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Distribution of cerebellar mossy fibers arising from neurons of the raccoon main cuneate nucleus
164
Brain Researci~..~23 / 1984~ 1{)4---1~7 Etsc~'ier
BRE 20497
Distribution of cerebellar mossy fibers arising from neurons of the raccoon main cuneate nucleus JOHN H. HARING l. SUSAN WARREN ~and M A R K J. ROWINSKI 3 1Department of Anatomy, St. Louis" University School of Medicine. St. Louis MO 63104: 2Department of Physiology and Biophysics, New York University School of Medicine, New York. N Y 10016 and 3Department of Physical Therapy, University of Wisconsin-LaCrosse. LaCrosse. W154601 gU.S.A. J
(Accepted July 17th, 1984) Key words: raccoon --- mare cuneate m, clcus
cuneocerebcllar tract - - [3H]leucinc
The present study demonstrates that fibers originating in the raccoon main cuneate nucleus are not segregated in specific cortical re~. gions of target cerebeltar folia as reported in studies of the cat cuneocerebellar tract. This ob~rvation supports the hypothesis that exteroceptive and proprioceptive divisions of the raccoon cuneocerebellar tract do not exist as distinct structural entities. The findings of this study do not preclude the possibility that projections from the external and main cuneate nuclei are segregated within the cerebellar cortex on the basis of functional modatity rather than nucleus of origin.
Studies conducted in several m a m m a l i a n species have demonstrated that n e u r o n s of the external cuneate nucleus (ECN) as well as cells of the polymorphic division of the main cuneate nucleus (MCN) u,12 give rise to the cuneocerebellar tract (CCT) 1,6-t0,14'16. The C C T in cats is composed of two, functional c o m p o n e n t s 2. The p r o p n o c e p t i v e component (P-CCT) originates in the ECN whereas the exteroceptive c o m p o n e n t (E-CCT) arises in the MCN. Anatomical], u and physiological5 observations suggest that these functional c o m p o n e n t s are also segregated within the cerebeUar cortex such that terminals of the P-CCT are located in the basal regions of the cerebellar folia and the E - C C T projects to the apices of the cerebellar folia. This segregation of the functional components of the CCT implies that the processing of the exteroceptwe and proprioceptive information is relegated to discrete regions of the cerebellar cortex. In the raccoon, MCN neurons having projections to the cerebellum are located in the ventrolateral polymorphic region at and rostral to the obex~L corresponding to the 'medial tongue' region of Johnson et al. u. These cells have b e e n found to relay mostly
deep information ~0 in contrast to findings in the cat. where the MCN has been identified as the source of the cutaneous cuneocerebellar retay 2. in addition, the retrograde transport of horseradish peroxidase does not reveal a differential distribution of cerebellar afferents from the MCN and ECN of the raccoon 9 In the cases studied, injections limited to the apices of folia in lobute V yielded labeling of neurons in both the MCN and ECN. The location of the labeled MCN cells 1H5 as well as the sensory modalities represented in the population of MCN n e u r o n s having terminals in the cerebellum suggests that cuneocerebellar n e u r o n s of the MCN could be functioning as a subnucleus of the ECN rather t h a n the origin of a distinct E-CCT. Thus the apparent tack of two separate functional c o m p o n e n t s in the raccoon C C T supports the observation that axons from both MCN and ECN have overlapping distributions within the cerebellar cortex. T h e present study was conducted using the anterograde labeling methods in order to further clarify the distribution of M C N axons within the raccoon cerebellum. Injections of [3H]leucine (50 #Ci/kd) were made m the ventral M C N in each of 6 adult raccoons using a
Correspondence: J. H. Haring, Department of Anatomy, St. Louis University School of Medicine, L402South Grand Boulevard. St Louis, MO 63104, U.S.A.
