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Adjustment of connectivity in rat neocortex after prenatal destruction of precursor cells of layers II-IV
Developmental Brain Research, 2 (1982) 425431
425
Elsevier/North-Holland Biomedical Press
Adjustment of connectivity in rat neocortex after prenatal destruction of precursor cells of layers II-IV
E. G. JONES, K. L. VALENTINO and J. W. FLESHMAN, Jr.* Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110 (U.S.A.)
(Accepted June 8th, 1981) Key words: cytotoxic drugs - - neocortical development - - microcephalic rats - - connections - -
morphogenesis
Prenatal administration of cytotoxic drugs during proliferation of precursor cells of neurons in granular and supragranular layers of the rat cerebral cortex prevents these layers from forming and causes malformations of some cells in the surviving layers. But it does not prevent the surviving layers from establishing normal efferent connections, nor does it prevent afferent fibers from colonizing the cortex and establishing a bilaminar pattern of synaptic connections, partly in an abnormal position. Thalamocortical and commissural fibers growing towards the sensorimotor cortex of the rat and cat reach the vicinity of their target early in development. They then accumulate in the developing white matter over a relatively protracted period of time before undergoing a renewed growth spurt which carries them into the overlying cortex10,21, 2a. While accumulating in the white matter the afferent fibers are traversed by recently generated neurons that are migrating to the superficial layers of the cortex. Presumably, many of these neurons will later receive synaptic connections from the invading afferent fibers. We wished to determine the fate of the two sets of afferents when deprived of any influences that they might normally receive from the growth through them of their ultimate target cells, and when, upon arrival in the cortex, they found a substantial proportion of their target cells absent. Pregnant Wistar rats, at 15, 16 or 17 days of gestation were injected intraperitoneally with 1-1.5 mg of 5-azacytidine or 6 mg of methylazoxymethanol acetate (MAM). These cytotoxic agents may be expected to kill all cells undergoing D N A synthesis in the neuroepithelium for a short time subsequent to the injectiong, 16. At the times injected this particularly includes the cells of origin of layers I I - I V of the neocortex. The cells of origin of layers V and VI have already undergone their final division and migrated towards their target layersL * Present address: Department of Surgery, The Jewish Hospital of St. Louis, St. Louis, MO 63110, U.S.A.
426 A p p r o x i m a t e l y 70 0/0 o f the pups born to injected mothers survived to m a t u r i t > The a p p e a r a n c e s and b e h a v i o r o f these rats were u n r e m a r k a b l e though the size., ,H~ their cerebral hemispheres were greatly reduced a n d their learning capacity, if tested a p p r o p r i a t e l y < la, should have been impaired. At 3-6 m o n t h s o f age the brains from some o f the a n i m a l s were prepared by Golgi m e t h o d s 14,1v. Other animals, under chloral h y d r a t e anesthesia, received small injections o f horseradish peroxidase or o f mixed [aH]proline and [aHJleucine in the p r e s u m p t i v e somatic sensory cortex, or stereotaxically in the v e n t r o p o s t e r i o r thalamic nucleus or p y r a m i d a l tract. Prior to m a k i n g the injections, restricted parts o f lhc cortex o r t h a l a m u s were m a p p e d with microelectrodes for units r e s p o n d i n g to naturally applied somesthetic stimuli. After 1-6 days, the animals were perfused with a p p r o p r i a t e a l d e h y d e fixatives and sections p r e p a r e d for enzyme histochemistry or autoradiography<7. The neocortex (Figs. I-3) is reduced in thickness and extent. A molecular layer is distinct but b e n e a t h it there are only two layers o f neurons. These (Fig. 1C) have all the histological a p p e a r a n c e s o f layers V a n d V1 o f n o r m a l animals, i.e. a h o m o g e n e o u s p o p u l a t i o n o f small to m e d i u m sized, pale, r o u n d cells in layer VI and a bistratified
Fig. 1. Figs. 1-3 are from animals in a single litter from a mother treated with MAM. A: distribution of autoradiographically labeled thalamocortical axons in layers V and VI of presumptive somatic sensory cortex of a MAM-treated rat after injection of [all]amino acids in ventroposterior thalamic nucleus; dark-field photomicrograph. M, molecular layer, V-VI, cellular layers. B: anterograde labeling of thalamocortical axons and retrograde labeling of corticothalamic cells in another treated animal injected with horseradish peroxidase in ventroposterior thalamic nucleus. C and D: adjacent sections showing in a Nissl stain (C) bilaminar nature of presumed somatic sensory cortex in MAMtreated rat, and distribution of major zone of thalamocortical fibers around apical parts of retrogradely labeled pyramidal tract neurons (D); injection of horseradish peroxidase in ventroposteriot thalamus gave thalamocortical fiber and corticothalamic cell labeling and second injection in pyramidal tract of same animal gave retrograde labeling of pyramidal tract neurons in layer V. Bars : 100/~m (A, B) 500 l*m (C, D).
