Organization of the visual pathways in the newborn kitten

628

Neuroscience Research, 3 (1986) 628-65~ Elsevier Scientific Publishers Ireland Ltd

NSR 00130

Organization of the Visual Pathways in the Newborn Kitten Z. Henderson* and Colin Blakemore University Laboratory of Physiology, Oxford OX1 3PT (U.K.) (Received March 3rd, 1986; Accepted March 5th, 1986) Key words: cat - - kitten - - retina - - superior colliculus - - lateral geniculate nucleus - - visual cortex - visual development

SUMMARY We have used retrograde and anterograde transport to examine the major visual pathways in newborn kittens. Retinal projections from both eyes to the dorsal lateral genieulate nucleus (dLGN) and superior colliculus (SC) are present and topographically organized. The dLGN projects topographically to areas 17 and 18 and receives reciprocal projections from cells in layer VI of areas 17, 18, 19 and suprasylvian cortex on the same side of the brain. Area 19 also has a sparse thalamic input but probably not from the dLGN. The laminar distribution of [3H]proline transported from dLGN to area 17 was quantified: label was spread through all layers, with a minimum at the border of layers I/II. Layer I was always labelled less heavily than IV. These results are critically compared with those based on other tracing techniques. Cells of layer V in areas 17, 18, 19 and the suprasytvian cortex project topographically to the superficial layers of the ipsilateral SC. Area 19 and the lateral suprasylvian cortex also send a crossed projection to restricted parts of the opposite SC. Thus these visual projections are not only present and topographically ordered on the day of birth, but, unlike certain highly exuberant interhemispheric and cortico-cortical projections, they are qualitatively remarkably mature, some days before the onset of visual activity. The major subcorticat projections to and from the visual cortex appear to be constructed without the benefit of visual experience and much of the activity-dependent plasticity of cortical cells may well involve only local modulation of synaptic input.

INTRODUCTION

Over the past two decades or so the kitten's visual system, especially the striate cortex, has become a model system for the study of neuronal plasticity (see recent reviews by Movshon and Van Sluyters 59, Sherman and Spear TM, Fregnac and Imbert 18

* Present address: Department of Physiology, University College, Cardiff CF1 1XL, U,K. Correspondence: C. Blakemore, University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, U.K. 0168-0102/86/$03,50 © 1986 Elsevier Scientific Publishers Ireland Ltd.

629 and Hirsch25). The ocular dominance 86, orientation preferences 7 and direction selectivity 13 of the population of neurons in the kitten's visual cortex can all quite rapidly be modified by appropriate forms of deprivation or selective visual experience during a sensitive period, starting at about 3 weeks of age. Despite all the evidence that the functional properties of cortical neurons are remarkably plastic, we know very little about any morphological alterations that might underlie these physiological changes. In many parts of the nervous system there is evidence for initial over-connection or "exuberance" of projection, followed by cell death or withdrawal of inappropriate axons to refine the pathway to its adult pattern ~°'3°'66. Some of the examples of functional plasticity in the visual system might conceivably depend on the selective elimination of initially exuberant projections. For instance, the most compelling correlation between structural and functional plasticity in the visual system remains the competitive redistribution of afferent axons in layer IV of the cortex that follows monocular deprivation and which seems to be responsible for the modification of ocular dominance for cortical neurons in that layer 73. However, even in that case, the rapid and more complete shifts in dominance in other layers are likely to depend on more subtle functional changes in intracortical interneuronal connectivity8. Obviously, an essential prerequisite for the elucidation of morphological changes associated with physiological plasticity in the visual system is a knowledge of the anatomical organization of the visual pathways before the onset of visual experience and plasticity. Equally, it is interesting to ask how the precise topographic and laminar patterns of projection in the normal adult visual system are achieved during development, and whether visually evoked activity in the pathway plays a role in the refinement of such patterns.

Retinal projections The axons of retinal ganglion cells start to invade the developing brain several weeks before birth in the fetal kitten, and these early projections to both the dorsal lateral geniculate nucleus (dLGN) ~1 and the superior colliculus (SC) s7 are exuberant, in that axons at first are abnormally diffuse in their distributions. In the dLGN, fibres from the two eyes are initially intermingled and they become segregated into their mature laminar pattern by about the time of birthTl: this process apparently involves the elimination of synapses that are already functional 72. On the other hand the patterns of decussation of different classes of ganglion cell axons seem to be at least grossly established early in fetal life52, and the sparse uncrossed retinal projection that characterizes the Siamese cat is evident at the earliest stages of development 45. However, as in rodents 36"3s, there is a small population of aberrant, ipsilaterally-projecting ganglion cells in the nasal retina of the newborn kitten, which are largely eliminated (presumably by cell death) by 10 days of age 37. The SC of the cat also initially receives a widespread, overlapping projection from both eyes, early in fetal life, but gradually fibres from the ipsilateral eye become restricted to a circumscribed field in the middle of the rostro-caudal extent of the nucleus. Some

630 days before birth, the ipsilateral terminals have become mainly restricted to patches, in the stratum griseum superficiale, corresponding to partial gaps in the dense crossed projection, just as in the adult cat 87.

Geniculo-cortical and cortico-geniculate projections In the adult cat, there are specific projections from the d L G N to several of the visual areas in the occipital cortex. Area 17 receives a quite strictly topographic projection from small, medium and large principal cells, corresponding to the physiological classes called W, X and Y cells, respectively (see Stone 79 for a recent review), in all laminae of the dLGN, as well as in the medial interlarninar nucleus (MIN). Area 18 receives mainly from the largest (Y) cells, while area 19 has a projection from small (W) cells in the C laminae and the MIN. Small cells in the MIN also project sparsely to the lateral suprasylvian cortex. In areas 17 and 18 of adult cats, the geniculate afferents terminate densely in layer IV and less heavily in layers I, lower III and VI 6s. The input to layer I comes from small (W) cells in the C laminae; terminals in lower III are derived from cells in all d L G N laminae and those in layers IV and VI come from laminae A and A148'51. Areas 17, 18 and 19, and parts of the suprasylvian cortex, in turn, send reciprocal cortico-geniculate projections, specifically from cells in layer VI, which innervate all the laminae of the d L G N and are in topographic register with the cells of origin of the geniculo-cortical pathways 21"83,84. Now, Anker z and Anker and Cragg 3, using anterograde degeneration techniques, suggested that the thalamic projections to areas 18 and 19 do not begin to arrive until 2 weeks after birth, but Henderson z3, using more sensitive axonal transport methods showed that there is a projection from d L G N to both areas 17 and 18, and from the MIN to 18 and 19 in the newborn kitten. Recently Kato and his colleagues 42,43 used both anterograde and retrograde transport of horseradish peroxidase (HRP) to examine the laminar pattern of termination of the afferent geniculate projection to area 17; they reported a dense, excessive termination in layer I in very young kittens, originating from an aberrant population of comparatively large neurons in the C laminae and even partly from cells in the A layers of the dLGN. A cortico-geniculate projection from cells of cortical layer VI is also present even in very young animals 9"42. Cortico-collicular projections In normal adult cats, a considerable proportion of cells in layer V of cortical areas 17, 18, 19 and the lateral suprasylvian areas project to the superficial laminae of the ipsilateral SC 2~'44'84. The projection from each cortical area covers the entire ipsilateral SC in a retinotopic pattern 55'84. There is a less extensive projection to the SC from the contrallateral cortex, which originates chiefly in area 19 and the cortex of the suprasylvian sulcus and terminates mainly in a restricted region at the rostral end of the S C4,6, 62" Previous anatomical studies have demonstrated the existence of a cortico-collicular pathway in kittens at or shortly after birth 9"67'76. Tsumoto et al.8o have recently shown