0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V
1(~5 micropipette attached to a 1 ul Hamilton syringe. The total volume of the injected amino acid solution ranged from 0.2 to 0.4 M in each MCN. A f t e r 4 days, animals were perfused intracardially with l()C/c neutral buffered formalin. Transverse frozen sections through the brainstem and sagittal sections through the cerebellum were coated with NTB-2 emulsion and exposed for 6 weeks~. The autoradiograms were then developed and the tissue sections counterstained through the emulsion with thionin. Sections were studied and p h o t o g r a p h e d on a Leitz dialux 20 microscope using either bright- or darkfield illumination. The sites of leucine injection were centered in the ventral MCN with no apparent tracer spread into the neighboring ECN (Fig. 1). A f t e r tracer injections of the ventral MCN, labeled axons were observed to exit the ventrolateral aspect of the MCN and traverse the ECN to enter the restiform body. Fibers ascend-
ed the brainstem in the restiform body and e n t e r e d the cerebellum via the inferior peduncle. Within the deep white matter of the cerebellum, axons from the MCN arched dorsally anterior to the deep cerebellar nuclei forming a fan-shaped array as fibers diverged to distribute to various cerebellar lobules. The m a i o r target of MCN afferents a p p e a r e d to be Iobule V, although substantial numbers of axons p r o j e c t e d to Iobules Ill and 1V. In addition, a small bundle of MCN fibers entered the paramedian Iobule and patterns of terminal labeling were seen within both the interposed and lateral cerebellar nuclei. Once the fibers had reached their respective cortical targets, terminals were distributed throughout the length of the individual folia from base to apex (Fig. 2). No difference was apparent in the relative densities of terminal fields seen in either the basal or apical regions of the target folia.
Fig. l. Brightfield photomicrograph of an injection site in the ventral MCN approximately 1 mm rostral to the obcx. ]'he center of the iniection (asterisk) is marked b x a dense accumulation of silver grains. The region surrounding the injection site is lcss heavily labeled and is seen to contain labeled neuronal perikarya (arrows) thus dcfining the zone of effective uptake of tracer. Note that the_'ECN is not mchlded within the injection site. x39.
Fig. 2. Darkfield photomicrographs of fibers t arrowheads) and terminals larrowsl seen in lobule V after rejections of the MCN. Note that labeled termipals are present in the granule cell layer of both the base (A) and the apical region (B/of Iobule V x 17"7
The present study has d e m o n s t r a t e d that the cerebellar projection of the M C N is directed primarily toward the anterior lobe wherein the m a j o r i t y of labeled fibers t e r m i n a t e in lobule V. Projections from the M C N to the p a r a m e d i a n lobule and deep cerebellar nuclei were also verified. These observations do not differ significantly from those o b t a i n e d in studies of the cat C C T 7. H o w e v e r , in contrast to findings in the cat, the labeled terminals of MCN axons were distributed throughout the length of the respective target folia. Thus, no segregation of M C N terminals from those of the ECN is likely. This result ts consistent with previous observations of r e t r o g r a d e labeling of the raccoon M C N after horseradish peroxidase injections of lobule V '). Our studies of cerebellar relay cells of the raccoon MCN indicate that the 'medial tongue" region ts both morphologically and functionally similar to the E C N 9Ao. Therefore, the raccoon C C T is concerned mainly with the conveying of information from d e e p receptors. A l t h o u g h there is a small cutaneous repre-
sentation in the MCN projection L~and the possibility of a similar transfer of cutaneous information through the ECN 4A3. the organization o f the raccoon CCT into distinct functional/anatomical c o m p o n e n t s is not evident. Rather. the ' m e d i a l tongue" region of the raccoon MCN appears to function as a subnucleus of the ECN, Because our evidence indicates that the ECN and MCN "medial tongue' may constitute an anatomical unit. the inability to d e m o n s t r a t e a regional segregation of terminals based u p o n cells of origin is not surprising. H o w e v e r . it is still possible that C C T projections are differentially distributed within the raccoon cerebellar cortex on the basis of sensory modality. We acknowledge the support and e n c o u r a g e m e n t of Dr. B. H. Pubols. Jr. and the excellent technical assistance of Christine H. Maliniak. This study was supported in part by Research G r a n t s NS-13418 and NS-19486 U S P H S .
