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The cerebellar nucleo-cortical projection in the rat studied by the retrograde fluorescent double-labelling method
Brain Research, 271 (1983) 141-144 Elsevier
141
The cerebellar nucleo-cortical projection in the rat studied by the retrograde fluorescent double-labelling method J. N. PAYNE Department of Anatomy and Cell Biology, The University of Sheffield, Sheffield SIO 2TN (U. K.) (Accepted March 22nd, 1983) Key words: cerebellum - - nucleo-corticalneurones - - retrograde fluorescent double-labelling
Injection of true blue into rat cerebellar cortex and nuclear yellow into either the superior cerebellar peduncle or the ventral thalamus produced double-labelled neurones in the cerebellar deep nuclei. This suggeststhat the nueleo-corticalprojection arises from collaterals of cerebellofugal fibres. The topography of this projection, and differences in collateralization between thalamic and other cerebellofugal fibres are discussed. The existence of a projection from the neurones in the deep cerebellar nuclei to the cerebellar cortex has been revealed using a variety of anatomical techniquesS,t4, 23. It appears to be topographically organized and to terminate as mossy fibres in the granule cell layerlS,27. There is electrophysiological evidence that the axons from the deep nuclear neurones to the cortex are collaterals of axons leaving the cerebellumt9,23,24; this is supported by Golgi 5, and intracellular horseradish peroxidase studies t9. Recently, anatomical methods have been developed to determine whether axonal branching occurs in a retrogradely labelled population of neurones2,18. The double fluorescent retrograde labelling technique has been used to show axonal branching in cerebellofugal projections I and in projections from precerebellar nuclei in the brainstem to the cerebellar cortex in rats 20. In this study, it is used to confirm that the deep nuclear projection to the cerebellar cortex is from collaterals of some cerebellofugal axons; the resuits also support earlier observations on this pathway in the rat6,16. Injections of 50-100 nl of a 2% suspension of true blue (TB) were made, using a horizontal stereotaxic approach, into the cerebellar cortex of a total of 13 albino rats. A 7102 Hamilton syringe connected to a micropipette with a tip diameter of 50-75/~m was used for the injections which were carried out over a 5 min period. One group of rats had a midvermal in0006-8993/83/$03.00 (~) 1983 Elsevier Science Pnbli~her~ R V
jection, while the second group had a similar injection 4.5 mm from the midsagittal plane into the lobulus simplex--Crus I junction. After 24 h, 10 animals had 200 nl of 1% nuclear yellow (NY) injected bilaterally into the decussation of the superior cerebellar peduncle, while the remaining 3 had a similar NY injection into V.A-V.L. thalamic nuclei contralateral to the TB injection. Eighteen hours later, each animal was perfused through the aorta, first with 0.9% saline, and then for 30 min with 10% phosphate-buffered formalin. All experimental manipulations were done under general anaesthesia. After removal, the brains were left for 6-12 h at 4 °C in the same fixative to which sucrose had been added to a concentration of 15%. Coronal sections of the cerebellum and brainstem were cut at 20/zm with a cryostat. The sections were air-dried and mounted in D.P.X. They were examined with a fluorescence microscope using an excitation wavelength of 360 nm. Dark ground white light illumination was used to identify the outline of the deep cerebellar nuclei, and the location of fluorescent-labelled neurones was marked on outline drawings of the nuclei. As previously described3,4,20,21, the TB injection sites in the cerebellum had concentric fluorescent zones and although the effective injection may be limited to the inner two zones4,2t, no animal was used if even the outer zone extended significantly into the
142 RO~'Rm.
Fig. 2. This diagram of coronal sections at 3 rostrocaudal levels represents the combined results from the different cerebellar injection sites. Only neurones (double) labelled from both the cerebellum and the superior peduncle or thalamus are shown. Asterisks, neurones labelled from vermal injections; squares, neurones labelled from lobulus simplex injections; circles, neurones labelled from Crus I injections. DN, dentate nucleus; NI, nucleus interpositus; FN fastiglal nucleus; dlh, dorsolateral hump; dmc, dorsomedial crest; dip, dorsolateral protruberance.