631 that, although the projection arises, as in the adult cat, from layer V of the striate cortex, it may include an aberrant component, in that a higher fraction of neurons in that layer appear to be labelled by HRP injections into the SC in very young kittens than in adults. The projection from the SC to the d L G N is also present and topographically organized at birth, but it terminates exuberantly in all layers of the d L G N and the MIN. Not until 3 weeks or so after birth is the terminal field restricted as in the adult cat, to the C laminae 77. By contrast, the projection from cortex to the pretectum is reported to be absent until 4 weeks after birth 7°. In the present study we have used a combination of retrograde and anterograde tracing methods to examine the state of organization of most of the major projections in the visual pathways of kittens on the day of birth, some days before the photoreceptors become mature 1~ and visual activity commences ~2. In particular, we wanted to know whether each projection is present and topographically organized. We were especially interested in whether any of the projections were exuberant either in terms of the extent or laminar distribution of the terminal field, or in terms of the distribution or number of the cells of origin.

MATERIALS AND METHODS Thirteen newborn kittens (80-140 g), not later than 24 h after birth, were injected with HRP conjugated to wheat germ agglutinin (HRP-WGA) and/or [ 3H]proline into the SC, dLGN, or visual cortex under halothane anesthesia (see Table I). The injections into the SC and d L G N were made by stereotaxic placement t5 and those into the cortex were made under visual control. For the d L G N injections, each of 5 animals was given a unilateral injection of 0.05-0.1 #1 of a solution of 25 #Ci [3H]proline and 100 #g HRP-WGA in 1 #1 distilled water, through a 32-gauge needle, over a period of 15-20 min. Diffusion up the injection track was reduced by pre-filling the needle with silicone oil, a very small quantity of which was expelled at the end of the tracer injection to seal the track, and by leaving the needle in place for 10 min before withdrawal. For comparison, one adult animal received two injections of a total of 2 #1 of HRP-WGA/[ 3H]proline solution into the d L G N under sodium pentobarbital anaesthesia. The d L G N was first located electrophysiologically by using a tungsten microelectrode to record neuronal responses to visual stimulation. The injections were made through a 10 #1 Hamilton syringe into the medial half of the dLGN, where the units recorded had receptive fields centred within a few degrees of the vertical meridian. For the SC injections, 0.05-0.1 #1 of 10~o HRP-WGA was injected in three animals through a stereotaxically guided needle which was advanced towards the SC at an angle of 25 ° to the vertical in order to avoid directly hitting areas 17 or 18. Since the tracer had a tendency to flow up the track, the needle tip was driven 1 mm deep or so into the SC in order to impregnate the superficial laminae well. In 5 animals tracer was injected unilaterally into the visual cortex. The injection site

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EXPERIMENTAL PROCEDURES

TABLE I

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25 #Ci/#l[ 3H]proline

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633 was selected at the time, by observation of the immature gyral pattern, and confirmed by subsequent histological examination of cytoarchitectonic appearance. In the fn'st two animals a single injection of 0.2 #1 of 25 #Ci/#l [ 3H]proline was made into the presumed 17/18 border through a micropipette with a 50 #m-wide tip. In the second pair of animals, several injections of[ 3H ]proline (total volume 0.5-1.0 #1) were made along the lateral gyrus on one side of the brain. In the fifth animal, four large injections of 10~o HRP-WGA, totalling 2 #1, were made into the lateral and suprasylvian gyri. Recovery from anaesthesia was rapid and the kitten was subsequently returned to the litter. Twenty-four hours after injection the animal was given a lethal dose of sodium pentobarbital and perfused through the heart for an hour with fixative: 2.5 ~o glutaraldehyde and 1~o paraformaldehyde in 0.1 M phosphate buffer at pH 7.4 for the animals injected with HRP-WGA alone; 4~o paraformaldehyde in the same buffer for those injected with [3H]proline. The brains and retinae were dissected out directly after perfusion and washed in phosphate buffer. The brains were blocked and put in 30~o sucrose until they sank; frozen sections were cut alternately at 50/tm for HRP histochemistry and at 25/~m for autoradiography. Sections were stained for HRP activity either with a cobalt intensification technique for the diaminobenzidine (DAB)-H20 2 reaction 1 or with the tetramethylbenzidine (TMB) method22'58, then mounted on gelatinized slides, dehydrated and cleared. To identify the location of labelled cells, some of the DAB-reacted sections were counterstained with thionin. Unstained sections were examined with dark-field illumination. The retinae were attached as flattened whole-mounts to double gelatinized slides and analyzed unstained; the coverslips were then removed and the retinae were counterstained with thionin for further examination. Drawings were made through a camera lucida during low-power examination with dark-field illumination under a Wild dissection microscope, and high-power dark-field work was done with a Zeiss photomicroscope under x 4, × 10 and × 40 objectives. For autoradiography, the 25 #m sections were mounted on clean gelatinized slides, defatted in alcohol and xylene, and coated with Ilford K5 emulsion using a loop method 39. With this technique, a film of emulsion was made by dipping a wire loop into a mixture composed of two parts of emulsion to one part of a 0.01 ~o soap (Dreft) solution kept at 45 °C in a water bath. The slides were coated with a film of emulsion using the loop, allowed to dry flat in total darkness, and then stored at 4 oC in light-proof boxes containing silica gel. After intervals of 3 weeks and 6 weeks, coated slides were developed in Kodak D19 for 3 min, fixed in 30~o sodium thiosulphate for 5 min, then washed, dehydrated and coverslipped. The sections were first analyzed under dark-field and then counterstained with thionin to assess the location of the label. For the measurement of grain density, a region of area 17 was selected where the labelling was at its maximum. The laminar distribution of label was determined by counting the density of silver grains in contiguous rectangular areas (ranging in size from 353 ktm2 to 5302 ktm2, depending on the overall density of label) extending along a radial strip through the depth of the cortex from the pia to the white matter, with the aid of a net eyepiece graticule.

