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Segregation of cortical and trigeminal afferents to the ventrobasal complex of the neonatal rat
Brain Research, 161 (1979) 527-532 © Elsevier/North-HollandBiomedicalPress
527
Segregation of cortical and trigeminal afferents to the ventrobasal complex of the neonatal rat
REBECCA M. AKERS and HERBERT P. KILLACKEY* Department of Psychobiology, University of California, Irvine, Calif. 92717 (U.S.A.)
(Accepted October 12th, 1978)
The somatosensory systems of rats, mice, and certain other rodents are specialized for processing discrete information from individual receptors of the whiskerpad. In these species, the mystacial vibrissae and sinus hairs are mapped in a topographical, non-overlapping fashion in the trigeminal ganglion ~° and nucleus 7,12, ventrobasal complex 14, and somatosensory cortex 15,16. The discrete functional organization of the trigeminal pathways in rodents has clear morphological correlates; in the young rat, afferents to the brain stem and thalamic somatosensory relay nuclei are organized in discontinuous clusters which are related to individual peripheral receptors e-4. The somatosensory cortex is comprised in part of cellular aggregates, or 'barrels', each of which constitutes the central representation of a single mystacial vibrissa or sinus hair and receives a discrete bundle of thalamocortical afferents5,6,19. The clustering of ascending somatosensory projections in discontinuous bundles clearly provides one mechanism by which the discreteness of responses to individual peripheral receptors may be maintained within the trigeminal pathways, but it is unclear what other factors may contribute to this specialized functional organization. Electrophysiological studies in a number of species have demonstrated that cortical afferents can exert potent effects on synaptic transmission within thalamic and brain stem relay nuclei 1,s, 11. We were interested, therefore, in the possibility that the afferents from the 'barrel field' cortex to the ventrobasal complex might also exhibit morphological specializations. Previous studies of corticothalamic projections in the adult rat have revealed no evidence of such specializationg, is, but a recent examination of the trigeminal pathways in rats has demonstrated that elaborate patterns of afferent fiber termination observed in the neonate may be obscured during postnatal development 2. Accordingly, in the present study we examined the organization of corticothalamic projections in rats during the first postnatal week. Injections of [3H]leucine, proline, or a combination of both were made in the parietal cortex of rat pups under cryogenic anesthesia. Injections ranged from 0.05-0.1 /~1 (3.3-6.6 #Ci) and were administered via a glass micropipette affixed to a 1 /~1 * To whom correspondence should be addressed.
528 Hamilton syringe. After survival times of 24 or 48 h, each animal was perfused intracardially with 0.9)~ saline followed by 10 % formal saline. Brains were embedded in paraffin, sectioned coronally at 10 #m, and processed for autoradiography with Kodak NTB-2 emulsion. Following exposures of 3-6 weeks, slides were developed in Kodak Dektol developer, fixed, and stained through the emulsion with 0.05°(, toluidine blue. Counts of grain distribution were made at 1000 >~ magnification under dark-field illumination, and background grain densities were determined by counts in the ipsilateral paleocortex. The resulting distribution of labeling in the ventrobasal complex is far from uniform. In Fig 1B, a dark-field photomicrograph of the ventrobasal complex of a 4-
A
Fig. 1. A: schematicrepresentation of the external (ext) and arcuate (arc) subdivisionsof the ventrobasal complex. Scale, 300/~m. B: dark-field photomicrograph of the ventrobasal complex of a 4-day-old rat followinga large injection of tritiated amino acids into the ipsilateral parietal cortex. C: light-field photomicrograph of the same section illustrated in B. D : photomicrograph of the ventrobasal complex of a 5-day-old rat processed for the localization of succinic dehydrogenase activity.
529
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Fig. 2. A: dark-field photomicrograph of transported label in the ventrobasal complex of a 5-day-old rat subsequent to a cortical injection of tritiated amino acids. The central bands of transported label are represented schematically in B. Box indicates area in which counts of grain density were made, as illustrated in C.
