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Trigemino-cerebellar projections in normal and reeler mutant mice
Neuroscience L-'ters, 6 (1977) 293--300 © Elsevier/North-Holland Scientific Publishers Ltd.
293
',PRIGEMINO-CEREBELLAR PROJECTIONS IN "NORMAL A N D R E E L E R M U T A N T MICE*
DENNIS A. STEINDLER** Department of Anatomy, School of Medicine, University of California, San Franscisco, San Franscisco, Calif. 94143 (U.S.A.) (Received M~Ly9th, 1977) (Revised version received September 6th, 1977) (Accepted ~eptember 10th, 1977)
SUMMARY
Retrogradely labeled neurons were found in the spinal trigemial (Spin V.) and principal sensory nucleus (Prin V.) and other regions following injections of horseraclish peroxidase (HRP) into normal mouse cerebellar hemisphere and vermis. HI~P injections into particular regions of the malformed cerebellum in the reeler mouse also resulted in retrograde labeling within regions of the trigeminal complex that are topographical!y similar to those seen in the normal. In association with a recently described cerebellar tactile representation of the head [17], the findings suggest that the neurons of origin of this somatosensory projection reside in portions of the trigeminal complex and possibly in other brainstem nuclei, in both normal mice and reeler mutants. These data also contribute to an understanding of afferent organization and the maintenance of appropriate patterns of connectivity within the malformed reeler cerebc~lum.
Evidence for trigemino~:erebellar (TC) projections has been presented in avian and mammalian species [14,27,9,16,8,26,3]. They were believed to originate from mesencephalic (Mes. V) [26,27] and sensory root fibers [26,27] and the principal sensory nucleus (Prin. V) [26,27] of the trigeminus in birds. A TC tract has also been reported in opossum [8] and in fetal human brains [14]. Whitlock [26] observed Marchi positive degeneration within the avian vermis after lesions of the Prin. V. He also saw chromatolytic changes within neurons of this nucleus following vermian lesions. Lesions of the interpolaris (interpol.) and oralis divisions of the spinal trigeminal nucleus (Spin. V) in the cat [3] produced preterminal degeneration within portions of the vermis. * Part of these results were presented at the 90th Session of the American Association of Anatomists (Anat. Rec., 187 (1977) 722). ** Present Address: C/o Dr. K.-M. Gottschaldt, Max-Planck-Institut fur Biophysikalische Chemie, Karl-Friedrich-Bonhoeffer'Institut, Postfach 968, D-3400 Gottingen, G.F R.
294
Recent contributions from anterograde degeneration studies suggest that there may be TC pathways in cat [ 3,22 ], but fail to elucidate major sites of origin and areas of termination of this system. The findings that appropriate somatosensory connections are achieved in the cytoarchitecturally abnormal forebrain o£ reeler mutant mice [ 1,2,20,21, 24,25] prompted the present study of somatosensory relationships in the normal and reeler mid- and hindbrain. A reassessment of TC pathways was undertaken using the retrograde horseradish peroxidase (HRP) transport technique [ 10 ]. Injections of 0.05--0.2 ~1 HRP* (Sigma, Type VI) were placed, using a Hamilton microsyringe, in various cerebellar regions of three normal and four homozygous reeler mice, (22--30 days old) bred in our laboratory from known heterozygotes**. After 16--24 h they were sacrificed by intracardiac perfusion of phosphate buffered (pH 7.2) 4% paraformaldehyde at 4 ° C over a period of 15 min. The brains were remove~, postfixed for 4 h in cold perfusate and immersed in cold 30% glucose in C.1 m phosphate buffer overnight. Frozen serial sections 30 and 90 ~m thick were cut in an oblique frontal plane and were reacted with diaminobenzidine and hydrogen peroxide [ 7,11]. Alternate sections were counterstained with cresyl violet. Sections were examined by bright- and dark-field microscopy. For cytoarchitectonics, ref. 18 was used.
* Some cases were combined horseradish peroxidase, tritiated amino acid injections (HAPTAA) [4 ] for studies of reciprocal afferent (HRP) and efferent (autoradiography) cerebellar projections. These findings will be discussed in a later report (Steindler, in preparation). ** C57BL/6J × C3H/HeJ hybrids originally obtained from The Jackson Laboratory, Bar Harbo-, Maine.