634 RESULTS The use of neuroanatomical tracing methods to examine projections in the immature brain is complicated by the greater diffusion of injected material. We tried to minimize this problem by using as the retrograde tracer HRP-WGA, which spreads much less than conventional HRP 65"75 and which may have the added advantage of being less readily taken up by damaged axons of passage TM. We also wished to avoid the difficulty of interpreting anterograde labelling when it occurs mixed with retrograde labelling by the same tracer, which is inevitable when HRP or WPA-HRP is used with a very sensitive reaction technique in a pathway (such as the geniculo-cortical pathway) that has reciprocal interconnections. We therefore chose to use [3H]proline as our main anterograde tracer, and some of the material in which HRP-WGA was used to examine retrograde transport was reacted with the less sensitive DAB method to minimize anterograde labelling.

Cytoarchitecture of LGN, SC and visual cortex in newborn kitten In the thalamus of the newborn kitten, the d L G N and its constituent laminae and some of the surrounding nuclei, including the MIN and the ventral lateral geniculate nucleus (vLGN), can be identified with relative ease, although the neuronal cell bodies are much more closely packed than in the adult, and the d L G N itself has not yet rotated fully to its adult position 4°. In the SC, the lamination pattern described for adult cat 41 can already be discerned (see Fig. 7B). Lamina I (stratum zonale) has relatively few cells, all of them very small. Lamina II (stratum griseum superficiale) is rich in small and medium-sized cell bodies. Lamina III (stratum opticum) is relatively cell-free; in adult cat it contains many myelinated fibres, but in kitten myelination is incomplete. Lamina IV (stratum griseum intermediale) is the deepest collicular layer included in Fig. 7B; it has an abundance of small and medium ceils, with a few very large cells that clearly distinguish this layer even in the newborn kitten. The combined depth of the superficial grey layers is approximately 600/am. In newborn kitten cortex, gyral patterns are just beginning to form; the primary sulci are evident, but relatively shallow, and the secondary sulci are absent 3. Fig. 1 is an illustration of Nissl-stalned sections of areas 17 and 18 in the newborn kitten. Even though the packing density of neurons is much higher than in the adult, pyramidal and stellate morphologies can often be differentiated: the exception is layer II, the lastformed cortical layer54, which is composed of small, very darkly staining and densely packed immature cell bodies. Another distinctive histological feature in the kitten is the presence of numerous interstitial cell bodies in the white matter. These cells, which we take to be neurons because of the abundant Nissl substance in the somata and in what appear to be proximal dendrites, are usually stellate-shaped and seem to be better differentiated and more mature than most neurons in the cortical plate. The constituent laminae of the cortex can be recognized in the neonate 3':3"63 and this

635

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Fig. 1. Thionin-stained sections of (A)area 17 and (B)area 18 of newborn kitten to show the lamination patterns, which are in most respects comparable to those of adult cat 2°. Layer IV in area 17 is relatively broad, occupying about a third of the depth of the cortex, and it is characterized by being populated mainly by small stellate cells. Layer IV in area 18 is narrower than it is in area 17 while layer V is slightly wider. Layer III in area 18 is broad and pale and it typically has a number of large dark pyramids in its lower half. Note the dense band of immature, closely-packed cell bodies in layer II. Roman numerals refer to cortical layers. Calibration bar = 200/~m. (C) is a diagram of the coronal section through the occipital cortex, showing the regions within areas 17 and 18 from which A and B were taken.

636

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C31 Fig. 2. Locations of retrogradely labelled ganglion cells in retinal whole=mounts (superior retina up) from newborn kittens that received single, localized injections of HRP-WGA into the right dLGN (A, B, C) or SC (D, E). On the drawings of the whole-mounts of the ipsilateral and contralateral retinae, the total area

637 allows the identification of areas 17, 18 and 19 to be made according to criteria similar to those applied in the adult 2°. In practice, though, it is difficult to discern the borders between areas as precisely as in mature animals. In the newborn kitten, cortical areas 17, 18 and 19 occupy more or less the same relative positions across the hemisphere as they do in adult cat, even though the gyral pattern is incomplete.

Topography of retrograde retinal labelling after injections of dLGN or SC Our main objective in examining the retinal whole-mounts was to be sure that the retino-geniculate and retino-collicular projections are at least roughly topographically organized in the neonatal kitten. Knowledge of the retinotopic location of the injections gained from the examination of retinal labelling then helped our interpretation of the position and extent of labelling in the cortex resulting from the same injections. Fig. 2 shows 5 examples of the patterns of retrograde labelling of ganglion cells in the ipsilaterai and contralateral retinae after injections of H R P - W G A into the right d L G N (Fig. 2 A - C ) and the right SC (Fig. 2D and E). There was no consistent difference in the intensity of labelling of individual cells in the two retinae, but stained cells covered a greater area of retina in the contralateral eye than in the ipsilateral. Undoubtedly the patterns of labelling were influenced not only by diffusion of tracer around the injection site, but also by some uptake of H R P - W G A by damaged axons of passage. Nevertheless the distributions of labelled ganglion cells in each case matched our expectation based on the relative position of the injection site in relation to the known retinotopy o f d L G N 69 and SC T M in the adult cat. On the drawings of the retinal whole-mounts in Fig. 2, the filled circle indicates the dense patch of most intensely labelled ganglion cells, while the hatched area bounded by an interrupted line shows the total distribution of labelled cells, which decreased in number and intensity of labelling towards the edge of this region. The first two examples (C33 and C19) show the results of injections at the medial edge of the LGN, which undoubtedly also involved MIN and probably the lateral posterior nucleus (LP) as well. In C33 (Fig. 2A) the injection was located in the medial part of the rostral tip of the d L G N , which in the adult animal would represent the lower part of the opposite visual hemifield, near the vertical meridian 69, and labelled ganglion cells were indeed seen in the upper, central parts of the retinae, In the ipsilateral eye of C33 the dense labelling of ganglion cells in the temporal half of the retina ended abruptly at the nasotemporal border (the vertical line passing through the area centralis) with very few labelled cells in the adjacent part of the nasal retina. In the contralateral containinglabelledganglioncells is hatched, while the filledcircleindicates the main concentrationof most heavily labelled cells. The optic disc is shown as an unfilledcircle, the area centralis as a filled star. The injection sites are reconstructed in the drawings on the right: for the LGN, a coronal section through the middle of the needletrack shows the total spread of HRP-WGAin relationto the layers(A, A1, C-complex) of the dLGN; for the SC, surface views of the colliculi(A = anterior, P = posterior) show the intensely stained region around the needle track (filledoval) and the surrounding halo of diffusedtracer (stippled area).