530 day-old rat pup reveals an elaborately patterned corticothalamic projection. Curvilinear rows of lightly labeled, block-like patches alternate with narrow bands of high grain density. Although labeling within both regions is clearly above background levels, the grain density within the narrow bands is 2 to 3 times greater than that within the intervening, lightly labeled patches (Fig. 2C). The densely labeled bands separating adjacent rows of lightly labeled patches are wider and more distinct than bands separating patches within a row. This segmentation of the corticothalamic projection is evident in both the arcuate and external subnuclei of the ventrobasal complex, and a strip of dense label forms a clear boundary between the two subdivisions. Within the arcuate nucleus, rows of lightly labeled patches are wide and sharply curved at the rostral tip, while more caudally, the rows are narrower, straighter, and oriented along the ventromedial-dorsolateral axis of the nucleus. Light-field photomicrographs (Fig. 1C) reveal that this pattern of corticothalamic fiber distribution is congruent with cytoarchitectonic specializations within the ventrobasal complex. Hollow clusters of cells are arranged in wide, curving rows separated by narrower, cell-sparse regions. Within a single row of clusters, the arrangement of cell somata is variable. Some clusters are completely surrounded by a zone of low cell density, while the cell-dense walls of other clusters appear to be directly apposed to one another. Bands of transported label resulting from cortical injections are localized within the cell-sparse regions which separate adjacent rows of cell clusters. Much narrower bands lie adjacent to the cell-dense walls which define individual clusters within a row, and in some instances, accumulations of silver grains can be seen extending over and outlining individual cell somata which lie within the cluster walls (Fig. 2A). The distribution of transported label in the ventrobasal complex also exhibits a clear relationship to the pattern of trigeminothalamic afferents. Fig. 1D illustrates the pattern of staining in the ventrobasal complex of a 5-day-old rat processed for the localization of succinic dehydrogenase (SD H) activity. Previous work in our laboratory has provided evidence that this technique reveals the distribution of the trigeminal afferents 2,4. Discrete patches of intense SDH activity are visible in both the arcuate and external subnuclei, and their arrangement in wide curvilinear rows forms a negative image of the pattern of transported corticothalamic label. Comparison of SDH and autoradiographic material suggests that the corticothalamic axons are preferentially localized in the narrow zones which separate adjacent patches of segmented trigeminal afferents. The ventrobasal complex of the neonatal rat thus exhibits an elaborate pattern of cellular organization and afferent distribution which is not readily apparent in the adult. It is interesting in this respect that Welt and Steindler 17 have reported a similar form of corticothalamic fiber distribution in more mature (30-day-old) reeler mutant mice. These authors proposed that the corticothalamic afferents project to the celldense 'lattices' of the thalamic 'barreloids', while our observations suggest that these fibers are preferentially localized in cell-sparse regions. Comparison of these data is complicated by the fact that the cytoarchitectonic specializations seen within the thalamus of the neonatal rat are not comparable to the 'barreloids' of the mouse
531
ventrobasal complexla. In the rat, clearly defined bands of cell sparsity separate adjacent rows of cell clusters, while the 'barreloids' consist of cell-sparse areas embedded in a matrix of high cell density. It remains to be determined whether the corticothalamic projections are truly distributed to different cellular elements in the rat and mouse, but both observations suggest that an individual cluster of thalamic neurons is surrounded by an equally discrete band of corticothalamic fibers. In addition, in the rat each cluster of thalamic neurons appears to receive input from a bundle of trigeminothalamic afferents preferentially distributed within the acellular core ~. The functional significance of this morphological specialization and the reasons for its disappearance in the adult rat are at present unclear. One possibility is that the initial segregation of cortical and trigeminal afferents to the ventrobasal complex is an important step in the formation of reciprocal connections between the cortex and thalamus. The development of a topographically organized map of the peripheral receptor surface requires a matching between a discrete bundle of trigeminal afferents, their appropriate target cells in the thalamus, and afferents derived from the corresponding portion of the cortical receptor map. The precise patterning of thalamic somata, trigeminal afferents, and corticothalamic projections in the neonate may set the stage for the establishment of such topographically organized neural connections. During subsequent maturation, terminal proliferation, synaptogenesis, and dendritic growth may obscure the morphological relationships seen in the neonate, but the functional connections which are established may still reflect this initial discreteness. Saporta and Kruger 10 have demonstrated that thalamic neurons projecting to a given locus in the somatosensory cortex of the rat are arranged in curvilinear arrays within the ventrobasal complex. The aggregates of thalamic neurons, corticothalamic axons, and trigeminothalamic afferents seen in the neonate may be the precursors of these mature, functional units. This research was s u p p o r t e d by N S F G r a n t N o . 42194.