427
Fig. 2. Golgi impregnations of abnormally inverted (B) and divaricated (D) pyramidal cells in layer V of MAM-treated rats. Comparable cells in A and C are retrogradely labeled by horseradish peroxidase injected into pyramidal tract. Large arrows indicate brain surface. Small arrows show in C a labeled dendrite and in D an intrinsic cortical neuron. Bars: 50/~m. layer V, with large pyramidal cells deeply and medium sized cells superficially. In some places, many of the medium-sized cells are arranged in clumps, but in no plane of section can the normal granule cell aggregations of the somatic sensory cortexm, 2~ be detected. The Golgi material shows normal appearing pyramidal cells in the remaining layers V and VI (Fig. 2B, D) and the presence of at least the larger class of non-pyramidal cells (the basket cells). Many of the pyramidal cells with somata in the superficial part of layer V have apical dendrites that divide quite close to the soma and, atypically, the branches divaricate extremely widely in the molecular layer (Fig. 2D). There are also many inverted pyramids (Fig. 2B). Other effects include the presence of abnormal islands of cells within the pyramidal layer of the hippocampus and in the white matter between the striatum and the neocortex (Fig. 3A,B). These may result from arrested migration. The corpus callosum is greatly reduced in thickness and rostrocaudal extent (Fig. 3A). All thalamic nuclei are clearly recognizable and their neuron density, though not yet quantified, appears substantially normal (Fig. 3B). Other parts of the brain, including the cerebellar granule cells whose precursors continue to proliferate after the time of the drug injection 1,1°, appear normal. Unit responses to natural somesthetic stimuli were easily recorded in a region corresponding to the somatic sensory cortex. Anterograde labeling of efferent fibers by autoradiography or with horseradish peroxidase following injections at this site revealed intact subcortical projections from this siteZ2,24. Labeled fibers could be traced as far as the spinal cord, with loci of labeled terminal ramifications detectable in striatum, thalamus, superior colliculus, pontine nuclei, dorsal column nuclei, and spinal gray matter. Injections of horseradish peroxidase in the thalamus or pyramidal
428 1 2 3
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D Pig. 3. A : aberrant cell islands (arrows) associated with the corpus callosum (CO) in a MAM-treated rat. We have shown that some of tie cells in tie islands send axons to the opposite cortex. I : anterolrade Iber and retrograde cellular labeling in central lateral (/L) and ventroposterior (VP) thalamic nuclei following injection of horseradish peroxidase in somatic sensory cortex of treated animal. LG> dorsal lateral geniculate nucleus ; arrow, aberrant cell island in hippocampus, lars" I l~m. C, D: partial somatotopic map of ventroposterior nucleus made from microelectrode recordings showing normal receptive field sequences of single units along 3 penetrations 1-3. For clarity, only every fourth or fifth receptive field is illustrated. tract revealed that the n o r m a l l a m i n a r origins of corticothalamic a n d pyramidal tract axons from cells respectively in layers VI a n d V22, 24 are preserved (Fig. 1B, D). M a n y of the labeled pyramidal tract n e u r o n s were the atypical forms in the superficial part of layer V (Fig. 2A, C). Anterograde fiber labeling could clearly be seen in the t h i n n e d corpus callosum, b u t relatively few retrogradely labeled commissural cells were seen in the contralateral cortex. These were largely confined to layer V, t h o u g h some were noted in the a b e r r a n t islands of cells in the subcortical white matter. The small n u m b e r s of callosal projection n e u r o n s a n d the reduction in size of the corpus
