Three main branches continued the direction of the trunk. Collaterals at the origin of these branches and those arising from the trunk distributed 305 boutons within a column (column a in Fig. 13) ~500 µm in diameter in the lateral part of area 18; boutons were in all layers but more numerous infragranularly. The three main branches continued medially, diverging slightly in both the horizontal and frontal planes. Two of them gave rise to a few collaterals distributing 73 boutons to supra- and infragranular layers (but not to layer IV) within a column (column b in Fig. 13) ~ 500 µm in diameter and separated from the lateral one by a space of ~ 300 µm. The three main branches gave rise to collaterals and terminal tufts which entered the grey matter near the 17/18 border, where they distributed in two columns ~ 500µm in diameter and ~200 µm apart. Both of the columns consisted of a complex combination of tufts and branches of different origins. In particular, a conspicuous collateral from column d formed a terminal tuft in column c (Figs 7 and 13). Each column received boutons supragranularly (103 in column c and 196 in column d) and infragranularly (43 in column c and 94 in column d) but none in layer

IV. Two collaterals extended further medially and established 50 boutons clearly outside the previously mentioned terminal columns. 16D (Fig. 14) showed a thus far unique architecture. This axon originated in area 18. Its 1.58 ± 0.23-µm-thick trunk formed two primary branches of unequal thickness (1.12 ± 0.23 and ~0.26 µm) in the white matter under the lateral part of area 18. These two branches ran in the white matter, parallel to the bottom of layer VI and with slightly diverging trajectories. Near this point the thicker branch gave rise to a collateral which first ascended to supragranular layers and then ran parallel to the pial surface (and to the deep branches). This branch established two sets of collaterals supplying two columns ~ 400 µm in diameter; columns a (59 boutons in layers III -VI) and b (55 boutons in layers II-III). It then continued medially to form a dense tuft distributing 471 boutons to the supragranular layers near the 17/18 border. The deeply running primary and secondary branches distributed collaterals in the deep layers, in register with the terminal supragranular tuft and terminated further medially in supra- and infragranular layers. In conclusion this axon established 706 boutons within at least four separate columns, two of which were supplied by separate branches in the supra- and infragranular layers.


Visual callosal axons 913

a

500 µm F IG. 14. Computer-aided 3-D reconstruction of axon 16D, using the same conventions as in Figure 6. In the top view separate terminal columns are encircled by dots and denoted by small letters. One of the columns is also shown in front view.

Discussion Matters of methods and terminology In agreement with previous studies (King et al., 1989; Lachica et al., 1991; Yoshioka et al., 1992; Assal and Innocenti, 1993) the anterograde transport of biocytin allowed a detailed visualization of individual axons of all calibres, impressively complex arbors, and fine morphological details including boutons. Even so, it remains difficult to decide if all the axons originating at the injection site were visualized and how completely. The tracer may selectively miss certain neuronal populations (Lachica et al., 1991) and/or may fail to be transported at branching points or more distally. In addition, the finest callosal axons or branches probably fall below light-microscopic resolution. Thus, some of the

arbors may have been incompletely visualized. In spite of these uncertainties, biocytin provided the most complete and detailed image of callosal axons available so far. No terminology seems to be universally accepted for the description of axonal morphology. The term ‘arbor’ has previously been used to designate an axon and all its branches or, alternatively, what we have called the ‘tuft’. The first usage has developmental implications (Schneider, 1973) and appears preferable. Here we have designated as ‘terminal arbor’ the set of branches an axon forms, from its first node onwards, in the contralateral hemisphere; this, however, is only the ‘distal terminal arbor’. These axons also form a ‘proximal terminal arbor’, i.e. a set of branches near their cell bodies (Innocenti, 1980). With ‘tuft’ we designated a part of an arbor characterized by densely