167 l Check, M. D., Rustiani, A. a n d T r e v i n o , D. L., Dorsal colunto nuclei projections to the ccrebellar cortex in cats as revealed by the use of the retrograde transport of horseradish pcroxidasc. J. temp. Neurol.. 164 (1975) 31-46. 2 Cook, J. D., Larson. B., Oscarsson, O. and Sjolund, B., Origin and termination ot cuneocerebellar tract, t','.V). Brain Re,s., 13 (1971) 339-358. 3 ('owan, M. W., Goltlieb, l). l., Hcndrickson, A. K., Price. ,l. L. and Woolscy, T. A., The autoradiographic demonstration el' ~txonal COllnCCti(ms in the central nerxous system, Brain Re,~earch. 37 (1072) 21-51. 4 Dykes. R. \~,'., R a s m u s s e n , 1)., Srctavan. D. and R e h m a n , I-I. B., SubmoduliD. segregation and receptive-field scquenccs in cuncatc, gracilc, and external cunealc nuclei of lhc cat. J. Neurophysiol., 47 { 1982) 389-416. 5 Ekcrot. C.-F. and l.arson, B., Differential tcrmination el the cxtcroccplivc and propriccptivc components of the cuncocercbcllar tract. Brain Research, 3() (1972) 420-424. 6 (i(mld, B. B., Organization of affcrents from brain stem nuclei to thc cercbellar cortex in the cat. Advanc. Anat. Emhrvol. ('ell Biol., (~2 (lt)Sl)) 1 90. 7 (}rant. (i., Projection of the external cuncate nucleus onto thc cerebellum in the cat: an experimental study using silver methods. Krp. Neurol., 5 (1%2) 179-195. 8 (}ray, 3. S., Hazier(, ,l. C. and Martin, G. F., Organization of projections, Iron3 the gracilc, medial cuneatc and lateral cuncatc nuclei m the North American opossum. Horseradish peroxidasc studx of cells projecting to the cerebellum.
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thalamus, and spinal cord, Brain Behav. Evol., 18 (19811 140-156. Haring, J. H. and Rowinski, M. J., A horseradish pert)xidase study of projections from the main and external cuncatc nuclei to the cerebellum of the North American raccoon. J. coral). Neuro[., 211 (1982) 3~33 37t3. Haring, J. H.. Rowinski, M. J. and Pubols, B. l l.. Electrophysiology ¢)1 raccoon cuncoccrebcl]ar neurons, ,~omffloven,sory Re.v, I (1984) 247-264. ,lohnson, J. 1., Wclkcr, W. 1. and Pubols, B. H.. Somatopic organization of raccoon dorsal c o h m m nuclei, J. temp. Neurol., 132 (1968) 1-44. Kuypers. H. G. J. M. m)d Tucrk, J. I)., I'hc distribution of the cortical fibers ~ithin the nuclei cuncalus and gracilis in the cal,J. Aria;. (Lend.), 98 (1964) 143-1(32. Raush. J., Electrophw'ioloKv (~/'1he K~termd ('urn'ate Nudeu.s in the Cat, Ph. D. dissertation, t;ni~crsitv of WashingIon, Seattle, W A , 1960. Rinvik, E. and Walbcrg. F.. Studies on the ccrcbellar pro]ections from main and external cuncatc nuclei in the cat bv means of retrograde axonal transport of horseradish peroxidasc, Brain Re.search. 95 (1975) 371-381. Rosen, l., Localization in caudal brain slcm and cervical spinal cord of neurons activated from forelimb group I afferents in the cat, Brain Research, 16 (1969) 55-71. Somana, R. and Walberg, F., A re-examination ol the ccrebellar projections from the gracilc, main and external cuneate nuclei in the cat, Brain Re,warch, IS6 (19811) 33 42.