429 callosum probably reflects destruction of the large proportion of callosal parent cells that normally reside in layer 11121. Retrograde labeling of afferent cells was particularly striking in the ventral and intralaminar nuclei of the thalamus after cortical injections of horseradish peroxidase (Fig. 3B). Anterograde labeling of the cortical terminations of the thalamic relay cells occurred in a dense band extending from the deep border of the molecular layer to just above the somata of the deeper stratum of large cells in layer V (Fig. 1A, B, D). It seemed thinner than in the normal but no less dense. Fingers of label commonly extended from it into the molecular layer (Fig. 1D). There was a second band of labeling in a normal position at the junction of layers V and VIa, 23. The superficial stratum of thalamocortical fiber terminations is placed closer to the somata of the corticopyramidal cells than normala, 23 (Fig. 1D). During our mapping of the thalamus to identify appropriate sites for injections, we found that the receptive fields of the neurons recorded from and the representation of body somatotopy were apparently little changed from normal (Fig. 3C, D). Units responsive to stimulation of the body surface were not difficult to find and the sizes of their receptive fields were normal. The parts of the body represented at different dorsoventral and mediolateral levels, as shown by the sequences of receptive fields encountered, wele also in a normal sequence 5,1s. Our more limited observations in the cortex suggested a similar preservation of somatotopy in the presumed first somatic sensory area. Our results show that the cytotoxic effect is short-lived and local. Remaining parts of the cerebral cortex and other parts of the brain directly or indirectly connected with the cortex, even if formed by later proliferating cells, appear substantially unaltered. The destruction of a considerable number of cortical target cells before their migration to the cortex through the waiting afferent fibers does not suppress any signal that might normally influence the fibers' second growth spurt into the cortex. Nor does the absence of the main target layers (III and IV) prevent colonization of the cortex by a significant number of thalamic afferents, many of which terminate on the intact layers V and/or VI cells. The development of a bilaminar distribution of thalamocortical axon terminals in a neocortex consisting of infragranular layers only may imply that synaptic connections have been made on inappropriate cells or in greater numbers than normal on a remaining set of cells. Somatotopy in the projection pattern is preserved. If this is normally imposed on the cortex from the thalamus ~3, it may reflect the preservation of the normally ordered lemniscal input to the thalamus, demonstrable in our microelectrode recordings. Cells that are migrating to, or that have already reached the cortex prior to the perturbation, form only appropriate laminae and only make connections appropriate to those laminae. One major efferent system, the callosal, has been substantially reduced by destruction of many of its parent cells normally resident in layer III. The few inappropriately placed callosally-projecting cells appear to be those whose migration has been interfered with. This seems to indicate that the establishment of efferent