Fig. 1. Normal 30-day-old mouse. (a) Frontal section through cerebellum; double HRP injection. One injection involved crura I and II, Iobuli ansiformis (cr 1 and cr 2) and portions of neighbouring folia in the left hemisphere. Another, smaller injection in the vermis (only needle tract seen at this tevel, arrow) involved portions of culmen (cu) and declive. Cresylviolet ,~ounterstain. Scale = 1 ram. pa, paramedian lobule; py, pyramis; uv, uvula; no, noduluts. (b) Drawing of 90 ~m frontal section through medulla following injection shown in (a). HRP-labeled neurons (dots) are in inferior olivary complex (inf. oliv.), reticular and raphe groups, and in interpolaris division of spinal trigeminal nucleus (interpol. v). XII, hypoglossal nucleus; iat. cu., lateral cuneate nucleus: (c) A cluster of HRP labeled neurons in the ipsilateral interpolaris spinal trigeminal nucleus (square in Fig. 1 (b)). Dark-field photomicrograph. Scale = 40 urn. (d) Drawing of 90 ~m frontal section through midbrain following injection shown in (a). Labeled neurons are seen in nucleus reticularis tegmenti pontis (rtp) and in principal se~sory trigeminal nucleus (prin. V). sup. collic., superior colliculus; mid. ped., middle cerebellar peduncle ; v. co., ventr~! cochlear nucleus; sup. oliv., superior olivary nucleus, trap., nucleus of trapezoid body; sup. ped., superior cerebellar peduncle; d. raphe, dorsal raphe; pag, periaqueductal gray; v. tegment., ventral tegmental nuc~eu~ of Von Gudden. (e) HRP-labeled neurons in the dorsal area of principal sensory V. nucleus (circle in (d)). Dark-field photomicrograph. Scale = 40 urn. (f) Dark-field photo-" micrograph of two HRP-hbeled neurons in ventral portion of principal sensory V. nucleus (squ~re in (d)). Scale = 40 pm.
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in the normal mouse, unilateral injections in the left hemisphere and vermis resulted in retrograde labeled neurons in several brainstem areas. Fig. 1 depicts a case in which two injections were made. The injections included portions of the vermis (culmen, declive and pyramis lobules) and of lobulus simplex, crura I, II and paramedian lobuie of the hemisphere of the left side. There was no spread of HRP tc the right side. Labeled cells were found in medullary reticular and raphe areas, pontine nuc!ei, prepositus hypoglossi nucleus, the inferior oiivary complex, Prin. V., interpol, and oralis divisions of the Spin. V, and in the nucleus reticularis tegmenti pontis. HitP labeled axons were seen in the restifonn body and entering the oralis and interpol, regions associated with clusters of retrogradely labeled cells. Similarly. labeled axons were seen in the middle cerebellar peduncle near labeled cells of Prin. V. TC neurGns give rise to a bilateral projection with two to three times the number of ipsilateral to contralateral cells. The majority of labeled neurons in the oralis division of Spin. V were located dorsally; labeled cells of the Prin. V and interpol, divisions avpear to be in dorsal and ventral regions of these nuclei. No labeled cells were found within the Mes. V nucleus. After injections of HRP in presumptive hemispheric regions of the undersized, afoliate and cytoarchitecturally abnormal cerebellum of the re,der mouse (Fig. 2), the dist_~ibutions of retrograde labeled brainstem neurons were comparable to the normal. Injections in non-hemispheric regions of the m u t a n t and normal cerebellum (e.g. the midline anterior and posterior spinocerebellar recipient zones) resulted in much less retrograde labeling in the interpol., oralis and Prin. V nucleus, particularly in the latter. Fig. 2 illustrates some of the results after two injections confined to the presumed lateral hemispheric l:egion of a reeler mouse. Results from the two cases depicted in Figs. I and 2, as well ~ other cases in this study in which some injections were confined to particular hemispheric (for example, crura I, II) or vermian (for example, centralis) lobules, suggest that" (1) In both normal and reeler mutant mice, bilateral TC projections from the interpol, and oralis Spin. V nuclei ascend into the cerebellum via the resti-
Fig. 2.30-day-old reeler mouse. (a) Frontal section through cerebellum; double injection case. Both HRP-TAA injections were confined to the presumed lateral hemispheric area and underlying deep nuclei. Cresyl-violet counterstain. Scale ffi 1 mm. (b) Drawing of 90 ~m frontal section through the medulla following injection shown in (a). HRP-lab~;led neurons are in inferior olivai~y complex, reticular formation, interpolaris division of spinal trigeminal nucleus and in prepositus hypoglossi (possibly nucleus intercalatus) (just above hypoglossal nucleus, XII). For other symbols, see Fig. l(b). (c) Bright-field photomicrograph of HRP !abeled neuron in the interpolaris spinal trigeminal nucleus (circle in (b)). Arrow indicates the unstained nucleus in this non-counterstained neuron. Scale = 10 ~m. (d) Drawing of 90 ~m frontal section through midbrain following injection shown in (a). HRP-labeled neurons are in principal sensory trigeminal nucleus (Prin. V). r.f., reticular formation; v. len'l, lat., ventral nucleus of the lateral lemniscus. For other symbols, see Fig. t(d). (e) Darkfield photomicrograph of two HRP labeled neurons in principal sensory V. nv cleus (circle in (d)). Scale ffi 10 ~ m.