Brain Researci~..~23 / 1984~ 1{)4---1~7 Etsc~'ier
BRE 20497
Distribution of cerebellar mossy fibers arising from neurons of the raccoon main cuneate nucleus JOHN H. HARING l. SUSAN WARREN ~and M A R K J. ROWINSKI 3 1Department of Anatomy, St. Louis" University School of Medicine. St. Louis MO 63104: 2Department of Physiology and Biophysics, New York University School of Medicine, New York. N Y 10016 and 3Department of Physical Therapy, University of Wisconsin-LaCrosse. LaCrosse. W154601 gU.S.A. J
(Accepted July 17th, 1984) Key words: raccoon --- mare cuneate m, clcus
cuneocerebcllar tract - - [3H]leucinc
The present study demonstrates that fibers originating in the raccoon main cuneate nucleus are not segregated in specific cortical re~. gions of target cerebeltar folia as reported in studies of the cat cuneocerebellar tract. This ob~rvation supports the hypothesis that exteroceptive and proprioceptive divisions of the raccoon cuneocerebellar tract do not exist as distinct structural entities. The findings of this study do not preclude the possibility that projections from the external and main cuneate nuclei are segregated within the cerebellar cortex on the basis of functional modatity rather than nucleus of origin.
Studies conducted in several m a m m a l i a n species have demonstrated that n e u r o n s of the external cuneate nucleus (ECN) as well as cells of the polymorphic division of the main cuneate nucleus (MCN) u,12 give rise to the cuneocerebellar tract (CCT) 1,6-t0,14'16. The C C T in cats is composed of two, functional c o m p o n e n t s 2. The p r o p n o c e p t i v e component (P-CCT) originates in the ECN whereas the exteroceptive c o m p o n e n t (E-CCT) arises in the MCN. Anatomical], u and physiological5 observations suggest that these functional c o m p o n e n t s are also segregated within the cerebeUar cortex such that terminals of the P-CCT are located in the basal regions of the cerebellar folia and the E - C C T projects to the apices of the cerebellar folia. This segregation of the functional components of the CCT implies that the processing of the exteroceptwe and proprioceptive information is relegated to discrete regions of the cerebellar cortex. In the raccoon, MCN neurons having projections to the cerebellum are located in the ventrolateral polymorphic region at and rostral to the obex~L corresponding to the 'medial tongue' region of Johnson et al. u. These cells have b e e n found to relay mostly
deep information ~0 in contrast to findings in the cat. where the MCN has been identified as the source of the cutaneous cuneocerebellar retay 2. in addition, the retrograde transport of horseradish peroxidase does not reveal a differential distribution of cerebellar afferents from the MCN and ECN of the raccoon 9 In the cases studied, injections limited to the apices of folia in lobute V yielded labeling of neurons in both the MCN and ECN. The location of the labeled MCN cells 1H5 as well as the sensory modalities represented in the population of MCN n e u r o n s having terminals in the cerebellum suggests that cuneocerebellar n e u r o n s of the MCN could be functioning as a subnucleus of the ECN rather t h a n the origin of a distinct E-CCT. Thus the apparent tack of two separate functional c o m p o n e n t s in the raccoon C C T supports the observation that axons from both MCN and ECN have overlapping distributions within the cerebellar cortex. T h e present study was conducted using the anterograde labeling methods in order to further clarify the distribution of M C N axons within the raccoon cerebellum. Injections of [3H]leucine (50 #Ci/kd) were made m the ventral M C N in each of 6 adult raccoons using a
Correspondence: J. H. Haring, Department of Anatomy, St. Louis University School of Medicine, L402South Grand Boulevard. St Louis, MO 63104, U.S.A.