430 c o n n e c t i o n s by layer V and VI cells is i n d e p e n d e n t of interactions with the granular' a n d s u p r a g r a n u l a r layers. The only overt change in the structure o f the r e m a i n i n g cortical layers occurs i~-~ the superficial p a r t o f layer V. In the preserved layers, therefore, basic p y r a m i d a l cell form, the d e v e l o p m e n t o f which n o r m a l l y occurs in parallel with the colonization of the cortex by afferent fibers ~9, seems to depend, like the establishment o f efferem connections, to a large extent on factors intrinsic to the cell. But it is possible that local factors o f an u n d e t e r m i n e d n a t u r e o p e r a t i n g at i n t e r l a m i n a r b o r d e r s may participate in the finer m o d e l l i n g o f the p y r a m i d a l cells. This idea comes from the observations t h a t p y r a m i d a l cells with s o m a t a in the vicinity o f the n o r m a l l y a p p e a r i n g layer V VI b o r d e r are substantially n o r m a l in a p p e a r a n c e , while those in the most superficial part o f the r e m a i n i n g cortex, where layer V meets n o t layer IV but a m o l e c u l a r layer, arc very atypical a n d m a y even be inverted. Similar concepts regarding the relative roles oF intrinsic and local influences have been expressed by others, particularly in the cerebelluml,a,11-13,25. The presence o f inverted forms o f p y r a m i d a l cell and o f forms with a b n o r m a l l y extensive dendritic trees m a y imply a response to an absence or a reduced c o n c e n t r a t i o n o f potential synaptic inputs in the anticipated d o m a i n o f the developing cells' apical dendrites. S u p p o r t e d b y N I H G r a n t s N S 15070 a n d T 3 2 - N S 0757. W e t h a n k Ms. Bertha M c C l u r e a n d Mr. R o n a l d Steiner for technical assistance. and Drs. R. P. Bunge, N. W. Daw, V. H a m b u r g e r , W. M. L a n d a u , and A. L. P e a r l m a n for helpful criticism.
1 Altman, J., Autoradiographic and histological studies of postnatal neurogenesis. 11I. Dating the time of production and onset of differentiation of cerebellar microneurons in rats, J. comp. Neurol., 136 (1969) 269-294. 2 Berry, M. and Rogers, A. W., The migration of neuroblasts in developing cerebral cortex, J. Anat. (Lond.), 99 (1965) 691-709. 3 Caviness, V. S., Jr., Patterns of cell and fiber distribution in the neocortex of the reeler mutant mouse, J. comp. Neurol., 170 (1976) 435-448. 4 Cowan, W. M., Gottlieb, D. I., Hendrickson, A. E., Price, J. L. and Woolsey, T. A., The autoradiographic demonstration of axonal connections in the central nervous system, Brah~ Res., 37 (1972) 21-51. 5 Emmers, R., Organization of the first and second somesthetic regions (SI and SII) in the rat thalamus, J. comp. Neurol., 124 (1965) 215-228. 6 Haddad, R. K., Rabe, A., Laquer, G. L., Spatz, M. and Valsamis, M. P., Intellectual deficit associated with transplacentally induced microcephaly in the rat, Science, 163 (1969) 88-90. 7 Hardy, H. and Heimer, L., A safer and more sensitive substitute for diaminobenzidine in the light microscopic demonstration of retrograde and anterograde axonal transport of HRP, Neurosci. Lett., 5 (1977) 235-240. 8 Herkenham, M., Laminar organization of thalamic projections to the rat neocortex, Science, 207 (1980) 532-534. 9 Johnston, M. V. and Coyle, J. T., Histological and neurochemical effects of fetal treatment with methylazoxymethanol on rat neocortex in adulthood, Brain Res., 170 (1979) 135-156. 10 Miale, I. and Sidman, R. L., An autoradiographic analysis of histogenesis in the mouse cerebellum, Exp. Neurol., 4 (1961) 277-296. 11 Rakic, P., Extrinsic cytological determinants of basket and stellate cell dendritic pattern in the cerebellar molecular layer, J. comp. Neurol., 146 (1972) 335-354.