297
form body. Bilateral TC projections from the Prin. V utilize the middle cerebellar peduncle. (2) In normal mice, it appears that the trigeminal complex, particularly Prin. V., projects most heavily upon the crura I, II and possibly other neighbouring hemispheric lobules. The interpol, and oralis Spin. V may project more heavily upon vermian regions. (3) In reeler mice, following injecw~f6
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298 tions into ntmlerous cerebellar regions, labeled neurons were found in the same divisions of the trigeminal complex as seen in the n grmal. The strongest TC projections also appear to be within the hemispheres. After lesions of the Spin. V nuclei in the cat, Carpenter and Hanna [ 3] observed preterminal degeneration entering the ipsilateral restiform body en route to the vermis and possibly the paramedian lobule. These projections were largely ipsilateral. Fibers of passage and some evidence for preterminal degeneration were found primarily in the dentate and interpositus nuclei. In the present study, some cerebellar HRP injections encroached upon the deep cerebellar nuclei (see Fig. 2a). The possibility of trigemino-deep nuclear projections remains to be elucidated by experiments utilizing injections confined to the deep cerebellar nuclei. However, TC projections, most likely mossy fibers, ~ e probably similar to other known cerebellar afferent systems [ 12] which send somatotopically organized collateral branches to the deep cerebellar nuclei before ascending to the cortex. In an experiment by Stewart and King [ 22] in which lesions were placed in the caudalis division of Spin. V in the cat, Nauta-Gygax stained matelial showed no degeneration within the cerebellum, mggesting that the interpol, and oralis divisions are the origins of the spinal TC projections. In the present study, no labelled cells were found in Meessen and Olszewski's [ 13 ] zonalis, gelatinosus or magnoceUularis divisions of spinal caudalis V. It seems that caudalis V does not contribute significantly to spinal TC pathways in rodents, as corroborated by Faull (pers. comm. ~ud [5] ) who observed marginal caadalis V neurons retrogradely labeled following cerebellar HRP injections in cat and monkey, but not in rat. In an electrophysiological study of the organization of tactile projections to the cerebellar cortex in rat, Shambes et al. [ 17] have recently recorded units that respond t:o tactile stimulation within the granule cell layer of the crura I, II and paramedian lobule. In their study, the crum and neighbouring lobuli were found to contain a somatotopic organization of the head. The present study suggests that the neurons of origin of these hemispheric (for example, crum I, II, paramedian and simplex lobules) projections, as well as vermian projections (for example, declive and uvula), reside in the Prin. V, and in interpol, and oralis [5,21]. In both normal and reeler mutant mice, other bminstem nuclei such as the reticular formation, raphe, prepositus hypoglossi and inferior olivary nuclei may also contribute to this somatotopic representation within the cerebellum. Of particular interest in the study of Shambes et al. were cerebellar units with receptive fields in the head, perioral and intraoral hahT skin, incisors and vibrissae. It would prove extremely interesting if similar neurons m the Prin. V that project to the cerebellum also project to thalamic sites containing the morphological units related to individual mystac~al vibrissae referred to as barreloids [23 ]. The reeler mutation produces defects in nevcortical areas of the nervous system such as the cerebellum [15,19] where lack of foliation and malposi-
299