Fig. 1. The diagram shows the location and extent of a vermal injection site, and of lateral injection sites in either the lobulus simplex (LS), or Crus I (C1). DN, cerebellar deep nuclei. The photomicrograph below is of the vermal injection site and comes from the area enclosed by the rectangle in the diagram.
cerebellar medulla. Two very superficial injections, in which only the outer zone entered the granule cell layer, failed to produce labelled neurones in either deep cerebeUar or pontine nuclei and only gave rise to labelled cells in the inferior olivary nucleus. Fig. 1 shows the location of the vermal and the lat-
eral TB injection sites in the cerebellum, and their typical microscopic appearance. The NY injections in the brainstem covered about half of the decussation of the superior cerebellar peduncle and all produced large numbers of N Y single-labelled neurones in the deep nuclei. These cells had a bright yellow nucleus and faint yellow cytoplasm; they were not surrounded by yellow glia. After vermal injections, TB-labelled neurones were found bilaterally in the fastigial nucleus and in the medial part of the nucleus interpositus posterior (Fig. 2). Of these TB neurones about a third were 'doublelabelled', having a blue cytoplasm from TB uptake and a yellow nucleus from NY uptake (Fig. 3), The lateral cerebellar injections produced single- and double-labelled neurones (in rougly equal numbers) only in the ipsilateral deep nuclei. Injection sites cen-
143
Fig. 3. A neurone from the nucleus interpositus which had blue cytoplasm and a yellow nucleus due to (double) labelling from TB and NY, respectively.
tered mainly in the lobulus simplex gave rise to labelled neurones in dorsomedial parts of the nucleus interpositus and dorsolateral protruberance of the fastigial nucleus, but none was seen in the dorsomedial crest or dorsolateral 'hump' regions17. Those injections centered in Crus I produced labelled neutones in the ventrolateral nucleus interpositus and the dentate nucleus (Fig. 2). NY injections in the thalamus produced few labelled neurones in the fastigial nucleus but large numbers in the other deep nuclei. These thalamic injections were only combined with lateral cerebellar TB injections. Neurones containing TB were found as before but, although they were intermingled with NY neurones in the deep nuclei, there were only very few double-labelled cells. Before attributing the presence of labelled neurones to the existence of a nucleo-cortical projection, it is important to consider the evidence that the effective injection sites were confined to the cortex. First,
true blue was chosen as the cerebellar tracer because its injection sites tend to be smaller and better localized than those from nuclear yellow 12. Secondly, no animal was used when the injection sites extended more than very superficially into the cerebellar medulla. Finally, injection sites only slightly more superficial labelled only inferior olivary cells whose axons end in the molecular layer as climbing fibres, and not pontine nucleus cells whose axons end in the granular layer as mossy fibres. A topographical pattern of nucleo-cortical projection, described here in the rat, is also seen in the cat 7-10A3 and monkey 25, but it is interesting to note that the projection from the dentate nucleus is greater in cats 7-10,13 and greater still in monkeys22,25. This may reflect the tendency for the importance of this nucleus to increase in higher animals 5, although cerebellar injections placed more laterally than described here may produce more labelled cells in the dentate nucleus. The double-labelling of deep nuclear neurones appears to confirm the electrophysiological work of Tolbert et al. 23,24, the Golgi studies of Chan-Palay 5 and the intracellular H R P work of McCrea et al. 19 who conclude that the axons which project to the cortex are collaterals of cerebellofugal axons from the deep nuclear neurones. As the proportion of the decussation of the superior cerebellar peduncle which is included in the NY injection site is approximately the same as the proportion of nucleo-cortical neurones which were double-labelled, there may be no nucleocortical fibres which are not axon collaterals. It is to be expected that fewer double-labelled neurones would be seen after thalamic NY injections because many deep nuclear neurones project to sites other than V.L. 1,11,26. Similarly Tolbert 26 has provided electrophysiological evidence that nucleo-cortical axons are collaterals of these non-thalamic axons. As, however, only very small numbers of double-labelled cells were found, it may be that fewer cerebellothalamic axons give rise to cortical collaterals. If this is so, it helps to confirm that TB-labelled neurones do have cortically directed axons. The alternative explanation, labelling by local spread of tracer directly to the cell bodies, should produce a similar proportion of double-labelled cells from thalamic and from superior cerebellar peduncle injections.