431 12 Rakic, P., Synaptic specificity in the cerebellar cortex: study of anomalous circuits induced by single gene mutations in mice, CoM Spring Harb. Symp. quant. Biol., 40 (1976) 333-346. 13 Rakic, P. and Sidman, R. L., Organization of cerebellar cortex secondary to deficit of granule cells in Weaver mutant mice, J. comp. Neurol., 152 (1973) 133-162. 14 Rethelyi, M., Cell and neuropil architecture of the intermediolateral (sympathetic) nucleus of cat spinal cord, Brain Res., 46 (1972) 203-213. 15 Rodier, P. M. and Reynolds, S. S., Morphological correlates of behavioral abnormalities in experimental congenital brain damage, Exp. Neurol., 57 (1977) 81-93. 16 Shimada, M. and Langman, J., Repair of the external granular layer of the hamster cerebellum after prenatal and postnatal administration of methylazoxymethanol, Teratology, 3 (1970) 119125. 17 Van der Loos, J., A 'controlled impregnation' modification of the Golgi-Cox technique. In Dendrodendritische Verbindingen in de Schors der Grote Hersenen, Thesis, University of Amsterdam, Haarlem: Stan, 1960. 18 Waite, P. M. E., Somatotopic organization of vibrissal responses in the ventrobasal complex of the rat thalamus, J. Physiol. (Lond.), 228 (1973) 527-540. 19 Wise, S. P., Fleshman, J. W., Jr. and Jones, E. G., Maturation of pyramidal cell form in relation to developing afferent and efferent connections of rat somatic sensory cortex, Neuroscience, 4 (1979) 1275-1297. 20 Wise, S. P., Hendry, S. H. C. and Jones, E. G., Prenatal development of sensorimotor cortical projection in cats, Brain Res., 138 (1977) 538-544. 21 Wise, S. P. and Jones, E. G., The organization and postnatal development of the commissural projection of the rat somatic sensory cortex, J. comp. Neurol., 163 (1976) 313-343. 22 Wise, S. P. and Jones, E. G.,Cells of origin and terminal distribution of descending projections of the rat somatic sensory cortex, J. comp. NeuroL, 175 (1977) 129-158. 23 Wise, S. P. and Jones, E. G., Developmental studies of thalamocortical and commissural connections in the rat somatic sensory cortex, J. comp. Neurol., 178 (1978) 187-208. 24 Wise, S. P., Murray, E. A. and Coulter, J. D., Somatotopic organization of corticospinal and corticotrigeminal neurons in the rat, Neuroscience, 4 (1979) 65-78. 25 Woodward, D. J., Bickett, D. and Chanda, R., Purkinje cell dendritic alterations after transient developmental injury of the external granular layer, Brain Res., 97 (1975) 195-214.
425
Elsevier/North-Holland Biomedical Press
Adjustment of connectivity in rat neocortex after prenatal destruction of precursor cells of layers II-IV
E. G. JONES, K. L. VALENTINO and J. W. FLESHMAN, Jr.* Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110 (U.S.A.)
(Accepted June 8th, 1981) Key words: cytotoxic drugs - - neocortical development - - microcephalic rats - - connections - -
morphogenesis
Prenatal administration of cytotoxic drugs during proliferation of precursor cells of neurons in granular and supragranular layers of the rat cerebral cortex prevents these layers from forming and causes malformations of some cells in the surviving layers. But it does not prevent the surviving layers from establishing normal efferent connections, nor does it prevent afferent fibers from colonizing the cortex and establishing a bilaminar pattern of synaptic connections, partly in an abnormal position. Thalamocortical and commissural fibers growing towards the sensorimotor cortex of the rat and cat reach the vicinity of their target early in development. They then accumulate in the developing white matter over a relatively protracted period of time before undergoing a renewed growth spurt which carries them into the overlying cortex10,21, 2a. While accumulating in the white matter the afferent fibers are traversed by recently generated neurons that are migrating to the superficial layers of the cortex. Presumably, many of these neurons will later receive synaptic connections from the invading afferent fibers. We wished to determine the fate of the two sets of afferents when deprived of any influences that they might normally receive from the growth through them of their ultimate target cells, and when, upon arrival in the cortex, they found a substantial proportion of their target cells absent. Pregnant Wistar rats, at 15, 16 or 17 days of gestation were injected intraperitoneally with 1-1.5 mg of 5-azacytidine or 6 mg of methylazoxymethanol acetate (MAM). These cytotoxic agents may be expected to kill all cells undergoing D N A synthesis in the neuroepithelium for a short time subsequent to the injectiong, 16. At the times injected this particularly includes the cells of origin of layers I I - I V of the neocortex. The cells of origin of layers V and VI have already undergone their final division and migrated towards their target layersL * Present address: Department of Surgery, The Jewish Hospital of St. Louis, St. Louis, MO 63110, U.S.A.