0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V
1(~5 micropipette attached to a 1 ul Hamilton syringe. The total volume of the injected amino acid solution ranged from 0.2 to 0.4 M in each MCN. A f t e r 4 days, animals were perfused intracardially with l()C/c neutral buffered formalin. Transverse frozen sections through the brainstem and sagittal sections through the cerebellum were coated with NTB-2 emulsion and exposed for 6 weeks~. The autoradiograms were then developed and the tissue sections counterstained through the emulsion with thionin. Sections were studied and p h o t o g r a p h e d on a Leitz dialux 20 microscope using either bright- or darkfield illumination. The sites of leucine injection were centered in the ventral MCN with no apparent tracer spread into the neighboring ECN (Fig. 1). A f t e r tracer injections of the ventral MCN, labeled axons were observed to exit the ventrolateral aspect of the MCN and traverse the ECN to enter the restiform body. Fibers ascend-
ed the brainstem in the restiform body and e n t e r e d the cerebellum via the inferior peduncle. Within the deep white matter of the cerebellum, axons from the MCN arched dorsally anterior to the deep cerebellar nuclei forming a fan-shaped array as fibers diverged to distribute to various cerebellar lobules. The m a i o r target of MCN afferents a p p e a r e d to be Iobule V, although substantial numbers of axons p r o j e c t e d to Iobules Ill and 1V. In addition, a small bundle of MCN fibers entered the paramedian Iobule and patterns of terminal labeling were seen within both the interposed and lateral cerebellar nuclei. Once the fibers had reached their respective cortical targets, terminals were distributed throughout the length of the individual folia from base to apex (Fig. 2). No difference was apparent in the relative densities of terminal fields seen in either the basal or apical regions of the target folia.
Fig. l. Brightfield photomicrograph of an injection site in the ventral MCN approximately 1 mm rostral to the obcx. ]'he center of the iniection (asterisk) is marked b x a dense accumulation of silver grains. The region surrounding the injection site is lcss heavily labeled and is seen to contain labeled neuronal perikarya (arrows) thus dcfining the zone of effective uptake of tracer. Note that the_'ECN is not mchlded within the injection site. x39.
Fig. 2. Darkfield photomicrographs of fibers t arrowheads) and terminals larrowsl seen in lobule V after rejections of the MCN. Note that labeled termipals are present in the granule cell layer of both the base (A) and the apical region (B/of Iobule V x 17"7
The present study has d e m o n s t r a t e d that the cerebellar projection of the M C N is directed primarily toward the anterior lobe wherein the m a j o r i t y of labeled fibers t e r m i n a t e in lobule V. Projections from the M C N to the p a r a m e d i a n lobule and deep cerebellar nuclei were also verified. These observations do not differ significantly from those o b t a i n e d in studies of the cat C C T 7. H o w e v e r , in contrast to findings in the cat, the labeled terminals of MCN axons were distributed throughout the length of the respective target folia. Thus, no segregation of M C N terminals from those of the ECN is likely. This result ts consistent with previous observations of r e t r o g r a d e labeling of the raccoon M C N after horseradish peroxidase injections of lobule V '). Our studies of cerebellar relay cells of the raccoon MCN indicate that the 'medial tongue" region ts both morphologically and functionally similar to the E C N 9Ao. Therefore, the raccoon C C T is concerned mainly with the conveying of information from d e e p receptors. A l t h o u g h there is a small cutaneous repre-
sentation in the MCN projection L~and the possibility of a similar transfer of cutaneous information through the ECN 4A3. the organization o f the raccoon CCT into distinct functional/anatomical c o m p o n e n t s is not evident. Rather. the ' m e d i a l tongue" region of the raccoon MCN appears to function as a subnucleus of the ECN, Because our evidence indicates that the ECN and MCN "medial tongue' may constitute an anatomical unit. the inability to d e m o n s t r a t e a regional segregation of terminals based u p o n cells of origin is not surprising. H o w e v e r . it is still possible that C C T projections are differentially distributed within the raccoon cerebellar cortex on the basis of sensory modality. We acknowledge the support and e n c o u r a g e m e n t of Dr. B. H. Pubols. Jr. and the excellent technical assistance of Christine H. Maliniak. This study was supported in part by Research G r a n t s NS-13418 and NS-19486 U S P H S .