144 1 Bentivoglio, M. and Kuypers, H. G. J. M., Divergent axon collaterals from rat cerebellar nuclei to diencephalon, mesencephalon, medulla oblongata and cervical cord. A fluorescent double retrograde labeling study, Exp. Brain Res., 46 (1982) 339-356. 2 Bentivoglio, M., Kuypers, H. G. J. M., Catsman-Berrevoets, C. E., Loewe, H. and Dann, O., Two fluorescent retrograde neuronal tracers which are transported over long distances, Neurosci. Lett., 18 (1980) 25-30. 3 Bharos, T. B., Kuypers, H. G. J. M., Lemon, R. N. and Muir, R. B., Divergent collaterals from deep cerebellar neurons to thalamus and tectum, and to medulla oblongata and spinal cord: retrograde fluorescent and electrophysiological studies, Exp. Brain Res., 42 (1981) 399--410. 4 Catsman-Berrevoets, C. E. and Kuypers, H. G. J. M., A search for corticospinal collaterals to thalamus and mesencephalon by means of multiple retrograde fluorescent tracers in cat and rat, Brain Research, 218 (1981) 15-33. 5 Chan-Palay, V., Cerebellar Dentate Nucleus. Organisation, Cytology and Transmitters, Springer-Verlag, Berlin. 1977. 6 Chan-Palay, V., Palay, S. L. and Wu, J.-Y., Gamma-aminobutyric acid pathways in the cerebellum studied by retrograde and anterograde transport of glutamic acid decarboxylase antibody after in vivo injections, Anat. Embryol., 157 (1979) 1-14. 7 Dietrichs, E., The cerebellar corticonuclear and nucleocortical projections in the cat as studied with anterograde and retrograde transport of horseradish peroxidase. III. The anterior lobe, Anat. Embryol., 162 (1981) 223-247. 8 Dietrichs, E., The cerebellar corticonuclear and nucleocortical projections in the cat as studied with anterograde and retrograde transport of horseradish peroxidase. IV. The paraflocculus, Exp. Brain Res., 44 (1981) 235-242. 9 Dietrichs, E. and Walberg, F., The cerebellar corticonuclear and nucleocortical projections in the cat studied with anterograde and retrograde transport of horseradish peroxidase. I. The paramedian lobule, Anat. Embryol., 158 (1979) 13-39. 10 Dietrichs, E. and Walberg, F., The cerebellar corticonuclear and nucleocortical projections in the cat as studied with anterograde and retrograde transport of horseradish peroxidase. II. Lobulus simplex, Crus I and Crus II, Anat. Embryol., 161 (1980) 83-103. 11 Flood, S. and Jansen, S., The efferent fibres of the cerebellar nuclei and their distribution on the cerebellar peduncles in the cat, Acta anat., 63 (1966) 137-166. 12 Gaarskjaer, F. B., The hippocampal mossy fibre system of the rat studied with retrograde tracing techniques. Correlation between topographic organisation and neurogenetic gradients, J. comp. Neurol., 203 (1981) 717-735. 13 Gould, B. B., The organization of afferents to the cerebel-
lar cortex in the cat: projections from the deep cerebellar nuclei, J. comp. Neurol., 184 (1979)27-42. 14 Gould, B. B. and Graybiel, A. M., Afferents to the cerebellar cortex in the cat: evidence for an intrinsic pathway leading from the deep nuclei to the cortex, Brain Research, 110 (1976) 601-611. 15 Hamori, J., Mezey, E. and Szent~igothai, J., Electron microscopic identification of cerebellar nucleo-cortical mossy terminals in the rat, Exp. Brain Res., 44 (1981) 97-100. 16 Hess, D. T., Cerebellar nucteo-cortical neurons projecting to the vermis of lobule VII in the rat, Brain Research, 248 (1982) 361-366. 17 Korneliussen, H. K., On the morphology and subdivision of the cerebellar nuclei in the rat, J. Hirnforsch., 10 (1968) 109--122. 18 Kuypers, H. G. J. M., Catsman-Berrevoets, C. E. and Padt, R. E., Retrograde axonal transport of fluorescent substances in the rat's forebrain, Neurosci. Lett., 6 (1977) 127-135. 