914 Visual callosal axons ramified and tightly distributed branches originating from a common stem. A ‘terminal column’ is a radially oriented volume in the cortex defined by a cluster or patch of preterminal branches and boutons belonging to one, or more often several, converging tufts and/or branches. Contrary to this work, the term ‘collateral’ has occasionally been used without implications concerning size or direction relative to the branch of origin. Other aspects of the terminology we employed -in particular the ranking order of nodes and branches-are similar to those used for dendritic analysis (Uylings et al., 1986). Since the analysis of axonal morphology will probably become more popular in the coming years, some efforts towards a unification of the terminology would avoid confusion. Topographical organization of callosal connections In agreement with previous anatomical and electrophysiological studies, we found that the corpus callosum interconnects corresponding locations along the transition between areas 17 and 18 of the two hemispheres (for references see Innocenti, 1986). However, the analysis of individual axons for the first time revealed the topographical mapping rules implemented by the connections at the cellular level. Firstly, the tangential extent of the territories covered by the terminal arbors of individual axons was found to vary. Some are restricted to no more than a few hundred µm2, while others distribute over territories spanning up to several mm2. The tangential extent of callosal axons may relate to the morphological type of the parent neuron or to the type of its receptive field. However, wider arbors were found caudally, where the efferent callosal zone (Innocenti, 1980) and the territory of termination of callosal axons (Payne and Siwek, 1991) are also broader. Thus the width of individual callosal arbors may be directly proportional to the retinal magnification factor and/or inversely proportional to the size of their receptive fields. Whatever the reason, contrary to what one would have expected, callosal connections only rarely implement a point-to-point mapping but rather implement a divergent, point-tosurface mapping between the hemispheres. Secondly, many visual callosal axons terminate with a disjunctive, columnar pattern. A similar pattern has been described in several areas and species (see Introduction, and Innocenti, 1986) but only exceptionally and inconstantly for visual callosal connections (Voigt et al., 1988; Payne and Siwek, 1991). One explanation for the previous negative findings could be that small injections of anterograde or retrograde tracer probably failed to achieve sufficient labelling while large injections obscured the pattern by simultaneously filling terminal columns of multiple axons. The patchy distribution of callosal axons appears to have different meanings in different species and areas. In the prefrontal cortex of the monkey it reflects, at least in part, the interdigitation of afferents of different origin, i.e. homolateral parietal cortex and contralateral prefrontal cortex (Goldman-Rakic and Schwartz, 1982). In the cat, callosal projections terminate in columns of left-ear/right-ear facilitation in the auditory areas (Imig and Brugge, 1978) and possibly in regions with bilateral representation of theperiphery in the somatosensory areas (Innocenti, 1986; Picard et al., 1990). Although they seldom stretch across the whole cortical thickness, the terminal columns we found in areas 17 and 18 probably correspond to orientation columns coding for the same orientation as the parent cell body. Indeed, electrophysiological studies in split-chiasm cats have repeatedly shown (Berlucchi et al., 1968; Leporé et al., 1986; Milleret and Buser, 1993) that neurons in areas 17 and 18 respond to the same orientation through the thalamocortical and callosal inputs. The diameter of the terminal columns, usually ~ 300 - 600 pm (without compensation for the 35-40% shrinkage), is close to that of orientation columns