tioned cerebe]lar cortical neurons result in an undersized adult cerebellum. The reeler cerebellum looks somewhat embryonic as a result of many Purkinje cells occupying positions beneath what appears to be an ectopic externa~ granule cell layer. After spinal cord lesions in normal and reeler mutant mice, anterograde degeneration studies [19] show that the reeler cerebellum contains a miniature, topographic spinocerebellar representation that can be mapped within presumptive vermian areas that appear to be homologous to the normal. There is sufficient data within this system to suggest that the cerebellum in reeler is cytoarchitecturally abnormal, but nonetheless containing afferent projections with modified axonal trajectories that are appropriately organized (also see refs. 1, 2, 6 and 20). Despite cytoarchitectural defects that obscure characteristic landmarks in the reeler cerebellum (Fig. 2a), the projections of brainstem nuclei such as the trigeminal upon particular regions of the reeler cerebellum allows an assignment of those cerebellar areas that correspond topographically to those present in the normal. Following injections of H R P into various regions of the mutant cerebellum, several brainstem nuclei displayed distributions of retrogradely labeled neurons similar to that seen in the normal. The finding of T C projections to lateral cerebellar regions in reeler (as depicted in Fig. 2) suggests that these regions can be considered a cerebellar hemisphere. Results from investigations on the relationship between cytoarchitecture and other aspects of neuronal organization such as connectivity in tne cerebral and cerebellar cortices of the reeler mutant mouse suggest that the afferent conveyance of somatotopy is not necessarily dependent upon, nor reflected by, cytoarchitecture. In other words, the malpositioK~ of neurons in the reeler mutant is an independent phenomenon from the topographic specificity of axonal connections. t
ACKI~,'OWLEDGEMENTS
I would like to thank Drs. Allan I. Basbaum, William R. Mehler and Henry J. Ralston, III for their criticaladvice during thc preparation of this manuscript. Mr. Dave Akers kindly assisted with expert photography. Special thanks to Janice J.U. Steindler and Joshua Nicholai Steindler w h o provided assistance and constant encoura~ement. Supported by grant NS-11614 from NIH. REFERENCES 1 Caviness, V.S., Jr., Frost, D.O. and Hayes, N.L., Barrels in the somatosensory cortex of normal and reeler mutant mice, Neuroscience Letters, 3 (1976) 7--14. 2 Caviness, V.S., Jr. and Yorke, C.H, Jr., Interhemispheric neocortical connections of the corpus callosum in the reeler mutant mouse: A study based on anterograde and retrograde methods, J. comp. Neurol., 170 (1976) 449--460. 3 Carpenter, M.B. and Hanna, G.R., Fiber projections from the spinal trigeminal nucleus in the cat, J. comp. Neurol., 117 (1961) 117--131. 4 Colwell, S.A., Thalamocortical-corticothalamic reciprocity: a combined anterograderetrograde tracer technique, Brain Res., 92 ( 1975) 443--449.
~00 5 F~ull, R.L.M., A comparative study of the cells of origin of cerebe[lar afferents in the rat, cat, and monkey studied with the horseradish peroxidase technique: I. The nonvestibular brainstem afferents, Anat. Rec., 187 (1977) 577. 6 Frost, D.O. and Caviness, V.S., Jr., Patterns of thalamic projections to the neocortex of normal and reeler mutant mice. In Program and Abstracts of the Fourth Annual Meeting of the Society of Neuroscience, St. Louis, Mo. (1974) 217. 7 Graham, Jr., R.C. and Karnovsky, M.J., The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem., 14 (1966) 291--302. 8 Larsell, O., The development and morphology of the cerebellum in ~he opossum. I. Early development, J. comp. Neurol., 63 (1935) 65--94.. 9 Larsell, O. The development and subdivisions of the cerebellum of birds, J. comp. Neurol., 89 (1948) 123--190. 10 LaVaii, J.H. and LaVail, M.M., Retrograde axonal transport in the central nervous system, Science, 176 (1972) 1416--1417. 11 LaVaii, J.H., Winston, K.R. and Tish, A., A method based on retrograde intraaxonal transport of protein for identification.of cell bodies of origin of axons terminating within the CNS, Brain Res., 58 (1973) 470--477. 12 Matsushita, M. and Ikeda, M., Spinal projections to the cerebellar nuclei in the cat, Exp. Brain Res., 10 (1970b.) 501--511. 13 Meessen, H. and Olszewski, J., A Cytoarchitectonic Atlas of the Rhombencephalon of the Rabbit, S. Karger, Basel, 1949. 14 F,:arson, A.A., Further observations on the mesencephalic root of the trigeminal n~,.rle, J. comp. Neurol., 91 (1949) 147--194. 15 Rahic, P., and Sidman, R.L., Synaptic organization of displaced and disoriented c~.rebellar cortical neurons in reeler mice, J. Neuropath. exp. Neurol., 31 (1972) 192. 