167 l Check, M. D., Rustiani, A. a n d T r e v i n o , D. L., Dorsal colunto nuclei projections to the ccrebellar cortex in cats as revealed by the use of the retrograde transport of horseradish pcroxidasc. J. temp. Neurol.. 164 (1975) 31-46. 2 Cook, J. D., Larson. B., Oscarsson, O. and Sjolund, B., Origin and termination ot cuneocerebellar tract, t','.V). Brain Re,s., 13 (1971) 339-358. 3 ('owan, M. W., Goltlieb, l). l., Hcndrickson, A. K., Price. ,l. L. and Woolscy, T. A., The autoradiographic demonstration el' ~txonal COllnCCti(ms in the central nerxous system, Brain Re,~earch. 37 (1072) 21-51. 4 Dykes. R. \~,'., R a s m u s s e n , 1)., Srctavan. D. and R e h m a n , I-I. B., SubmoduliD. segregation and receptive-field scquenccs in cuncatc, gracilc, and external cunealc nuclei of lhc cat. J. Neurophysiol., 47 { 1982) 389-416. 5 Ekcrot. C.-F. and l.arson, B., Differential tcrmination el the cxtcroccplivc and propriccptivc components of the cuncocercbcllar tract. Brain Research, 3() (1972) 420-424. 6 (i(mld, B. B., Organization of affcrents from brain stem nuclei to thc cercbellar cortex in the cat. Advanc. Anat. Emhrvol. ('ell Biol., (~2 (lt)Sl)) 1 90. 7 (}rant. (i., Projection of the external cuncate nucleus onto thc cerebellum in the cat: an experimental study using silver methods. Krp. Neurol., 5 (1%2) 179-195. 8 (}ray, 3. S., Hazier(, ,l. C. and Martin, G. F., Organization of projections, Iron3 the gracilc, medial cuneatc and lateral cuncatc nuclei m the North American opossum. Horseradish peroxidasc studx of cells projecting to the cerebellum.
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10
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thalamus, and spinal cord, Brain Behav. Evol., 18 (19811 140-156. Haring, J. H. and Rowinski, M. J., A horseradish pert)xidase study of projections from the main and external cuncatc nuclei to the cerebellum of the North American raccoon. J. coral). Neuro[., 211 (1982) 3~33 37t3. Haring, J. H.. Rowinski, M. J. and Pubols, B. l l.. Electrophysiology ¢)1 raccoon cuncoccrebcl]ar neurons, ,~omffloven,sory Re.v, I (1984) 247-264. ,lohnson, J. 1., Wclkcr, W. 1. and Pubols, B. H.. Somatopic organization of raccoon dorsal c o h m m nuclei, J. temp. Neurol., 132 (1968) 1-44. Kuypers. H. G. J. M. m)d Tucrk, J. I)., I'hc distribution of the cortical fibers ~ithin the nuclei cuncalus and gracilis in the cal,J. Aria;. (Lend.), 98 (1964) 143-1(32. Raush. J., Electrophw'ioloKv (~/'1he K~termd ('urn'ate Nudeu.s in the Cat, Ph. D. dissertation, t;ni~crsitv of WashingIon, Seattle, W A , 1960. Rinvik, E. and Walbcrg. F.. Studies on the ccrcbellar pro]ections from main and external cuncatc nuclei in the cat bv means of retrograde axonal transport of horseradish peroxidasc, Brain Re.search. 95 (1975) 371-381. Rosen, l., Localization in caudal brain slcm and cervical spinal cord of neurons activated from forelimb group I afferents in the cat, Brain Research, 16 (1969) 55-71. Somana, R. and Walberg, F., A re-examination ol the ccrebellar projections from the gracilc, main and external cuneate nuclei in the cat, Brain Re,warch, IS6 (19811) 33 42.