426 A p p r o x i m a t e l y 70 0/0 o f the pups born to injected mothers survived to m a t u r i t > The a p p e a r a n c e s and b e h a v i o r o f these rats were u n r e m a r k a b l e though the size., ,H~ their cerebral hemispheres were greatly reduced a n d their learning capacity, if tested a p p r o p r i a t e l y < la, should have been impaired. At 3-6 m o n t h s o f age the brains from some o f the a n i m a l s were prepared by Golgi m e t h o d s 14,1v. Other animals, under chloral h y d r a t e anesthesia, received small injections o f horseradish peroxidase or o f mixed [aH]proline and [aHJleucine in the p r e s u m p t i v e somatic sensory cortex, or stereotaxically in the v e n t r o p o s t e r i o r thalamic nucleus or p y r a m i d a l tract. Prior to m a k i n g the injections, restricted parts o f lhc cortex o r t h a l a m u s were m a p p e d with microelectrodes for units r e s p o n d i n g to naturally applied somesthetic stimuli. After 1-6 days, the animals were perfused with a p p r o p r i a t e a l d e h y d e fixatives and sections p r e p a r e d for enzyme histochemistry or autoradiography<7. The neocortex (Figs. I-3) is reduced in thickness and extent. A molecular layer is distinct but b e n e a t h it there are only two layers o f neurons. These (Fig. 1C) have all the histological a p p e a r a n c e s o f layers V a n d V1 o f n o r m a l animals, i.e. a h o m o g e n e o u s p o p u l a t i o n o f small to m e d i u m sized, pale, r o u n d cells in layer VI and a bistratified
Fig. 1. Figs. 1-3 are from animals in a single litter from a mother treated with MAM. A: distribution of autoradiographically labeled thalamocortical axons in layers V and VI of presumptive somatic sensory cortex of a MAM-treated rat after injection of [all]amino acids in ventroposterior thalamic nucleus; dark-field photomicrograph. M, molecular layer, V-VI, cellular layers. B: anterograde labeling of thalamocortical axons and retrograde labeling of corticothalamic cells in another treated animal injected with horseradish peroxidase in ventroposterior thalamic nucleus. C and D: adjacent sections showing in a Nissl stain (C) bilaminar nature of presumed somatic sensory cortex in MAMtreated rat, and distribution of major zone of thalamocortical fibers around apical parts of retrogradely labeled pyramidal tract neurons (D); injection of horseradish peroxidase in ventroposteriot thalamus gave thalamocortical fiber and corticothalamic cell labeling and second injection in pyramidal tract of same animal gave retrograde labeling of pyramidal tract neurons in layer V. Bars : 100/~m (A, B) 500 l*m (C, D).