1 Anderson, P., Junge, K. and Sveen, V., Cortico-thalamic facilitation of somatosensory impulses, Nature (Lond.), 214 (1967) 1011-1012. 2 Belford, G. R. and Killackey, H. P., Vibrissae representation in subcortical trigeminal centers of the neonatal rat, J. comp. Neurol., (1978) in press. 3 Belford, G. R., Development of peripherally-related segmentation in the ventrobasal complex of the rat, Anat. Rec., 190 (1978) 336. 4 Belford, G. R. and Killackey, H. P., Anatomical correlates of the forelimb in the ventrobasal complex and cuneate nucleus of the neonatal rat, Brain Research, (1978) in press. 5 Killackey, H. P., Anatomical evidence for cortical subdivisions based on vertically discrete thalamic projections from the ventral posterior nucleus to cortical barrels in the rat, Brain Research, 51 (1973) 326-331. 6 Killackey, H. P. and Leshin, S., The organization of specific thalamocortical projections to the posteromedial barrel subfield of the rat somatic sensory cortex, Brain Research, 86 (1975) 469-472. 7 Nord, S. G., Receptor field characteristics of single cells in the rat spinal trigeminal complex, Exp. Neurol., 21 (1968) 236-243. 8 Ogden, T. E., Cortical control of thalamic somatosensory relay nuclei, Electroenceph. clin. NeurophysioL, 12 (1960) 621-634. 9 Price, T. R. and Webster, K. E., The corticothalamic projection from the primary somatosensory cortex of the rat, Brain Research, 44 (1972) 63~640.
532 10 Saporta, S. and Kruger, L., The organization ofthalamocortical relay neurons in the rat ventrobasal complex studied by retrograde transport of horseradish peroxidase, J. comp. Neurol., 174 (1977) 187-208. l l Shimazu, H., Yanagisawa, N. and Garoutte, B., Cortico-pyramidal influences on t halamic somatosensory transmission in the cat, Jap. J. Physiol., 15 (1965) 101-124. 12 Shipley, M. T., Response characteristics of single units in the rat's trigeminal nuclei to vibrissa displacement, J. Neurophysiol., 37 (1974) 73-90. 13 Van der Lops, H., Barreloids in mouse somatosensory thalamus, Neurosci. Lett., 2 (1976) 1-6. 14 Waite, P. M.E.,Theresponsesofcellsintheratthalamustomechanicalmovementsofthewhiskers, J. Physiol. (Lond.), 228 (1973) 541-561. 15 Welker, C., Microelectrode delineation of fine grain somatotopic organization of Sml cerebral neocortex in albino rat, Brain Research, (1971) 259-275. 16 Welker, C., Receptive fields of barrels in the somatosensory neocortex of the rat, J. comp. Neurol., 166 (1976) 173-190. 17 Welt, C. and Steindler, D. A., Somatosensory cortical barrels and thalamic barreloids in reeler mutant mice, Neuroscience, 2 (1977) 755-765. 18 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. 19 Woolsey, T. A. and Van der Lops, H., The structural organization of layer IV in the somatosensory region (SI) of mouse cerebral cortex. The description of a cortical field composed of discrete cytoarchitectonic units, Brain Research, 17 (1970) 205-242. 20 Zucker, E. and Welker, W. I., Coding of somatic sensory input by vibrissae neurons in the rat's trigeminal ganglion, Brain Research, 12 (1969) 138-156.