638 eye the region of densest labelling lay in the upper nasal retina, c o r r e s p o n d i n g to the pattern seen in the ipsilateral eye. However, as in the adult cat 29, the n a s o t e m p o r a l division was less sharply defined than in the ipsilateral eye; a considerable proportion o f ganglion cells was labelled in the u p p e r t e m p o r a l retina, over a 1-2 m m wide strip a d j a c e n t to the vertical meridian. In C19 (Fig. 2B) the injection was in the medial part o f the c a u d a l pole of the d L G N and, in agreement with the expected t o p o g r a p h y , labelled retinal ganglion cells were seen in the lower retina, with the m o s t intensely stained cells localized near the decussation line. In C13 (Fig. 2C) the injection lay in the lateral portion o f the rostral half of the d L G N a n d the central p a t c h o f strongly labelled cells was seen in the upper, peripheral parts o f the two retinae. The two remaining examples in Fig. 2 show the results of injections in the right SC. The injection in C32 (Fig. 2 D ) involved the rostral pole o f the SC (which, in adults, represents the middle of the visual field, including part o f the ipsilateral hemifield o f the contralateral e y C ) : ganglion cells o f the area centralis were densely labelled in both eyes and in the contralateral eye the area o f less heavily labelled cells extended far into the t e m p o r a l retina (ipsilateral visual hemifield) as well as the n a s a l retina. F o r the ipsilateral eye, just as in the adult cat 85, the distinct patch o f labelled ganglion cells e n d e d quite abruptly j u s t to the nasal side o f the vertical meridian passing through the area centralis: there a p p e a r e d to be no substantial u n c r o s s e d projection from the n a s a l retina o f the ipsilateral eye. In C31 (Fig. 2E) the injection lay more posterior, in that p a r t o f the SC representing the middle o f the contralateral hemifield: as expected, the p a t c h o f densely labelled ganglion cells lay a r o u n d the horizontal meridian in the middle o f the t e m p o r a l retina o f the ipsilateral eye and the nasal retina o f the contralateral eye.

Fig. 3. A: dark-field autoradiograph showing anterograde label in area 17 of adult cat (C85) after an • injection of [SH]proline into the dLGN. There is a prominent band of labelling over layer IV and a fainter band over layer VI. (The light scattering in layer I on the right hand side of the section is an artefact, not associated with silver grains). Roman numerals refer to cortical layers. Calibration bar = 200#m. B: montage of an autoradiograph showing anterograde labelling in the visual cortex of kitten C30, which received an injection of [SH]proline into the medial edge of the dLGN, with probable involvement of MIN and LP. Label is seen in and around layer IV not only in the expected positions in areas 17 and 18, around the 17/18 border but also near the lateral boundary of area 19, as indicated by white arrows. There is also obvious label in upper layer I, which extends over the whole of areas 17, 18 and 19 in this section but ends abruptly at the medial border of area 17, in the fundus of the splenial sulcus (filled arrow). The inferior bank of the sulcus (parasplenial gyrus) is rendered almost invisible by the absence of label. The bright, crumpled oval in the white matter is an artefact. Calibration bar = 1 mm. C and D: dark-field micrographs of cortical sections from kitten C 13, which had received an injection of HRP-WGA and [3H]proline in the lateral half of the dLGN (see Fig. 6B). C: shows the distribution of retrogradely labelled cells in layer VI in areas 17 and 18. D: an autoradiograph of a nearby section showing the dense anterograde label in layer IV in the same regions of 17 and 18. Note the diffuse distribution of label through the grey matter, with secondary peaks in layers VI and I of area 17. The layer I labelling is much more restricted in this case than in C30 (B), although inspection of the entire series of sections showed it to be more widespread than the labelling in layer IV. Calibration bar = 1 ram.

640 Even in the adult cat the uptake of HRP-WGA by damaged axons of passage complicates the results of injections near the rostral pole of the SC, since optic tract fibres on their way from the brachium to more caudal parts of the nucleus enter the SC at its antero-lateral margin and are therefore prone to damage during injections in the rostral region aS. The total distribution of labelled ganglion cells in C32 spread as far into the temporal periphery of the ipsilateral eye and the nasal periphery of the contralateral eye as did the patch of labelled cells in C31, even though the injection site for the latter animal lay much further caudal in the SC. Presumably it was the involvement of axons of passage through the more rostral injection site in C32 that led to this peripheral labelling.

The thalamocortical projections Despite the considerable diffusion of tracer that occurs in the immature brain, the injections of [ 3H]proline into the LGN of newborn kittens were restricted to different sectors of the nucleus. In each case the injection involved all of the laminae of the dLGN but the degree of invasion of the C laminae varied between animals. Anterograde labelling was found only in the ipsilateral visual cortex and it showed that there are definitely projections from dLGN to areas 17 and 18 at birth (Figs. 3, 4 and 6). In two animals with injections of [3H]proline into the medial part of the dLGN, where there was clear staining of MIN and LP, there was also unequivocal evidence of anterograde labelling of thalamic afferents in area 19 (see Fig. 3B). However, in animals with injections in the lateral part of the dLGN there was no visible sign of anterograde labelling in area 19, even though the injections had clearly involved the C laminae (see Fig. 5). None of our thalamic injections produced any detectable anterograde labelling in the cortex of the opposite hemisphere. The geniculo-cortical projections to areas 17 and 18, present in the newborn,

Fig. 4. Laminar distributions of anterograde labelling in area 17 after injection of [3H]proline into the I1~ dLGN, determined by grain counts along a radial strip through the middle of the region of densest label. A: results for the adult cat (C85), which show a characteristic peak of label in layer IV, with a secondary band in VI and very little in I. The interrupted line is background label measured at the corresponding point in the other hemisphere. B: similar analysis for the newborn kitten C13 (see Fig. 3D). Label is much more diffusely spread through all layers, with a minimum at the II/I border. Although there is a clear peak in upper layer I, it is less dense than the labelling in IV and lower VI. Note the 5-fold difference in scale for the ordinates in A and B. Although it is difficult to attach significance to differences in density of labelling between animals, it is worth pointing out that the absolute density in layer I was less in the newborn than in the adult. It is also important to mention that the injection in C13 was lateral in the dLGN but certainly involved all layers (see Fig. 6B). C: comparative distributions for three more kittens (C33, 30 and 16: see Table I) in which grain counts were made over larger, contiguous reetangies, each covering 47 #m of cortical depth. For ease of comparison, the distributions have been normalized to the highest count (given a value of 100%), which was, in each ease, in layer IV. The graphs have been aligned on the abscissa at the junction between layer VI and the white matter (WM), which is marked with filled arrows. Data from successive animals are displaced on the ordinate for clarity. For each animal, horizontal bars indicate the position and thickness of layers I and IV.