16 S:mders, E.B., A consideration of certain bulbar, midbrain, and cerebellar centers and fiber tracts in birds, J. comp. Neurol., 49 (1929) 155--222. 17 Shambes, G.M., Gibson, J.M. and Welker, W., Somatotopic organization of rat cerebellar tsctile cortex using micromapping methods, Neurosci. Abs., lI (1976) 117. !8 Sidman, R.L., Angevine, J.B., Jr., and Pierce, E.T., Atlas of the Mouse Brain and Spinal Cor,cl, Harvard University Press, Cambridge, Mass., 1971, 261 pp. 19 Steindler, D.A., Distribution of spinal afferents in the cerebellum of the reeler mutant mouse, Neurosci. Abs., 1 (1975) 516. 20 Steindler, D.A., and Colweil, S.A., Reeler mutant mouse: maintenance of appropriate and reciprocal connections in the cerebral cortex and thalamus, Brain Res., 113 (1976) 386--393. 21 Steindler, D.A., Cortical oar~01s, thalamic barreloids, and trigemino~erebellar projections in normal and reeler mutant mice. Anat. Rec., 187 (1977) 722. 22 Stewart, W.A., and King, R.G., Fiber projections from the nucleus caudalis of the spinal trigeminal nucleus, J. comp. N~.,urol., 121 (1963) 271--286. 23 Van der Loos, H., Barreloids in mouse somatosensory thalamus, Neuroscience Letters, 2 (1976) 1--6. 24 Welker, C., Cytoarehitectural organization of the somatosensory (SmI) cortical "barrel field" in reeler mutant mice, Anat. Rec., 184 (1976) 560--561. 25 Welt, C., and Steindler, D.A., Somato~ensory cortical barrels and thalamic barreloids in reeler mutant mice, Neuroscience (1977)In press. 26 Whitlock, D.G., A neurohistological and neurophysiological study of afferent fiber tracts and receptive areas of the avian cerebellum, J. comp. Neuroi., 97 (1952) 567--636. 27 Woodburne, R.T., A ptwlogenetic consideration of the primary and secondary centers and connection of the trigeminal complex in a series of vertebrates, J. comp. Neurol., 65 (1936) 403--502.
293
',PRIGEMINO-CEREBELLAR PROJECTIONS IN "NORMAL A N D R E E L E R M U T A N T MICE*
DENNIS A. STEINDLER** Department of Anatomy, School of Medicine, University of California, San Franscisco, San Franscisco, Calif. 94143 (U.S.A.) (Received M~Ly9th, 1977) (Revised version received September 6th, 1977) (Accepted ~eptember 10th, 1977)
SUMMARY
Retrogradely labeled neurons were found in the spinal trigemial (Spin V.) and principal sensory nucleus (Prin V.) and other regions following injections of horseraclish peroxidase (HRP) into normal mouse cerebellar hemisphere and vermis. HI~P injections into particular regions of the malformed cerebellum in the reeler mouse also resulted in retrograde labeling within regions of the trigeminal complex that are topographical!y similar to those seen in the normal. In association with a recently described cerebellar tactile representation of the head [17], the findings suggest that the neurons of origin of this somatosensory projection reside in portions of the trigeminal complex and possibly in other brainstem nuclei, in both normal mice and reeler mutants. These data also contribute to an understanding of afferent organization and the maintenance of appropriate patterns of connectivity within the malformed reeler cerebc~lum.
Evidence for trigemino~:erebellar (TC) projections has been presented in avian and mammalian species [14,27,9,16,8,26,3]. They were believed to originate from mesencephalic (Mes. V) [26,27] and sensory root fibers [26,27] and the principal sensory nucleus (Prin. V) [26,27] of the trigeminus in birds. A TC tract has also been reported in opossum [8] and in fetal human brains [14]. Whitlock [26] observed Marchi positive degeneration within the avian vermis after lesions of the Prin. V. He also saw chromatolytic changes within neurons of this nucleus following vermian lesions. Lesions of the interpolaris (interpol.) and oralis divisions of the spinal trigeminal nucleus (Spin. V) in the cat [3] produced preterminal degeneration within portions of the vermis. * Part of these results were presented at the 90th Session of the American Association of Anatomists (Anat. Rec., 187 (1977) 722). ** Present Address: C/o Dr. K.-M. Gottschaldt, Max-Planck-Institut fur Biophysikalische Chemie, Karl-Friedrich-Bonhoeffer'Institut, Postfach 968, D-3400 Gottingen, G.F R.