527
Segregation of cortical and trigeminal afferents to the ventrobasal complex of the neonatal rat
REBECCA M. AKERS and HERBERT P. KILLACKEY* Department of Psychobiology, University of California, Irvine, Calif. 92717 (U.S.A.)
(Accepted October 12th, 1978)
The somatosensory systems of rats, mice, and certain other rodents are specialized for processing discrete information from individual receptors of the whiskerpad. In these species, the mystacial vibrissae and sinus hairs are mapped in a topographical, non-overlapping fashion in the trigeminal ganglion ~° and nucleus 7,12, ventrobasal complex 14, and somatosensory cortex 15,16. The discrete functional organization of the trigeminal pathways in rodents has clear morphological correlates; in the young rat, afferents to the brain stem and thalamic somatosensory relay nuclei are organized in discontinuous clusters which are related to individual peripheral receptors e-4. The somatosensory cortex is comprised in part of cellular aggregates, or 'barrels', each of which constitutes the central representation of a single mystacial vibrissa or sinus hair and receives a discrete bundle of thalamocortical afferents5,6,19. The clustering of ascending somatosensory projections in discontinuous bundles clearly provides one mechanism by which the discreteness of responses to individual peripheral receptors may be maintained within the trigeminal pathways, but it is unclear what other factors may contribute to this specialized functional organization. Electrophysiological studies in a number of species have demonstrated that cortical afferents can exert potent effects on synaptic transmission within thalamic and brain stem relay nuclei 1,s, 11. We were interested, therefore, in the possibility that the afferents from the 'barrel field' cortex to the ventrobasal complex might also exhibit morphological specializations. Previous studies of corticothalamic projections in the adult rat have revealed no evidence of such specializationg, is, but a recent examination of the trigeminal pathways in rats has demonstrated that elaborate patterns of afferent fiber termination observed in the neonate may be obscured during postnatal development 2. Accordingly, in the present study we examined the organization of corticothalamic projections in rats during the first postnatal week. Injections of [3H]leucine, proline, or a combination of both were made in the parietal cortex of rat pups under cryogenic anesthesia. Injections ranged from 0.05-0.1 /~1 (3.3-6.6 #Ci) and were administered via a glass micropipette affixed to a 1 /~1 * To whom correspondence should be addressed.
528 Hamilton syringe. After survival times of 24 or 48 h, each animal was perfused intracardially with 0.9)~ saline followed by 10 % formal saline. Brains were embedded in paraffin, sectioned coronally at 10 #m, and processed for autoradiography with Kodak NTB-2 emulsion. Following exposures of 3-6 weeks, slides were developed in Kodak Dektol developer, fixed, and stained through the emulsion with 0.05°(, toluidine blue. Counts of grain distribution were made at 1000 >~ magnification under dark-field illumination, and background grain densities were determined by counts in the ipsilateral paleocortex. The resulting distribution of labeling in the ventrobasal complex is far from uniform. In Fig 1B, a dark-field photomicrograph of the ventrobasal complex of a 4-
A
Fig. 1. A: schematicrepresentation of the external (ext) and arcuate (arc) subdivisionsof the ventrobasal complex. Scale, 300/~m. B: dark-field photomicrograph of the ventrobasal complex of a 4-day-old rat followinga large injection of tritiated amino acids into the ipsilateral parietal cortex. C: light-field photomicrograph of the same section illustrated in B. D : photomicrograph of the ventrobasal complex of a 5-day-old rat processed for the localization of succinic dehydrogenase activity.
529
ii iiii ,,ii il !iiiii~i iiiiiiliii~i~iii~ iii¸i!i~iI¸~ii i:~i~ ~!~~~iiiii!iiii!il iii~i!~il ~i! !?!~:ii~!~(i!i i !~i~ii iiiiiiiiii~i!iiiil
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Fig. 2. A: dark-field photomicrograph of transported label in the ventrobasal complex of a 5-day-old rat subsequent to a cortical injection of tritiated amino acids. The central bands of transported label are represented schematically in B. Box indicates area in which counts of grain density were made, as illustrated in C.