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642 appeared to be topographically organized as in the adult, at least to the level of accuracy that our techniques allowed. The anterograde label in the grey matter (except in layer I: see below) was always restricted in extent; it occupied a position in each area retinotopically appropriate to the location of the injection site in the d L G N and it extended over a region that correlated roughly with the fraction of the d L G N involved in the injection. In those kittens in which the injection track ran through the lateral part of the dLGN, patches of anterograde label were seen within area 17 in the medial bank of the lateral gyrus, and in the lateral part of area 18, near the border with 19, corresponding, in each case, to the presumptive representation of the peripheral visual field81,s2 (see Fig. 6). Equally, injections involving the medial dLGN, which represents the central visual field69 produced anterograde labelling in areas 17 and 18, close to the boundary between the two areas (Fig. 3B), where the vertical meridian is represented in the adult 8~,~2. The rostro-caudal position of the concentration of label also depended on the rostro-caudal location of the injection in the dLGN. The dark-field autoradiographs in Fig. 3 show the laminar distributions of anterograde label in three animals - the adult (Fig. 3A), a kitten with a medial dLGN injection (C30: Fig. 3B) and one with a lateral injection with no involvement of MIN or LP (C 13: Fig. 3D). In the adult (Fig. 3A), where the injection had apparently impregnated all the laminae in the medial part of the dLGN, most of the anterograde label was restricted to layer IV and there was a minor band of labelling in layer VI, just as previously described 48. In all the newborn animals examined, even those with injections apparently confined entirely to the lateral dLGN, there was not only a relatively dense band of silver grains over layer IV in areas 17 and 18, as in adults, but also a distinct band of label over upper layer I, and a relatively high level of diffuse labelling over layers III, V and VI, and in the white matter just beneath the cortical plate (see Fig. 4). The band of labelling in the white matter was sometimes seen in continuity with heavily labelled fibre fascicles of the optic radiation (Fig. 3B and D). Interestingly, examination of the entire series of cortical sections showed that the band of label in layer I always extended over a wider region than that in layer IV, sometimes covering virtually the whole medio-lateral extent of areas 17, 18 and 19 (Fig. 3B). Therefore, towards the edge of the patch of anterograde labelling in layer IV, layer I was actually more heavily labelled than IV, although, in every case, the maximum density of label in layer IV appeared to exceed that in layer I in the middle of the labelled region. Anterograde label in area 18 and, when it occurred, in 19 too was sparser but its laminar distribution was similar to that of area 17. We have quantified the laminar distribution of anterograde label by counting silver grains in the autoradiographs along a strip running through the entire depth of the cortex, in the middle of the most densely labelled region of area 17. The distribution for the adult (Fig. 4A) shows the dense, sharp peak of label in layer IV, the secondary concentration in layer VI and the low level of label, just above background, in layer I. For the newborn animal, C13, analyzed in detail in Fig. 4B (see also Fig. 3D), which had received an injection into the lateral d L G N involving all layers (see Fig. 6B), there

643 was a broad spread of label through the full depth of the cortex, with the lowest density of silver grains over the upper part of layer II. Labelling was relatively higher in layer I for the kitten than for the adult but in none of our kittens did the density of grains in layer I exceed that in IV: Fig. 4C shows distributions of relative grain density for three more animals; labelling was always densest in layer IV.

The cortico-geniculate projection The injections of HRP-WGA into the dLGN of newborn kittens all produced retrograde labelling in the visual cortex, but only on the ipsilateral side of the brain. Most of the labelled cells lay in layer VI (Fig. 5) just as in the adult cat, but in the kittens they were more densely packed. None of the interstitial cells in the white matter below layer VI was labelled. A few labelled neurons were found in layer V, but we think that this unusual labelling was largely, if not entirely, due to uptake by fibres of passage of the cortico-tectal pathway (see below) and does not represent an aberrant immature projection from layer V to the thalamus. We draw this conclusion because: (1)the

I

II III

VI

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A R E A 17:HRP

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Fig. 5. A: dark-field photograph of HRP-labelled cells (DAB-cobalt reaction) in layer VI of area 17 after an H R P - W G A injection into the d L G N of C 13. The HRP-labelled cells are pyramidal, but they are more densely packed than in the adult, and have an immature appearance. There are no labelled cells in layer V in this example. B: thionin-stained section adjacent to that in A, to show cortical lamination. Calibration bar = 200/am.

644 sparse patch of cells in layer V was often substantially displaced from that in VI in the same cortical area (and from the region of anterograde labelling of geniculate afferents), suggesting a different topographic order in the two sets of retrogradely-labetled cells; (2) the extent of labelling in layer V varied very considerably between animals in a way that was not correlated with the intensity of labelling in layer VI (in fact in one animal, C13, which had a lateral d L G N injection, there were very few cells at all labelled in layer V); and (3)in other experiments we have occasionally seen indirect layer V labelling of this type after thalamic injections in adult cells. The labelled cortico-geniculate cells, which were very closely packed in layer VI, were pyramidal in shape but the filling with reaction product was not extensive enough to reveal the whole shape of the dendritic tree. Fig. 5 shows adjacent HRP-reacted and Nissl-stained sections through area 17 in C13, which had received an injection in the lateral half of the d L G N (see Fig. 6B). We deliberately chose to use the DAB reaction (which does not reveal anterogradely transported H R P very strongly) for this material so as to avoid the problem of interpreting a mixture of dense anterograde and retrograde H R P label. Nevertheless a slight mist of fine labelling, presumably in geniculate axon terminals, is evident over layer IV directly above the labelled cell bodies of layer VI in Fig. 5A. The light band at the pial surface of layer I is mainly artefactual light scatter, which often occurs at the surface of cortical sections under dark-field illumination, rather than H R P reaction product (see also Figs. 7C and 9). There was clearly a topographic organization in the cortico-geniculate projection. The labelled cells of layer VI were mostly distributed in three dense bands, in retinotopically-corresponding positions in areas 17, 18 and 19. There was also a sparser scattering of labelled cells in layer VI of the suprasylvian gyrus. In these experiments [ 3H]proline and H R P - W G A had been injected together into the d L G N and, in areas 17 and 18, the band of retrogradely-filled cells in layer VI always lay in register with the patch of anterograde labelling of thalamic afferents. Fig. 3C is a dark-field micrograph showing areas 17 and 18 in an HRP-reacted coronal section from C13, through the region of maximum density of labelled cells in layer VI. The cells are seen in two main groups, in retinotopically corresponding parts of areas 17 and 18. These locations, in the mature cat, would represent an intermediate eccentricity in the visual field, as predicted by the site of the injection in the lateral half of the d L G N (see Fig. 6B). A less dense scattering of labelled cells is also seen extending along layer VI, further down the medial bank of the lateral gyrus (which corresponds to more peripheral visual field): this pattern is presumably due to diffusion of injected tracer to the lateral margin of the d L G N (see Fig. 6B). Comparison of Fig. 3C with 3D (an autoradiograph of an adjacent section from the same animal) shows the good registration of the regions of highest densities of retrograde and anterograde labelling in both areas 17 and 18. Fig. 6A gives a more complete indication of the distributions of anterograde labelling in layer IV (dotted lines) and retrograde labelling of cells in layer VI (continuous lines), and of the degree of correspondence between them, in a series of coronal sections of the cortex in C13. The injection site, reconstructed in Fig. 6B, fully involved all laminae