19 McCrea, R. A., Bishop, G. A. and Kitai, S. T., Electrophysiological and horseradish peroxidase studies of precerebellar afferents to the nucleus interpositus anterior. II. Mossy fibre system, Brain Research, 122 (1977) 215-228. 20 Payne, J. N., Axonal branching in the projections from precerebellar nuclei to the lobulus simplex in the rat's cerebellum investigated by retrograde fluorescent double labeling, J. comp. Neurol., 213 (1983) 233--240. 21 Sawchenko, P. E. and Swanson, L. W., A method for tracing biochemically defined pathways in the central nervous system using combined fluorescence retrograde transport and immunohistochemical techniques, Brain Research, 210 (1981)31-51. 22 Tolbert, D. L. and Bantli, H., An HRP and autoradiographic study of cerebellar corticonuclear-nucleocortical reciprocity in the monkey, Exp. Brain Res., 36 (1979) 563-571. 23 Tolbert, D. L., Bantli, H. and Bloedel, J. R., Anatomical and physiological evidence for a cerebellar nucleocortical projection in the cat, Neuroscience, 1 (1976) 205-217. 24 Tolbert, D. L., Bantli, H. and Bloedel, J. R., The intracerebellar nucleocortical projection in a primate, Exp. Brain ICes., 30 (1977) 425-434. 25 Tolbert, D. L., Bantli, H. and Bloedel, J. R., Organisational features of the cat and monkey cerebellar nucleocortical projection, J. comp. Neurol., 182 (1978) 39-56. 26 Tolbert, D. L., Bantli, H. and Bloedel, J. R., Multiple branching of cerebellar efferent projections in cats, Exp. Brain Res., 31 (1978) 305-316. 27 Tolbert, D. L., Kultas-Ilinsky, K. and Ilinsky, I., EM-autoradiography of cerebellar nucleocortical terminals in the cat, Anat. Embryol., 161 (1980) 215-223.
141
The cerebellar nucleo-cortical projection in the rat studied by the retrograde fluorescent double-labelling method J. N. PAYNE Department of Anatomy and Cell Biology, The University of Sheffield, Sheffield SIO 2TN (U. K.) (Accepted March 22nd, 1983) Key words: cerebellum - - nucleo-corticalneurones - - retrograde fluorescent double-labelling
Injection of true blue into rat cerebellar cortex and nuclear yellow into either the superior cerebellar peduncle or the ventral thalamus produced double-labelled neurones in the cerebellar deep nuclei. This suggeststhat the nueleo-corticalprojection arises from collaterals of cerebellofugal fibres. The topography of this projection, and differences in collateralization between thalamic and other cerebellofugal fibres are discussed. The existence of a projection from the neurones in the deep cerebellar nuclei to the cerebellar cortex has been revealed using a variety of anatomical techniquesS,t4, 23. It appears to be topographically organized and to terminate as mossy fibres in the granule cell layerlS,27. There is electrophysiological evidence that the axons from the deep nuclear neurones to the cortex are collaterals of axons leaving the cerebellumt9,23,24; this is supported by Golgi 5, and intracellular horseradish peroxidase studies t9. Recently, anatomical methods have been developed to determine whether axonal branching occurs in a retrogradely labelled population of neurones2,18. The double fluorescent retrograde labelling technique has been used to show axonal branching in cerebellofugal projections I and in projections from precerebellar nuclei in the brainstem to the cerebellar cortex in rats 20. In this study, it is used to confirm that the deep nuclear projection to the cerebellar cortex is from collaterals of some cerebellofugal axons; the resuits also support earlier observations on this pathway in the rat6,16. Injections of 50-100 nl of a 2% suspension of true blue (TB) were made, using a horizontal stereotaxic approach, into the cerebellar cortex of a total of 13 albino rats. A 7102 Hamilton syringe connected to a micropipette with a tip diameter of 50-75/~m was used for the injections which were carried out over a 5 min period. One group of rats had a midvermal in0006-8993/83/$03.00 (~) 1983 Elsevier Science Pnbli~her~ R V