530 day-old rat pup reveals an elaborately patterned corticothalamic projection. Curvilinear rows of lightly labeled, block-like patches alternate with narrow bands of high grain density. Although labeling within both regions is clearly above background levels, the grain density within the narrow bands is 2 to 3 times greater than that within the intervening, lightly labeled patches (Fig. 2C). The densely labeled bands separating adjacent rows of lightly labeled patches are wider and more distinct than bands separating patches within a row. This segmentation of the corticothalamic projection is evident in both the arcuate and external subnuclei of the ventrobasal complex, and a strip of dense label forms a clear boundary between the two subdivisions. Within the arcuate nucleus, rows of lightly labeled patches are wide and sharply curved at the rostral tip, while more caudally, the rows are narrower, straighter, and oriented along the ventromedial-dorsolateral axis of the nucleus. Light-field photomicrographs (Fig. 1C) reveal that this pattern of corticothalamic fiber distribution is congruent with cytoarchitectonic specializations within the ventrobasal complex. Hollow clusters of cells are arranged in wide, curving rows separated by narrower, cell-sparse regions. Within a single row of clusters, the arrangement of cell somata is variable. Some clusters are completely surrounded by a zone of low cell density, while the cell-dense walls of other clusters appear to be directly apposed to one another. Bands of transported label resulting from cortical injections are localized within the cell-sparse regions which separate adjacent rows of cell clusters. Much narrower bands lie adjacent to the cell-dense walls which define individual clusters within a row, and in some instances, accumulations of silver grains can be seen extending over and outlining individual cell somata which lie within the cluster walls (Fig. 2A). The distribution of transported label in the ventrobasal complex also exhibits a clear relationship to the pattern of trigeminothalamic afferents. Fig. 1D illustrates the pattern of staining in the ventrobasal complex of a 5-day-old rat processed for the localization of succinic dehydrogenase (SD H) activity. Previous work in our laboratory has provided evidence that this technique reveals the distribution of the trigeminal afferents 2,4. Discrete patches of intense SDH activity are visible in both the arcuate and external subnuclei, and their arrangement in wide curvilinear rows forms a negative image of the pattern of transported corticothalamic label. Comparison of SDH and autoradiographic material suggests that the corticothalamic axons are preferentially localized in the narrow zones which separate adjacent patches of segmented trigeminal afferents. The ventrobasal complex of the neonatal rat thus exhibits an elaborate pattern of cellular organization and afferent distribution which is not readily apparent in the adult. It is interesting in this respect that Welt and Steindler 17 have reported a similar form of corticothalamic fiber distribution in more mature (30-day-old) reeler mutant mice. These authors proposed that the corticothalamic afferents project to the celldense 'lattices' of the thalamic 'barreloids', while our observations suggest that these fibers are preferentially localized in cell-sparse regions. Comparison of these data is complicated by the fact that the cytoarchitectonic specializations seen within the thalamus of the neonatal rat are not comparable to the 'barreloids' of the mouse
531
ventrobasal complexla. In the rat, clearly defined bands of cell sparsity separate adjacent rows of cell clusters, while the 'barreloids' consist of cell-sparse areas embedded in a matrix of high cell density. It remains to be determined whether the corticothalamic projections are truly distributed to different cellular elements in the rat and mouse, but both observations suggest that an individual cluster of thalamic neurons is surrounded by an equally discrete band of corticothalamic fibers. In addition, in the rat each cluster of thalamic neurons appears to receive input from a bundle of trigeminothalamic afferents preferentially distributed within the acellular core ~. The functional significance of this morphological specialization and the reasons for its disappearance in the adult rat are at present unclear. One possibility is that the initial segregation of cortical and trigeminal afferents to the ventrobasal complex is an important step in the formation of reciprocal connections between the cortex and thalamus. The development of a topographically organized map of the peripheral receptor surface requires a matching between a discrete bundle of trigeminal afferents, their appropriate target cells in the thalamus, and afferents derived from the corresponding portion of the cortical receptor map. The precise patterning of thalamic somata, trigeminal afferents, and corticothalamic projections in the neonate may set the stage for the establishment of such topographically organized neural connections. During subsequent maturation, terminal proliferation, synaptogenesis, and dendritic growth may obscure the morphological relationships seen in the neonate, but the functional connections which are established may still reflect this initial discreteness. Saporta and Kruger 10 have demonstrated that thalamic neurons projecting to a given locus in the somatosensory cortex of the rat are arranged in curvilinear arrays within the ventrobasal complex. The aggregates of thalamic neurons, corticothalamic axons, and trigeminothalamic afferents seen in the neonate may be the precursors of these mature, functional units. This research was s u p p o r t e d by N S F G r a n t N o . 42194.