645 A

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Fig. 6. A: location of HRP-labeUed cells in layer VI (continuous thick lines) and anterogradely labelled terminals (dotted lines) in cortex al~er an injection of HRP-WGA and [3H]proline into the dLGN. The distance of each successive section in a caudal direction from the first one at the top is shown on the left hand side of each section. Retrogradely labelled cells were located in retinotopically equivalent regions of areas 17, 18 and 19, and in more lateral, suprasylvian cortical areas. Anterograde labeUing was restricted to areas 17 and 18 in this animal (C13). B: coronal sections through the posterior thalamus to show the injection needle track through the rostro-lateral part of the dLGN, with the dense staining and diffuse halo of HRP around the track. In this case there appeared to be no spread at all beyond the medial borders of the nucleus.

646 of the d L G N but not the MIN or LP. Notice the way in which anterograde label in this case was restricted to areas 17 and 18, whereas retrograde labelling was also seen in the retinotopically appropriate part of area 19 (adjacent to the border with 18) and in part of the suprasylvian cortex.

The cortico-collicular projection We examined the pathway from visual cortex to SC in newborn kittens by means of anterograde transport of [ 3H]proline or HRP-WGA after injections into the cortex, as well as by retrograde transport of HRP-WGA injected into the SC.

Anterograde labelling In two animals (C17, C20) a single small injection of [ 3H]proline was made into the area 17/18 border region of the visual cortex, where the rnidline of the visual field would be represented, at the junction between the lateral and posterior gyri. In both of these kittens there was dense anterograde labelling in a small spot in the rostral half of the SC, which is also the presumptive representation of the vertical meridian 5,16. Fig. 7A shows the pattern of label in the SC of C20: it was concentrated in the stratum zonale (lamina I) and in the lower part of the stratum griseurn superliciale (lamina II), and there was only sparse labelling in the upper part of lamina II and in lamina III (stratum opticum). No label was seen in the contralateral colliculus after these single injections restricted to the 17/18 border. Two other animals (C29, C82) received multiple small injections of [ 3H]proline in the visual cortex: the injected marker had spread into most of the lateral gyrus (areas 17, 18 and 19) in C29 and into the medial bank of the lateral gyrus (areas 17 and 18) in C82. Labelling in the ipsilateral colliculus showed the same laminar distribution as described above for C17 and C20 but it covered a wider area of the SC. In C29 there was also very sparse labelling in stratum griseum superficiale in the rostral pole of the contralateral colliculus. In C75, in which extensive multiple injections of HRP-WGA had been made into the visual cortex, the label at the injection site had spread into all the cortical regions that project to the SC in adult cats 21"44's4, i.e. all of the lateral, posterior, and suprasylvian gyri and the medial bank of the ectosylvian gyrus (Fig. 8A). In the ipsilateral side of the brain of C75 there was dense anterograde HRP labelling in laminae I, II and III over the whole of the SC (Fig. 8B). Labelling in laminae I and II was fine and dust-like and most likely belonged to axon terminals or growth cones. Labelling in lamina III was coarse and appeared to be associated with fibre bundles of the cortico-tectal pathway. There was some faint and diffuse labelling in the intermediate laminae of the SC. There was also labelling in the SC on the contralateral side of the brain after this extensive injection (Fig. 8B) but it was much fainter than on the ipsilateral side and was restricted to specific parts of the SC. Label was present throughout much of the lateral brachium of the contralateral SC and within the tectal commissure, but within the contralateral SC itself, label, which was restricted to the

647

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Fig. 7. A: dark-field photomicrograph of a coronal section through the rostral part of the S C, showing the distribution of anterograde label after a restricted injection of [3H]proline into the 17/18 border region of the ipsilateral visual cortex (experiment C20). There is strong anterograde labelling in lamina I and in lower lamina II (stratum zonale and stratum griseum superficiale). Calibration bar = 500 #m. B: adjacent thioninstained section of SC to show the lamination pattern (see text). The Roman numerals refer to the laminae of the SC 41, C: dark-field photomicrograph of HRP-labelled cells (DAB-cobalt reaction) in layer V of area 17 after an HRP-WGA injection into ipsilateral SC (experiment C31). Calibration bar = 200#m. D: adjacent thionin-stained section showing cortical lamination. Roman numerals indicate layers of the cortex.

648 |cm m

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Fig. 8. A: surfaceviewof an extensive,multipleHRP-WGAinjectionsite(hatchedarea)in the cortex (experimentC75).B:distributionofanterogradelabelling(TMBreaction)in ipsilateralandcontralateral SC al~erthis largeinjectionof HRP-WGAinto visualcortex.The relativeintensityof labellingin contralateralSChasbeenexaggeratedto showits distributionclearly(seetext).Theexcessiveshrinkage that occurredin thisseriesof sectionswascausedbythe fixationand reactionprocedurefor HRP.

649 stratum griseum superficiale (lamina II), was seen only at the rostral pole, and, to a lesser extent, at the extreme caudal pole of the SC.