294
Recent contributions from anterograde degeneration studies suggest that there may be TC pathways in cat [ 3,22 ], but fail to elucidate major sites of origin and areas of termination of this system. The findings that appropriate somatosensory connections are achieved in the cytoarchitecturally abnormal forebrain o£ reeler mutant mice [ 1,2,20,21, 24,25] prompted the present study of somatosensory relationships in the normal and reeler mid- and hindbrain. A reassessment of TC pathways was undertaken using the retrograde horseradish peroxidase (HRP) transport technique [ 10 ]. Injections of 0.05--0.2 ~1 HRP* (Sigma, Type VI) were placed, using a Hamilton microsyringe, in various cerebellar regions of three normal and four homozygous reeler mice, (22--30 days old) bred in our laboratory from known heterozygotes**. After 16--24 h they were sacrificed by intracardiac perfusion of phosphate buffered (pH 7.2) 4% paraformaldehyde at 4 ° C over a period of 15 min. The brains were remove~, postfixed for 4 h in cold perfusate and immersed in cold 30% glucose in C.1 m phosphate buffer overnight. Frozen serial sections 30 and 90 ~m thick were cut in an oblique frontal plane and were reacted with diaminobenzidine and hydrogen peroxide [ 7,11]. Alternate sections were counterstained with cresyl violet. Sections were examined by bright- and dark-field microscopy. For cytoarchitectonics, ref. 18 was used.
* Some cases were combined horseradish peroxidase, tritiated amino acid injections (HAPTAA) [4 ] for studies of reciprocal afferent (HRP) and efferent (autoradiography) cerebellar projections. These findings will be discussed in a later report (Steindler, in preparation). ** C57BL/6J × C3H/HeJ hybrids originally obtained from The Jackson Laboratory, Bar Harbo-, Maine.
Fig. 1. Normal 30-day-old mouse. (a) Frontal section through cerebellum; double HRP injection. One injection involved crura I and II, Iobuli ansiformis (cr 1 and cr 2) and portions of neighbouring folia in the left hemisphere. Another, smaller injection in the vermis (only needle tract seen at this tevel, arrow) involved portions of culmen (cu) and declive. Cresylviolet ,~ounterstain. Scale = 1 ram. pa, paramedian lobule; py, pyramis; uv, uvula; no, noduluts. (b) Drawing of 90 ~m frontal section through medulla following injection shown in (a). HRP-labeled neurons (dots) are in inferior olivary complex (inf. oliv.), reticular and raphe groups, and in interpolaris division of spinal trigeminal nucleus (interpol. v). XII, hypoglossal nucleus; iat. cu., lateral cuneate nucleus: (c) A cluster of HRP labeled neurons in the ipsilateral interpolaris spinal trigeminal nucleus (square in Fig. 1 (b)). Dark-field photomicrograph. Scale = 40 urn. (d) Drawing of 90 ~m frontal section through midbrain following injection shown in (a). Labeled neurons are seen in nucleus reticularis tegmenti pontis (rtp) and in principal se~sory trigeminal nucleus (prin. V). sup. collic., superior colliculus; mid. ped., middle cerebellar peduncle ; v. co., ventr~! cochlear nucleus; sup. oliv., superior olivary nucleus, trap., nucleus of trapezoid body; sup. ped., superior cerebellar peduncle; d. raphe, dorsal raphe; pag, periaqueductal gray; v. tegment., ventral tegmental nuc~eu~ of Von Gudden. (e) HRP-labeled neurons in the dorsal area of principal sensory V. nucleus (circle in (d)). Dark-field photomicrograph. Scale = 40 urn. (f) Dark-field photo-" micrograph of two HRP-hbeled neurons in ventral portion of principal sensory V. nucleus (squ~re in (d)). Scale = 40 pm.