427
Fig. 2. Golgi impregnations of abnormally inverted (B) and divaricated (D) pyramidal cells in layer V of MAM-treated rats. Comparable cells in A and C are retrogradely labeled by horseradish peroxidase injected into pyramidal tract. Large arrows indicate brain surface. Small arrows show in C a labeled dendrite and in D an intrinsic cortical neuron. Bars: 50/~m. layer V, with large pyramidal cells deeply and medium sized cells superficially. In some places, many of the medium-sized cells are arranged in clumps, but in no plane of section can the normal granule cell aggregations of the somatic sensory cortexm, 2~ be detected. The Golgi material shows normal appearing pyramidal cells in the remaining layers V and VI (Fig. 2B, D) and the presence of at least the larger class of non-pyramidal cells (the basket cells). Many of the pyramidal cells with somata in the superficial part of layer V have apical dendrites that divide quite close to the soma and, atypically, the branches divaricate extremely widely in the molecular layer (Fig. 2D). There are also many inverted pyramids (Fig. 2B). Other effects include the presence of abnormal islands of cells within the pyramidal layer of the hippocampus and in the white matter between the striatum and the neocortex (Fig. 3A,B). These may result from arrested migration. The corpus callosum is greatly reduced in thickness and rostrocaudal extent (Fig. 3A). All thalamic nuclei are clearly recognizable and their neuron density, though not yet quantified, appears substantially normal (Fig. 3B). Other parts of the brain, including the cerebellar granule cells whose precursors continue to proliferate after the time of the drug injection 1,1°, appear normal. Unit responses to natural somesthetic stimuli were easily recorded in a region corresponding to the somatic sensory cortex. Anterograde labeling of efferent fibers by autoradiography or with horseradish peroxidase following injections at this site revealed intact subcortical projections from this siteZ2,24. Labeled fibers could be traced as far as the spinal cord, with loci of labeled terminal ramifications detectable in striatum, thalamus, superior colliculus, pontine nuclei, dorsal column nuclei, and spinal gray matter. Injections of horseradish peroxidase in the thalamus or pyramidal
428 1 2 3
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D Pig. 3. A : aberrant cell islands (arrows) associated with the corpus callosum (CO) in a MAM-treated rat. We have shown that some of tie cells in tie islands send axons to the opposite cortex. I : anterolrade Iber and retrograde cellular labeling in central lateral (/L) and ventroposterior (VP) thalamic nuclei following injection of horseradish peroxidase in somatic sensory cortex of treated animal. LG> dorsal lateral geniculate nucleus ; arrow, aberrant cell island in hippocampus, lars" I l~m. C, D: partial somatotopic map of ventroposterior nucleus made from microelectrode recordings showing normal receptive field sequences of single units along 3 penetrations 1-3. For clarity, only every fourth or fifth receptive field is illustrated. tract revealed that the n o r m a l l a m i n a r origins of corticothalamic a n d pyramidal tract axons from cells respectively in layers VI a n d V22, 24 are preserved (Fig. 1B, D). M a n y of the labeled pyramidal tract n e u r o n s were the atypical forms in the superficial part of layer V (Fig. 2A, C). Anterograde fiber labeling could clearly be seen in the t h i n n e d corpus callosum, b u t relatively few retrogradely labeled commissural cells were seen in the contralateral cortex. These were largely confined to layer V, t h o u g h some were noted in the a b e r r a n t islands of cells in the subcortical white matter. The small n u m b e r s of callosal projection n e u r o n s a n d the reduction in size of the corpus
429 callosum probably reflects destruction of the large proportion of callosal parent cells that normally reside in layer 11121. Retrograde labeling of afferent cells was particularly striking in the ventral and intralaminar nuclei of the thalamus after cortical injections of horseradish peroxidase (Fig. 3B). Anterograde labeling of the cortical terminations of the thalamic relay cells occurred in a dense band extending from the deep border of the molecular layer to just above the somata of the deeper stratum of large cells in layer V (Fig. 1A, B, D). It seemed thinner than in the normal but no less dense. Fingers of label commonly extended from it into the molecular layer (Fig. 1D). There was a second band of labeling in a normal position at the junction of layers