jection, while the second group had a similar injection 4.5 mm from the midsagittal plane into the lobulus simplex--Crus I junction. After 24 h, 10 animals had 200 nl of 1% nuclear yellow (NY) injected bilaterally into the decussation of the superior cerebellar peduncle, while the remaining 3 had a similar NY injection into V.A-V.L. thalamic nuclei contralateral to the TB injection. Eighteen hours later, each animal was perfused through the aorta, first with 0.9% saline, and then for 30 min with 10% phosphate-buffered formalin. All experimental manipulations were done under general anaesthesia. After removal, the brains were left for 6-12 h at 4 °C in the same fixative to which sucrose had been added to a concentration of 15%. Coronal sections of the cerebellum and brainstem were cut at 20/zm with a cryostat. The sections were air-dried and mounted in D.P.X. They were examined with a fluorescence microscope using an excitation wavelength of 360 nm. Dark ground white light illumination was used to identify the outline of the deep cerebellar nuclei, and the location of fluorescent-labelled neurones was marked on outline drawings of the nuclei. As previously described3,4,20,21, the TB injection sites in the cerebellum had concentric fluorescent zones and although the effective injection may be limited to the inner two zones4,2t, no animal was used if even the outer zone extended significantly into the
142 RO~'Rm.
Fig. 2. This diagram of coronal sections at 3 rostrocaudal levels represents the combined results from the different cerebellar injection sites. Only neurones (double) labelled from both the cerebellum and the superior peduncle or thalamus are shown. Asterisks, neurones labelled from vermal injections; squares, neurones labelled from lobulus simplex injections; circles, neurones labelled from Crus I injections. DN, dentate nucleus; NI, nucleus interpositus; FN fastiglal nucleus; dlh, dorsolateral hump; dmc, dorsomedial crest; dip, dorsolateral protruberance.
Fig. 1. The diagram shows the location and extent of a vermal injection site, and of lateral injection sites in either the lobulus simplex (LS), or Crus I (C1). DN, cerebellar deep nuclei. The photomicrograph below is of the vermal injection site and comes from the area enclosed by the rectangle in the diagram.
cerebellar medulla. Two very superficial injections, in which only the outer zone entered the granule cell layer, failed to produce labelled neurones in either deep cerebeUar or pontine nuclei and only gave rise to labelled cells in the inferior olivary nucleus. Fig. 1 shows the location of the vermal and the lat-
eral TB injection sites in the cerebellum, and their typical microscopic appearance. The NY injections in the brainstem covered about half of the decussation of the superior cerebellar peduncle and all produced large numbers of N Y single-labelled neurones in the deep nuclei. These cells had a bright yellow nucleus and faint yellow cytoplasm; they were not surrounded by yellow glia. After vermal injections, TB-labelled neurones were found bilaterally in the fastigial nucleus and in the medial part of the nucleus interpositus posterior (Fig. 2). Of these TB neurones about a third were 'doublelabelled', having a blue cytoplasm from TB uptake and a yellow nucleus from NY uptake (Fig. 3), The lateral cerebellar injections produced single- and double-labelled neurones (in rougly equal numbers) only in the ipsilateral deep nuclei. Injection sites cen-
143
Fig. 3. A neurone from the nucleus interpositus which had blue cytoplasm and a yellow nucleus due to (double) labelling from TB and NY, respectively.