295
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in the normal mouse, unilateral injections in the left hemisphere and vermis resulted in retrograde labeled neurons in several brainstem areas. Fig. 1 depicts a case in which two injections were made. The injections included portions of the vermis (culmen, declive and pyramis lobules) and of lobulus simplex, crura I, II and paramedian lobuie of the hemisphere of the left side. There was no spread of HRP tc the right side. Labeled cells were found in medullary reticular and raphe areas, pontine nuc!ei, prepositus hypoglossi nucleus, the inferior oiivary complex, Prin. V., interpol, and oralis divisions of the Spin. V, and in the nucleus reticularis tegmenti pontis. HitP labeled axons were seen in the restifonn body and entering the oralis and interpol, regions associated with clusters of retrogradely labeled cells. Similarly. labeled axons were seen in the middle cerebellar peduncle near labeled cells of Prin. V. TC neurGns give rise to a bilateral projection with two to three times the number of ipsilateral to contralateral cells. The majority of labeled neurons in the oralis division of Spin. V were located dorsally; labeled cells of the Prin. V and interpol, divisions avpear to be in dorsal and ventral regions of these nuclei. No labeled cells were found within the Mes. V nucleus. After injections of HRP in presumptive hemispheric regions of the undersized, afoliate and cytoarchitecturally abnormal cerebellum of the re,der mouse (Fig. 2), the dist_~ibutions of retrograde labeled brainstem neurons were comparable to the normal. Injections in non-hemispheric regions of the m u t a n t and normal cerebellum (e.g. the midline anterior and posterior spinocerebellar recipient zones) resulted in much less retrograde labeling in the interpol., oralis and Prin. V nucleus, particularly in the latter. Fig. 2 illustrates some of the results after two injections confined to the presumed lateral hemispheric l:egion of a reeler mouse. Results from the two cases depicted in Figs. I and 2, as well ~ other cases in this study in which some injections were confined to particular hemispheric (for example, crura I, II) or vermian (for example, centralis) lobules, suggest that" (1) In both normal and reeler mutant mice, bilateral TC projections from the interpol, and oralis Spin. V nuclei ascend into the cerebellum via the resti-
Fig. 2.30-day-old reeler mouse. (a) Frontal section through cerebellum; double injection case. Both HRP-TAA injections were confined to the presumed lateral hemispheric area and underlying deep nuclei. Cresyl-violet counterstain. Scale ffi 1 mm. (b) Drawing of 90 ~m frontal section through the medulla following injection shown in (a). HRP-lab~;led neurons are in inferior olivai~y complex, reticular formation, interpolaris division of spinal trigeminal nucleus and in prepositus hypoglossi (possibly nucleus intercalatus) (just above hypoglossal nucleus, XII). For other symbols, see Fig. l(b). (c) Bright-field photomicrograph of HRP !abeled neuron in the interpolaris spinal trigeminal nucleus (circle in (b)). Arrow indicates the unstained nucleus in this non-counterstained neuron. Scale = 10 ~m. (d) Drawing of 90 ~m frontal section through midbrain following injection shown in (a). HRP-labeled neurons are in principal sensory trigeminal nucleus (Prin. V). r.f., reticular formation; v. len'l, lat., ventral nucleus of the lateral lemniscus. For other symbols, see Fig. t(d). (e) Darkfield photomicrograph of two HRP labeled neurons in principal sensory V. nv cleus (circle in (d)). Scale ffi 10 ~ m.
297
form body. Bilateral TC projections from the Prin. V utilize the middle cerebellar peduncle. (2) In normal mice, it appears that the trigeminal complex, particularly Prin. V., projects most heavily upon the crura I, II and possibly other neighbouring hemispheric lobules. The interpol, and oralis Spin. V may project more heavily upon vermian regions. (3) In reeler mice, following injecw~f6
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298 tions into ntmlerous cerebellar regions, labeled neurons were found in the same divisions of the trigeminal complex as seen in the n grmal. The strongest TC projections also appear to be within the hemispheres. After lesions of the Spin. V nuclei in the cat, Carpenter and Hanna [ 3] observed preterminal degeneration entering the ipsilateral restiform body en route to the vermis and possibly the paramedian lobule. These projections were largely ipsilateral. Fibers of passage and some evidence for preterminal degeneration were found primarily in the dentate and interpositus nuclei. In the present study, some cerebellar HRP injections encroached upon the deep cerebellar nuclei (see Fig. 2a). The possibility of trigemino-deep nuclear projections remains to be elucidated by experiments utilizing injections confined to the deep cerebellar nuclei. However, TC projections, most likely mossy fibers, ~ e probably similar to other known cerebellar afferent systems [ 12] which send somatotopically organized collateral branches to the deep cerebellar nuclei before ascending to the cortex. In an experiment by Stewart and King [ 22] in