shown, for example, with 2-deoxyglucose (Löwel et al., 1987; Gilbert and Wiesel, 1989) or in vivo optical imaging (Bonhoeffer and Grinvald, 1993). Smaller terminal columns as well as the variable spacing between them might be a result of irregularities in the lay-out of orientation columns (Bonhoeffer and Grinvald, 1993) or the fact that callosal axons avoid certain orientation columns totally or in part. However, considering that callosal connections might contribute to binocularity in areas 17 and 18 (Payne, 1986; but see also Minciacchi and Antonini, 1984), the possibility that callosal terminal columns may also be related to ocular dominance should remain open. Specificity and heterogeneity of callosal axons The present findings on the morphology of callosal axons can only be compared with a limited number of incompletely reconstructed axons in the parietal cortex of the mouse (Hartenstein and Innocenti, 1981) and in the visual cortex of the hamster (Fish et al., 1991). In both species individual axons were found to terminate in infra- and supragranular layers. In addition, in mice some callosal axons have a partially tangential trajectory and therefore probably activate different cortical columns. In rodents, Ramon y Cajal (1894) described axons distributing to widely separate contralateral sites, as well as axons reaching contralateral sites and ipsilateral cortical or subcortical targets. In contrast, callosal axons originating in areas 17 and 18 and directed to contralateral homotopic areas seem to be specific for the latter since they do not appear to project elsewhere in the contralateral hemisphere. Thus, with caution due to the small sample and the possibility that some axonal branches may have been missed by the tracer, the present results confirm previous, double retrograde-labelling studies in adult cats where no neurons in areas 17 and 18 were found to project to both contralateral areas 17/18 and PMLS; neurons projecting to both contralateral and ipsilateral visual areas were found only in area PMLS, not in area 17 or 18 (Segraves and Innocenti, 1985). Callosal axons, however, form local initial collaterals in both cat visual cortex (Innocenti, 1980) and rat somatosensory cortex (Czeiger and White, 1993). Species or area differences may exist that further studies with biocytin could clarify. As predicted by the hypothesis that callosal and intra-areal connections may have similar functions (Hubel and Wiesel, 1967; Innocenti, 1986), we found similarities in the organization of interhemispheric and intra-areal axons of area 17 (Gilbert and Wiesel, 1979; Martin and Whitteridge, 1984) and 18 (Boyd and Matsubara, 1991). Like callosal axons, intra-areal axons originating in layers III, IV and VI (the same layers where callosal projections originate) were found to spread considerable tangential distances and often to form clustered terminations. As we suggest for callosal axons, they appear to link mainly or exclusively columns with similar orientation (Gilbert and Wiesel, 1989; but see also Matsubara et al., 1985; Wörgötter and Eysel, 1991). Intra-areal axons originating from the supragranular layers also resemble callosal axons in their laminar pattern of termination, being mainly distributed to layers I -III, V and VI, and most often avoiding layer IV. However, unlike callosal axons, intra-areal axons project more heavily to layer V than to layer VI. Similarities between these two systems of axons are not surprising since a subset of intracortical axons are initial collaterals of callosal axons (see above), which they resemble in the choice of their postsynaptic target (Czeiger and White, 1993). Other intracortical axons may belong to neurons which lost a callosal axon in development (Innocenti et al., 1986). From several points of view the axons connecting visual areas 17 and 18 appear to be a heterogeneous population. Firstly, they differ in their diameter, confirming the previously reported scatter in the conduction velocity of callosal axons (Innocenti,


Visual callosal axons 915 1980; McCourt et al., 1990). In the present study axon diameters were estimated to range between 0.46 and 2.25 Âľm. Axons of this calibre are probably myelinated (Berbel and Innocenti, 1988). Ultrastructural work (in progress) will indicate whether thinner, unmyelinated callosal axons also exist between areas 17 and 18. Secondly, callosal axons differ in their laminar termination. On the whole, the present study confirms that they contact mainly the supragranular layers, notably layer III (reviewed in Innocenti, 1986). However, the pattern of termination is more complex than previously thought. Most axons have a site of heavier termination with more extensive laminar distribution and one or more sites of lighter termination restricted to supragranular or, more rarely, to infragranular layers. In addition, individual axons differ with respect to their termination in layer IV. While most of them avoid it by terminating supra- and infragranularly (bilaminar projections) others either terminate selectively in this layer (granular projections) or include it in their projection (transgranular projection). The target neurons of callosal axons are unknown, although most of them terminate on spines in the visual cortex of mice (Czeiger and White, 1993) and in other areas and species (for references see Innocenti, 1986; Czeiger and White, 1993). It now appears probable that a callosal axon may contact different types of target neurons in different parts of its terminal territory and that in this respect individual axons may differ from each other. The bilaminar, granular and transgranular patterns of termination resemble respectively the M, F and C types of termination identified by Felleman and Van Essen (1991) in their hierarchical taxonomy of visual areas in the monkey. However, so far we have not found the type of ascending axon with tangential trajectories in layer I, or branching in layers I and II (but scarcely elsewhere), which was described in the feedback projection from V2 to Vl in the monkey (Rockland and Virga, 1989). Nor have we found the heavy projection to layer IV described in feed-forward connections from Vl to V2, V2 to V4, or Vl to middle temporal area (Rockland, 1992). Indeed, one would expect areas 17 and 18 in the two hemispheres to be at the same hierarchical level of visual processing, but even so, the connectivity deviates from the lateral connectivity described for the monkey (Fellernan and Van Essen, 1991). A third aspect of the heterogeneity of axonal morphology revealed by the present study is the broad range of geometries which appear to exist among callosal axons. Firstly, as mentioned above, the degree of tangential divergence varies among axons. Secondly, axons differ in terms of parallel versus serial architecture of their arbors. The parallel architecture is particularly intriguing since it appears to double, over several millimetres, the amount of axoplasm which, on purely geometrical considerations, would seem to be sufficient for the connection. This architecture could be appropriate for producing synchronous activation of spatially separate cortical points or, if branches differ in their calibre, to introduce delays between the activations. The second purpose, however, could be better fulfilled by serial architecture, as in brainstem auditory nuclei (Carr and Konishi, 1990). Two considerations suggest that the structure of callosal axons may indeed reflect temporal constraints in cortical processing (for further discussion see Innocenti et al., 1994). Activity appears to be involved in the development of callosal connections (for review see Innocenti, 1991); therefore the morphology of callosal axons may be shaped according to Hebbian-like temporal rules. On the other hand, the geometry of callosal axons may reflect their role in the synchronous activation of separate cortical columns (Engel et al., 1991). It is unclear how the heterogeneity of callosal axons may relate to structural variables of their parent cell bodies. Callosally projecting neurons from areas 17 and 18 originate most heavily from layer III,