1 Anderson, P., Junge, K. and Sveen, V., Cortico-thalamic facilitation of somatosensory impulses, Nature (Lond.), 214 (1967) 1011-1012. 2 Belford, G. R. and Killackey, H. P., Vibrissae representation in subcortical trigeminal centers of the neonatal rat, J. comp. Neurol., (1978) in press. 3 Belford, G. R., Development of peripherally-related segmentation in the ventrobasal complex of the rat, Anat. Rec., 190 (1978) 336. 4 Belford, G. R. and Killackey, H. P., Anatomical correlates of the forelimb in the ventrobasal complex and cuneate nucleus of the neonatal rat, Brain Research, (1978) in press. 5 Killackey, H. P., Anatomical evidence for cortical subdivisions based on vertically discrete thalamic projections from the ventral posterior nucleus to cortical barrels in the rat, Brain Research, 51 (1973) 326-331. 6 Killackey, H. P. and Leshin, S., The organization of specific thalamocortical projections to the posteromedial barrel subfield of the rat somatic sensory cortex, Brain Research, 86 (1975) 469-472. 7 Nord, S. G., Receptor field characteristics of single cells in the rat spinal trigeminal complex, Exp. Neurol., 21 (1968) 236-243. 8 Ogden, T. E., Cortical control of thalamic somatosensory relay nuclei, Electroenceph. clin. NeurophysioL, 12 (1960) 621-634. 9 Price, T. R. and Webster, K. E., The corticothalamic projection from the primary somatosensory cortex of the rat, Brain Research, 44 (1972) 63~640.
532 10 Saporta, S. and Kruger, L., The organization ofthalamocortical relay neurons in the rat ventrobasal complex studied by retrograde transport of horseradish peroxidase, J. comp. Neurol., 174 (1977) 187-208. l l Shimazu, H., Yanagisawa, N. and Garoutte, B., Cortico-pyramidal influences on t halamic somatosensory transmission in the cat, Jap. J. Physiol., 15 (1965) 101-124. 12 Shipley, M. T., Response characteristics of single units in the rat's trigeminal nuclei to vibrissa displacement, J. Neurophysiol., 37 (1974) 73-90. 13 Van der Lops, H., Barreloids in mouse somatosensory thalamus, Neurosci. Lett., 2 (1976) 1-6. 14 Waite, P. M.E.,Theresponsesofcellsintheratthalamustomechanicalmovementsofthewhiskers, J. Physiol. (Lond.), 228 (1973) 541-561. 15 Welker, C., Microelectrode delineation of fine grain somatotopic organization of Sml cerebral neocortex in albino rat, Brain Research, (1971) 259-275. 16 Welker, C., Receptive fields of barrels in the somatosensory neocortex of the rat, J. comp. Neurol., 166 (1976) 173-190. 17 Welt, C. and Steindler, D. A., Somatosensory cortical barrels and thalamic barreloids in reeler mutant mice, Neuroscience, 2 (1977) 755-765. 18 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. 19 Woolsey, T. A. and Van der Lops, H., The structural organization of layer IV in the somatosensory region (SI) of mouse cerebral cortex. The description of a cortical field composed of discrete cytoarchitectonic units, Brain Research, 17 (1970) 205-242. 20 Zucker, E. and Welker, W. I., Coding of somatic sensory input by vibrissae neurons in the rat's trigeminal ganglion, Brain Research, 12 (1969) 138-156.