tered mainly in the lobulus simplex gave rise to labelled neurones in dorsomedial parts of the nucleus interpositus and dorsolateral protruberance of the fastigial nucleus, but none was seen in the dorsomedial crest or dorsolateral 'hump' regions17. Those injections centered in Crus I produced labelled neutones in the ventrolateral nucleus interpositus and the dentate nucleus (Fig. 2). NY injections in the thalamus produced few labelled neurones in the fastigial nucleus but large numbers in the other deep nuclei. These thalamic injections were only combined with lateral cerebellar TB injections. Neurones containing TB were found as before but, although they were intermingled with NY neurones in the deep nuclei, there were only very few double-labelled cells. Before attributing the presence of labelled neurones to the existence of a nucleo-cortical projection, it is important to consider the evidence that the effective injection sites were confined to the cortex. First,
true blue was chosen as the cerebellar tracer because its injection sites tend to be smaller and better localized than those from nuclear yellow 12. Secondly, no animal was used when the injection sites extended more than very superficially into the cerebellar medulla. Finally, injection sites only slightly more superficial labelled only inferior olivary cells whose axons end in the molecular layer as climbing fibres, and not pontine nucleus cells whose axons end in the granular layer as mossy fibres. A topographical pattern of nucleo-cortical projection, described here in the rat, is also seen in the cat 7-10A3 and monkey 25, but it is interesting to note that the projection from the dentate nucleus is greater in cats 7-10,13 and greater still in monkeys22,25. This may reflect the tendency for the importance of this nucleus to increase in higher animals 5, although cerebellar injections placed more laterally than described here may produce more labelled cells in the dentate nucleus. The double-labelling of deep nuclear neurones appears to confirm the electrophysiological work of Tolbert et al. 23,24, the Golgi studies of Chan-Palay 5 and the intracellular H R P work of McCrea et al. 19 who conclude that the axons which project to the cortex are collaterals of cerebellofugal axons from the deep nuclear neurones. As the proportion of the decussation of the superior cerebellar peduncle which is included in the NY injection site is approximately the same as the proportion of nucleo-cortical neurones which were double-labelled, there may be no nucleocortical fibres which are not axon collaterals. It is to be expected that fewer double-labelled neurones would be seen after thalamic NY injections because many deep nuclear neurones project to sites other than V.L. 1,11,26. Similarly Tolbert 26 has provided electrophysiological evidence that nucleo-cortical axons are collaterals of these non-thalamic axons. As, however, only very small numbers of double-labelled cells were found, it may be that fewer cerebellothalamic axons give rise to cortical collaterals. If this is so, it helps to confirm that TB-labelled neurones do have cortically directed axons. The alternative explanation, labelling by local spread of tracer directly to the cell bodies, should produce a similar proportion of double-labelled cells from thalamic and from superior cerebellar peduncle injections.
144 1 Bentivoglio, M. and Kuypers, H. G. J. M., Divergent axon collaterals from rat cerebellar nuclei to diencephalon, mesencephalon, medulla oblongata and cervical cord. A fluorescent double retrograde labeling study, Exp. Brain Res., 46 (1982) 339-356. 2 Bentivoglio, M., Kuypers, H. G. J. M., Catsman-Berrevoets, C. E., Loewe, H. and Dann, O., Two fluorescent retrograde neuronal tracers which are transported over long distances, Neurosci. Lett., 18 (1980) 25-30. 3 Bharos, T. B., Kuypers, H. G. J. M., Lemon, R. N. and Muir, R. B., Divergent collaterals from deep cerebellar neurons to thalamus and tectum, and to medulla oblongata and spinal cord: retrograde fluorescent and electrophysiological studies, Exp. Brain Res., 42 (1981) 399--410. 4 Catsman-Berrevoets, C. E. and Kuypers, H. G. J. M., A search for corticospinal collaterals to thalamus and mesencephalon by means of multiple retrograde fluorescent tracers in cat and rat, Brain Research, 218 (1981) 15-33. 