V and VIa, 23. The superficial stratum of thalamocortical fiber terminations is placed closer to the somata of the corticopyramidal cells than normala, 23 (Fig. 1D). During our mapping of the thalamus to identify appropriate sites for injections, we found that the receptive fields of the neurons recorded from and the representation of body somatotopy were apparently little changed from normal (Fig. 3C, D). Units responsive to stimulation of the body surface were not difficult to find and the sizes of their receptive fields were normal. The parts of the body represented at different dorsoventral and mediolateral levels, as shown by the sequences of receptive fields encountered, wele also in a normal sequence 5,1s. Our more limited observations in the cortex suggested a similar preservation of somatotopy in the presumed first somatic sensory area. Our results show that the cytotoxic effect is short-lived and local. Remaining parts of the cerebral cortex and other parts of the brain directly or indirectly connected with the cortex, even if formed by later proliferating cells, appear substantially unaltered. The destruction of a considerable number of cortical target cells before their migration to the cortex through the waiting afferent fibers does not suppress any signal that might normally influence the fibers' second growth spurt into the cortex. Nor does the absence of the main target layers (III and IV) prevent colonization of the cortex by a significant number of thalamic afferents, many of which terminate on the intact layers V and/or VI cells. The development of a bilaminar distribution of thalamocortical axon terminals in a neocortex consisting of infragranular layers only may imply that synaptic connections have been made on inappropriate cells or in greater numbers than normal on a remaining set of cells. Somatotopy in the projection pattern is preserved. If this is normally imposed on the cortex from the thalamus ~3, it may reflect the preservation of the normally ordered lemniscal input to the thalamus, demonstrable in our microelectrode recordings. Cells that are migrating to, or that have already reached the cortex prior to the perturbation, form only appropriate laminae and only make connections appropriate to those laminae. One major efferent system, the callosal, has been substantially reduced by destruction of many of its parent cells normally resident in layer III. The few inappropriately placed callosally-projecting cells appear to be those whose migration has been interfered with. This seems to indicate that the establishment of efferent
430 c o n n e c t i o n s by layer V and VI cells is i n d e p e n d e n t of interactions with the granular' a n d s u p r a g r a n u l a r layers. The only overt change in the structure o f the r e m a i n i n g cortical layers occurs i~-~ the superficial p a r t o f layer V. In the preserved layers, therefore, basic p y r a m i d a l cell form, the d e v e l o p m e n t o f which n o r m a l l y occurs in parallel with the colonization of the cortex by afferent fibers ~9, seems to depend, like the establishment o f efferem connections, to a large extent on factors intrinsic to the cell. But it is possible that local factors o f an u n d e t e r m i n e d n a t u r e o p e r a t i n g at i n t e r l a m i n a r b o r d e r s may participate in the finer m o d e l l i n g o f the p y r a m i d a l cells. This idea comes from the observations t h a t p y r a m i d a l cells with s o m a t a in the vicinity o f the n o r m a l l y a p p e a r i n g layer V VI b o r d e r are substantially n o r m a l in a p p e a r a n c e , while those in the most superficial part o f the r e m a i n i n g cortex, where layer V meets n o t layer IV but a m o l e c u l a r layer, arc very atypical a n d m a y even be inverted. Similar concepts regarding the relative roles oF intrinsic and local influences have been expressed by others, particularly in the cerebelluml,a,11-13,25. The presence o f inverted forms o f p y r a m i d a l cell and o f forms with a b n o r m a l l y extensive dendritic trees m a y imply a response to an absence or a reduced c o n c e n t r a t i o n o f potential synaptic inputs in the anticipated d o m a i n o f the developing cells' apical dendrites. S u p p o r t e d b y N I H G r a n t s N S 15070 a n d T 3 2 - N S 0757. W e t h a n k Ms. Bertha M c C l u r e a n d Mr. R o n a l d Steiner for technical assistance. and Drs. R. P. Bunge, N. W. Daw, V. H a m b u r g e r , W. M. L a n d a u , and A. L. P e a r l m a n for helpful criticism.
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