Retrograde labelling As expected from the patterns o f anterograde labelling, injections o f H R P - W G A into the SC of newborn kittens retrogradely labelled large numbers o f cells in the ipsilateral cortex and a few on the contralateral side. Fig. 7C shows the population o f labelled cells in area 17 o f C31 (see Fig. 1E for a reconstruction of the injection site). As far as we could tell, all o f the labelled cells in the cortex were pyramidal and they were exclusively in layer V. In all the specimens examined, heavily labelled cells were found in areas 17, 18 and 19 and in the cortex o f the suprasylvian sulcus on the ipsilateral side of the brain (Figs. 9 and 10). On the contralateral side a few faintly labelled cells were found in

Fig. 9. Low-power dark-field photomicrograph of HRP-labelled cells (TMB reaction) in layer V of areas 17, 18 and 19 and in the lateral suprasylvian gyrus (LSG) after an injection of HRP-WGA in the caudal part of the ipsilateral SC, which represents the periphery of the contralateral visual field (experiment C31). The patches of labelled cells within areas 17, 18 and 19 lie in the presumptive representations of this part of the field. Note the intense light scattering (not HRP labelling) in layer I, which is a common feature of dark-field photomicrographs. (The white oblique streak in the white matter is a fold in the section). Calibration bar = 1 mm.

650

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Fig. 10. A: location of bands of HRP-labelled cells (continuous thick lines) in layer V of visual cortical areas after an injection of H R P - W G A into the centre of the SC, the presumptive representation of the middle of the contralateral visual hemifield (experiment C22). The distance of each section in a caudal direction from the first one at the top is indicated at the left hand side. On the ipsilateral side of the brain, retrogradely labelled cells are found in cortical areas 17, 18 and 19, and in the cortex of the suprasylvian sulcus. Note that the patch of labelled cell bodies within areas 17, 18 and 19 lies in the middle of the medio-lateral extent of each area, where the central part of the contralateral visual hemifield would be represented. On the contralateral side of the brain, labelled cells are seen in area 19 and in the suprasylvian sulcus. B: injection site in the SC, shown in coronal sections. The filled area indicates the extent of dense staining for H R P - W G A around the needle track and the hatched area is the diffuse halo of staining around the main injection site.

651 area 19 and in the suprasylvian cortex (Fig. 10). Cortico-collicular projections from areas 17, 18 and 19 on the ipsilateral side of the brain were topographically organized: labelled cells in these areas occurred in discrete patches, the localization of which varied systematically according to where the injections had been made in the SC, corresponding to the representations of the visual field in these same structures in the adult cat5,16,55.84. In C75 and in other preliminary experiments involving large injections of HRP or HRP-WGA into the visual cortex we found no evidence for an aberrant direct projection from the SC to the cortex in neonatal kittens. DISCUSSION The interpretation of anatomical tracing experiments in the immature brain is fraught with problems. Even in adult animals some pathways appear not to transport certain retrograde tracers effectively6°, axonal transport could be more capricious or selective in young animals since the very immaturity of axons and terminals may render them incapable of taking up or transporting label. Developing projections may also be very sparse and therefore hard to detect reliably. The excessive diffusion of some tracers (especially unconjugated HRP) after injection into the immature nervous system is also a serious obstacle to studies of the precision and topography of projections. HRP-WGA and [3H]proline offer certain advantages for this kind of work because they not only provide great sensitivity for the detection of weak projections but also appear to spread less excessively around the injection site than do some other tracers. In this study we have gained further information about the organization of the visual pathways in the newborn kitten, before the onset of visual activity and of functional plasticity in the striate cortex. Our results add to the growing evidence for a considerable level of maturity in most of the major visual l~rojections at the time of birth. Retinal projections

The projections from both eyes to dLGN and SC are certainly present and topographically organized at birth. The characteristics of the decussation patterns for both crossed and uncrossed retino-geniculate and retino-collicular projections also seem to be at least grossly mature at birth, though there is good evidence for a small population of aberrant ganglion cells in the nasal retina with uncrossed axons, which becomes much reduced in number by 10 days or so of age 37. It will obviously be of interest to examine the morphological appearance of ganglion cells that project to dLGN or to SC and the details of their distributions and decussation patterns during development. Interconnections between thalamus and cortex

It is clear from this and previous w o r k 3'9'23'42"43"50 that there are well-developed connections from dLGN to areas 17 and 18 in very young kittens. What we have added

652 here is further evidence that these afferent projections are topographically organized and are in good register with the reciprocal cortico-geniculate projections, which arise, as in the adult 21"s3"84, from pyramidal cells of layer VI in areas 17 and 18, as well as from area 19 and the lateral suprasylvian area. We detected no major aberrant pathways to or from ipsilateral cortical areas that are not normally connected to the dLGN, and there were no exuberant contralateral geniculo-cortical interconnections. The only major component of the mature reciprocal circuit between d L G N and cortex that could not be detected in the neonatal kitten was the projection from the C laminae to area 1926'51'56. Only when the injection of [3H]proline lay near the medial edge of the dLGN, with a high probability of involvement of MIN and LP, was there clear anterograde label in area 19 (e.g. Fig. 3B). It is possible, then, that the geniculocortical projection to area 19 develops later than the reverse projection from 19 to the dLGN, which could be seen in the newborn kitten even after injections of HRP-WGA in the lateral d L G N (Fig. 6). Many of the examples of functional plasticity that have been described for kitten cortical neurons 1s,25,59 might depend on changes in the distribution of afferent axons. The shift in ocular dominance (at least of cells in layer IV) induced by monocular deprivation is certainly associated with a competitive, activity-dependent re-arrangement of geniculo-cortical terminals in layer IV 5°'73. It would be very valuable then to know just how precisely organized the geniculo-cortical projection is before the onset of plasticity. Unfortunately it is not practicable to compare the exact degree of topographic order in kittens and adults with the techniques used here, because of uncontrollable potential differences in the spread and uptake of tracer, described above. However, visualization of the arborizations of individual afferent axons reveals no gross difference between kittens 46'49 and adult cats 17"27"2s'49 in terms of lateral spread of afferent fibres in the cortex. What is certain is that the laminar distribution of anterograde labelling after geniculate injections is more diffuse in kittens than in adults. Instead of very dense label in layer IV, and a secondary peak in VI, the kitten has a broad spread of label through all layers, with a minimum in layer II (Fig. 4). Of course, light-microscopic examination did not allow us to identify precisely where the anterogradely-transported label lay. In adults, it accumulates primarily in synaptic terminals 24, but in the newborn kitten, where there are few synapses ~'s8, and axons are presumably still invading the cortex, much of the label may be present in axons and growth cones. This may account for some of the diffuse nature of the labelling, especially in the lower cortical layers and even in the white matter. There is also the possibility of some transneuronal transport, within the cortex, even after the 24 h survival time that we used. Anterograde degeneration techniques 2'47, Golgi impregnation 57, transneuronal transport of [3H]proline after injection of one eye5°, and anterograde transport of unconjugated HRP from the d L G N 42'43 have all revealed a relatively excessive projection to layer I in young kittens. Our material, involving anterograde transport of