5 Chan-Palay, V., Cerebellar Dentate Nucleus. Organisation, Cytology and Transmitters, Springer-Verlag, Berlin. 1977. 6 Chan-Palay, V., Palay, S. L. and Wu, J.-Y., Gamma-aminobutyric acid pathways in the cerebellum studied by retrograde and anterograde transport of glutamic acid decarboxylase antibody after in vivo injections, Anat. Embryol., 157 (1979) 1-14. 7 Dietrichs, E., The cerebellar corticonuclear and nucleocortical projections in the cat as studied with anterograde and retrograde transport of horseradish peroxidase. III. The anterior lobe, Anat. Embryol., 162 (1981) 223-247. 8 Dietrichs, E., The cerebellar corticonuclear and nucleocortical projections in the cat as studied with anterograde and retrograde transport of horseradish peroxidase. IV. The paraflocculus, Exp. Brain Res., 44 (1981) 235-242. 9 Dietrichs, E. and Walberg, F., The cerebellar corticonuclear and nucleocortical projections in the cat studied with anterograde and retrograde transport of horseradish peroxidase. I. The paramedian lobule, Anat. Embryol., 158 (1979) 13-39. 10 Dietrichs, E. and Walberg, F., The cerebellar corticonuclear and nucleocortical projections in the cat as studied with anterograde and retrograde transport of horseradish peroxidase. II. Lobulus simplex, Crus I and Crus II, Anat. Embryol., 161 (1980) 83-103. 11 Flood, S. and Jansen, S., The efferent fibres of the cerebellar nuclei and their distribution on the cerebellar peduncles in the cat, Acta anat., 63 (1966) 137-166. 12 Gaarskjaer, F. B., The hippocampal mossy fibre system of the rat studied with retrograde tracing techniques. Correlation between topographic organisation and neurogenetic gradients, J. comp. Neurol., 203 (1981) 717-735. 13 Gould, B. B., The organization of afferents to the cerebel-
lar cortex in the cat: projections from the deep cerebellar nuclei, J. comp. Neurol., 184 (1979)27-42. 14 Gould, B. B. and Graybiel, A. M., Afferents to the cerebellar cortex in the cat: evidence for an intrinsic pathway leading from the deep nuclei to the cortex, Brain Research, 110 (1976) 601-611. 15 Hamori, J., Mezey, E. and Szent~igothai, J., Electron microscopic identification of cerebellar nucleo-cortical mossy terminals in the rat, Exp. Brain Res., 44 (1981) 97-100. 16 Hess, D. T., Cerebellar nucteo-cortical neurons projecting to the vermis of lobule VII in the rat, Brain Research, 248 (1982) 361-366. 17 Korneliussen, H. K., On the morphology and subdivision of the cerebellar nuclei in the rat, J. Hirnforsch., 10 (1968) 109--122. 18 Kuypers, H. G. J. M., Catsman-Berrevoets, C. E. and Padt, R. E., Retrograde axonal transport of fluorescent substances in the rat's forebrain, Neurosci. Lett., 6 (1977) 127-135. 19 McCrea, R. A., Bishop, G. A. and Kitai, S. T., Electrophysiological and horseradish peroxidase studies of precerebellar afferents to the nucleus interpositus anterior. II. Mossy fibre system, Brain Research, 122 (1977) 215-228. 20 Payne, J. N., Axonal branching in the projections from precerebellar nuclei to the lobulus simplex in the rat's cerebellum investigated by retrograde fluorescent double labeling, J. comp. Neurol., 213 (1983) 233--240. 21 Sawchenko, P. E. and Swanson, L. W., A method for tracing biochemically defined pathways in the central nervous system using combined fluorescence retrograde transport and immunohistochemical techniques, Brain Research, 210 (1981)31-51. 22 Tolbert, D. L. and Bantli, H., An HRP and autoradiographic study of cerebellar corticonuclear-nucleocortical reciprocity in the monkey, Exp. Brain Res., 36 (1979) 563-571. 23 Tolbert, D. L., Bantli, H. and Bloedel, J. R., Anatomical and physiological evidence for a cerebellar nucleocortical projection in the cat, Neuroscience, 1 (1976) 205-217. 24 Tolbert, D. L., Bantli, H. and Bloedel, J. R., The intracerebellar nucleocortical projection in a primate, Exp. Brain ICes., 30 (1977) 425-434. 25 Tolbert, D. L., Bantli, H. and Bloedel, J. R., Organisational features of the cat and monkey cerebellar nucleocortical projection, J. comp. Neurol., 182 (1978) 39-56. 26 Tolbert, D. L., Bantli, H. and Bloedel, J. R., Multiple branching of cerebellar efferent projections in cats, Exp. Brain Res., 31 (1978) 305-316. 27 Tolbert, D. L., Kultas-Ilinsky, K. and Ilinsky, I., EM-autoradiography of cerebellar nucleocortical terminals in the cat, Anat. Embryol., 161 (1980) 215-223.