but also originate from layers IVa, II and VI (Innocenti, 1980; Vercelli and Innocenti, 1993). Callosal axons with bilaminar projections, and those which include layer IV in their termination, might originate from layer III pyramids as well as from the spiny stellate cells in layer IV. The rare arbors with exclusive termination in layer IV might originate from the rare layer VI neurons with a callosal axon. This interpretation is consistent with the laminar pattern of intra-area1 projections in cat area 17 and with the notion that callosal axons resemble intra-area1 axons (see below). While most of the callosal axons originate from excitatory cortical neurons, the possibility that some smooth stellate cells may send an inhibitory axon through the corpus callosum was recently raised (Buhl and Singer, 1989; Peters et al., 1990). This study cannot bear on this issue although the short and sparse preterminal branches of the axon 16W (Fig. 4) would be difficult to distinguish from those described for basket cells by Fairen et al. (1984). In conclusion, the present results strongly suggest that inter-hemispheric communications between the primary visual areas use channels with different morphological and presumably functional properties. Future studies may reveal whether and how the heterogeneity of callosal axons may relate, possibly through developmental mechanisms, to the well-documented heterogeneity of thalamocortical axons (Ferster and Levay,1978; Blasdel and Lund, 1983; Humphrey et al., 1985; Freund et al., 1985).

Acknowledgements We are grateful to Laurence Decorte, Danièle Thiesson and Marceline Robert-Tissot for histological assistance, Eric Bemardi and Mireille Chat for the illustrations, Patricia Lehmann, Rudolf Kraftsik and Laurent Tettoni for computer assistance, and Peter Clarke for useful comments on the manuscript. Supported by European Training Programme in Brain and Behaviour Research twinning grant no. 9158 and Swiss National Science Foundation Grant no. 3129948.90.

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; i

B

Appendix The procedure for the identification of terminal columns using the Maxsim software (Fig. 15) consisted of: (i) Rotation of the sections around the z-axis, aligning to the vertical the radius of the cortex in the region containing the boutons (Fig. 15A). (ii) Rotation of the sections around the x-axis until optimal superposition of pial and white matter borders respectively was obtained (Fig. 15D). (iii) A Cartesian frame was placed on the sections, with its y-axis perpendicular to the pial surface in the region containing the boutons. (iv) 90º rotation of the sections around the x-axis of the Cartesian frame.

C

F

:

FIG. 15. Effects of angle of view on separation of terminal columns. (A) Schematized sections and terminal boutons (dots) viewed perpendicular to the plane of section. (B) and (C) Medial and top views respectively after 90º rotations around the original y- and x-axes. (D) View following a rotation of (A) which achieves maximal superposition of section contours. (E) and (F) Medial and top views respectively after 90º rotations around the y- and x-axes of (D). In This procedure achieved a view perpendicular to the cortical surface on which (B) and (E) dashed lines represent sections, continuous lines contours of pial separation of clusters of boutons was judged irrespective of the original angle surface and white matter. See text for description. Arrows in (B) and (E) show of sectioning (Fig. 15F). directions of view in parts (A), (C), (D) and (F).


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