which lesions were placed in the caudalis division of Spin. V in the cat, Nauta-Gygax stained matelial showed no degeneration within the cerebellum, mggesting that the interpol, and oralis divisions are the origins of the spinal TC projections. In the present study, no labelled cells were found in Meessen and Olszewski's [ 13 ] zonalis, gelatinosus or magnoceUularis divisions of spinal caudalis V. It seems that caudalis V does not contribute significantly to spinal TC pathways in rodents, as corroborated by Faull (pers. comm. ~ud [5] ) who observed marginal caadalis V neurons retrogradely labeled following cerebellar HRP injections in cat and monkey, but not in rat. In an electrophysiological study of the organization of tactile projections to the cerebellar cortex in rat, Shambes et al. [ 17] have recently recorded units that respond t:o tactile stimulation within the granule cell layer of the crura I, II and paramedian lobule. In their study, the crum and neighbouring lobuli were found to contain a somatotopic organization of the head. The present study suggests that the neurons of origin of these hemispheric (for example, crum I, II, paramedian and simplex lobules) projections, as well as vermian projections (for example, declive and uvula), reside in the Prin. V, and in interpol, and oralis [5,21]. In both normal and reeler mutant mice, other bminstem nuclei such as the reticular formation, raphe, prepositus hypoglossi and inferior olivary nuclei may also contribute to this somatotopic representation within the cerebellum. Of particular interest in the study of Shambes et al. were cerebellar units with receptive fields in the head, perioral and intraoral hahT skin, incisors and vibrissae. It would prove extremely interesting if similar neurons m the Prin. V that project to the cerebellum also project to thalamic sites containing the morphological units related to individual mystac~al vibrissae referred to as barreloids [23 ]. The reeler mutation produces defects in nevcortical areas of the nervous system such as the cerebellum [15,19] where lack of foliation and malposi-
299
tioned cerebe]lar cortical neurons result in an undersized adult cerebellum. The reeler cerebellum looks somewhat embryonic as a result of many Purkinje cells occupying positions beneath what appears to be an ectopic externa~ granule cell layer. After spinal cord lesions in normal and reeler mutant mice, anterograde degeneration studies [19] show that the reeler cerebellum contains a miniature, topographic spinocerebellar representation that can be mapped within presumptive vermian areas that appear to be homologous to the normal. There is sufficient data within this system to suggest that the cerebellum in reeler is cytoarchitecturally abnormal, but nonetheless containing afferent projections with modified axonal trajectories that are appropriately organized (also see refs. 1, 2, 6 and 20). Despite cytoarchitectural defects that obscure characteristic landmarks in the reeler cerebellum (Fig. 2a), the projections of brainstem nuclei such as the trigeminal upon particular regions of the reeler cerebellum allows an assignment of those cerebellar areas that correspond topographically to those present in the normal. Following injections of H R P into various regions of the mutant cerebellum, several brainstem nuclei displayed distributions of retrogradely labeled neurons similar to that seen in the normal. The finding of T C projections to lateral cerebellar regions in reeler (as depicted in Fig. 2) suggests that these regions can be considered a cerebellar hemisphere. Results from investigations on the relationship between cytoarchitecture and other aspects of neuronal organization such as connectivity in tne cerebral and cerebellar cortices of the reeler mutant mouse suggest that the afferent conveyance of somatotopy is not necessarily dependent upon, nor reflected by, cytoarchitecture. In other words, the malpositioK~ of neurons in the reeler mutant is an independent phenomenon from the topographic specificity of axonal connections. t
ACKI~,'OWLEDGEMENTS
I would like to thank Drs. Allan I. Basbaum, William R. Mehler and Henry J. Ralston, III for their criticaladvice during thc preparation of this manuscript. Mr. Dave Akers kindly assisted with expert photography. Special thanks to Janice J.U. Steindler and Joshua Nicholai Steindler w h o provided assistance and constant encoura~ement. Supported by grant NS-11614 from NIH. REFERENCES 1 Caviness, V.S., Jr., Frost, D.O. and Hayes, N.L., Barrels in the somatosensory cortex of normal and reeler mutant mice, Neuroscience Letters, 3 (1976) 7--14. 2 Caviness, V.S., Jr. and Yorke, C.H, Jr., Interhemispheric neocortical connections of the corpus callosum in the reeler mutant mouse: A study based on anterograde and retrograde methods, J. comp. Neurol., 170 (1976) 449--460. 3 Carpenter, M.B. and Hanna, G.R., Fiber projections from the spinal trigeminal nucleus in the cat, J. comp. Neurol., 117 (1961) 117--131. 4 Colwell, S.A., Thalamocortical-corticothalamic reciprocity: a combined anterograderetrograde tracer technique, Brain Res., 92 ( 1975) 443--449.
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