653 [ 3H]proline directly from the dLGN, shows the same general pattern, but quantitative analysis of grain density through all layers of the cortex (Fig. 4) produced a slightly different impression from that seen in Kato et al.'s 42'43 results with anterogradely transported HRP. None of our animals had a higher level of label in layer I than in layer IV and the density and spread of label in layer I varied substantially between animals. In particular, more lateral injections in the dLGN (certainly involving the C complex but not the visual thalamic nuclei medial to the dLGN) produced only modest labelling in layer I, restricted to the same general regions of cortex as the denser label in layer IV (e.g. C13; Figs. 3D, 4B and 6B). More medial injections, with spread of tracer to MIN and possibly LP, tended to produce very widespread labelling in layer I, covering most of the medio-lateral extent of areas 17 and 18, though still less dense than the centre of the patch of label in layer IV (e.g. C30; Fig. 3B). A possible interpretation of this f'mding is that, while the dLGN itself contributes a small, topographically organized input to layer I, a very diffuse projection to I arises from cells (or axons) lying medial to the dLGN. The reason for the differences in relative densities of labelling in layers I and IV seen after anterograde transport of HRP 42'43 and of [ 3H]proline is not immediately obvious. Kato et al.42.43 did not quantify their results but the clear impression was that the density of HRP label in layer I in young kittens was not only much higher than that in IV but was absolutely greater than that in layer I of the adult cat. This would, of course, imply early exuberance (and subsequent withdrawal) of the layer I input, rather than mere precocity of maturation. It must be said that Kato et al.42 recognize that interpretation of HRP labelling in layer I may be complicated by retrogradely transported label in the apical dendrites of layer VI cells. There may also be other technical differences, including the possibility of accumulation of HRP in glial cells in layer I. Paradoxically, studies of single impregnated afferent axons have not revealed substantial numbers of unusual fibres specifically targeted on layer I in kittens 46,49. On the other hand, Kato et al.42 have shown that superficial injections of HRP into area 17 in young kittens label a small but specific population of relatively large cells in the C laminae and even a few in the A layers of the dLGN. LeVay et al. 5° also interpret the pattern of transneuronal label in area 17 of young kittens in terms of precocious development of the C laminae, which would be compatible with the fact that W-cells in the C laminae appear to be morphologically relatively mature at an early age 19. However, the C laminar projection to area 19 seems, from our experiments, to be undetectable at birth. The large cells described by Kato et al. 42 may, then, be a special population whose subsequent fate, after birth, is well worthy of further study.

The cortico-collicular pathway Again, our main impression from study of the projection from visual cortex to SC is that it seems to be very mature on the day of birth. Just as in the adult cat 21"44'84, the cortical projections to the ipsilateral SC of newborn kittens appear to arise exclusively from cells of layer V in areas 17, 18 and 19, and in the cortex around the

654 suprasylvian sulcus; and the projection terminates in a topographic pattern over thc whole SC, mainly in the stratum zonale and stratum griseum superficiale. Recently, Tsumoto et al.8o, who also used retrograde labelling methods to reveal the presence of the cortico-coUicular pathway in very young kittens, suggested that there may initially be an aberrant element in this projection, since the fraction of layer V cells labelled after a collicular injection appeared to decrease over the first 2 weeks of l':fe. Although our material was prepared in much the same way as theirs, we cannot bc certain that an excessive proportion of cortical cells was labelled: the small size and very dense packing of neurons in the immature cortex makes it extremely difficult to assess with accuracy the fraction of labelled cells in 50 #m sections. In any case, interpretation of the proportion of cells labelled in layer V is complicated by the fact that a substantial population of neurons in that layer with aberrant cortico-cortical projections may be dying during the first 3 weeks of life65. There is now ample evidence in the adult cat for a restricted projection from the visual cortex to the opposite SC: it arises from cells in layer V of area 19 and the lateral suprasylvian gyrus and terminates mainly in the medial and lateral margins of the rostral part of the contralateral SC 4,6'62 where the part of the ipsilateral visual hemifield adjacent to the vertical meridian is represented. This crossed cortico-coUicular pathway is present in 4-day-old kittens 9 and it seems mature in its distribution even in newborn kittens. With very extensive cortical injections of HRP-WGA (Fig. 8) we observed anterograde label not only in the rostral part of the contralateral SC but also at the caudal pole. However, we assume that this sparse projection to the posterior part of the opposite SC is not an abnormal, immature pathway because it was also seen in adult cat by Berman and Payne 6. Although the crossed cortico-collicular projection does not, then, appear to be exuberant in terms of the distribution of either its cells of origin or its terminal field, there remains the possibility that the restricted pattern evident at birth results from selective elimination of a more diffuse early projection in utero.

CONCLUSIONS Apart from the possibility of an aberrant early projection to layer I of the c o r t e x 42 and the existence of a small population of nasal ganglion cells with uncrossed axons 37, studies of the major visual pathways into and between the cortex and the SC in young kittens reveal no gross errors or deficits in organization. To be sure, there are still many signs of immaturity (e.g. the continuing death of ganglion cells 37'61, the unusual orientation of the d L G N 4°, the incomplete differentiation of cortical layer I154'63, the general sparseness ofgeniculate termination in the cortex, especially in area 19, and the small number of synaptic contacts per cortical ce1111'88). But the main pathways have been built, in a topographic and systematic fashion, by the time of birth. All of this remarkable state of order (including any prenatal elimination of exuberant projections) must be achieved without the benefit of visual activity.

655 The remarkable physiological plasticity o f cortical neurons during the second month of life, which certainly does depend on activity, must involve subtle morphological changes. It is already k n o w n that geniculate axon arborizations in layer IV are locally redistributed under the influence o f visual activity 49"5°, but much of the normal physiological maturation of cortical cells as well as their plasticity may well depend partly on functional modulation o f existing synaptic inputs, or o f inhibitory networks within the cortex. The high degree o f precision in the laminar and areal distributions and the topography o f projection o f cortico-geniculate and cortico-tectal neurons in neonatal kitten contrasts with the florid exuberance that has been described for interhemispheric and association projections14'3°-33"64'65: the selective elimination of at least some of these cortico-cortical projections does seem to be influenced by abnormal forms of visual experience 34'35'53. Perhaps there is a general rule here. Cortical projections to subcortical structures m a y be more precisely specified and more rigidly determined by the time of birth in the cat than onward projections to other cortical areas.

ACKNOWLEDGEMENTS This work was supported by Programme G r a n t s (Nos. G979/49 and G7900491) from the Medical Research Council. We are grateful to D u n c a n Fleming, Laurence Waters and J o h n Eldridge for their technical help.

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