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A double-labelling study of reticular collaterals to the spinal cord and cerebellum of the North American opossum
Neuroscience Letters, 47 (1984) 185-191
185
Elsevier Scientific Publishers Ireland Ltd. NSL 02742 A DOUBLE-LABELLING STUDY OF RETICULAR COLLATERALS TO THE SPINAL CORD AND CEREBELLUM OF THE NORTH AMERICAN OPOSSUM
G.F. MARTIN and R.P. WALTZER Department of Anatomy, College of Medicine, The Ohio State University, 1645 Neil A venue, Columbus, OH 43210 (U.S.A.)
(Received July 12th, 1983; Revised version received January 23rd, 1984; Accepted March 13th, 1984)
Key words: reticular formation - spinal cord - cerebellum - collaterals
Injections of horseradish peroxidase into either the spinal cord or cerebellum label neurons in the gigantocellular and lateral reticular nuclei of the North American opossum. In order to determine if neurons which project to the spinal cord and cerebellum are intermingled in these two nuclei and if single neurons provide collaterals to both areas, we have employed fluorescent markers in double-labelling experiments. Our results show that reticular neurons innervating either the spinal cord or cerebellum are often close together and that a few provide collaterals to both areas. Neurons providing such collaterals are rare, however, comprising 2% or less of those innervating either target alone.
T h e results o f retrograde degeneration studies in kittens [1, 18] have suggested that little overlap exists in the location o f reticular neurons which innervate the spinal c o r d and cerebellum. In the course o f studies using the N o r t h A m e r i c a n o p o s s u m , however, we have m a d e injections o f horseradish peroxidase into the spinal cord [11, 13] and cerebellum [8, 9] and have been impressed by the degree to which neurons are labelled in some o f the same reticular nuclei. This observation has led us to investigate the question o f overlap m o r e directly using the doublelabelling technique described in Kuypers et al. [7]. This technique has also allowed us to determine whether single reticular neurons innervate b o t h the spinal cord and cerebellum, p r e s u m a b l y via axonal collaterals. Eight adult o p o s s u m s were used for o u r experiments. Five o f t h e m received injections o f True Blue (TB) in the rostral cervical cord, followed 6-7 days later by injections o f Nuclear Yellow (NY) in the cerebellum. In two cases the markers were reversed. In one control experiment, b o t h TB and NY were injected into the cerebellum. All o f the injections were intentionally large and those in the spinal cord o f t e n involved reticular axons in the white matter as well as terminal fields [11, 13]. Since H R P studies showed that injections o f the anterior lobe and posterior lobe vermis were particularly effective for labelling reticular neurons, m o s t o f the 0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.
186
cerebellar injections were made into those areas (6 animals). In one animal injections were made into the lobus simplex, crus I and II as well as the paramedian lobule. All of the cerebellar injections were limited to the cortex. The markers were injected at different times because of their different rates of transport [7] and in all cases the second injection was made ipsilateral to the first on the basis of the predominant laterality of the connections in question. Neurons are labelled contralateral to both spinal or cerebellar injection, but we wished to maximize the potential for overlap in the double-labelling experiments. All animals were anesthetized with sodium pentobarbitol (40 mg/kg body wt.) for the surgical exposures and injections. After stabilization in either a spinal frame or head holder the injections were made using a micropipette attached to a Hamilton syringe. Both markers were dissolved in distilled water (2-10o70 solutions) and delivered in volumes varying from 1.0 to 4.5 #1. Twenty-four hours or less after the NY injection(s) the animals were reanesthetized and sacrificed. The survival time for NY transport was adjusted so as to minimize glial labelling. In two cases the animals were unperfused. Their brains and spinal cords were removed under deep anesthesia, frozen immediately with dry ice and sectioned at 32/zm on a cryostat. The remaining 5 animals were perfused with 30°70 formalin buffered to a pH of 7.2 with a cacodylate buffer after Huisman et al. [5]. In the perfused cases the removed brain and injected segment of the spinal cord were sectioned at 40/~m on a freezing microtome. All sections were mounted immediately, coverslipped using a nonfluorescent mounting medium and examined using a Leitz microscope equipped for epifluorescence. Both markers were viewed using an excitation wavelength of 360 nm and the positions of neurons labelled by TB or NY alone or by both markers were recorded using an x - y plotter interfaced with the microscope stage by potentiometers. The terminology used for the opossum's brainstem was taken from Oswaldo-Cruz and Rocha-Miranda [15]. Counts of labelled neurons were taken from evenly spaced (1 out of 4) sections. Fig. 1 shows the medullary labelling present in a representative case subjected to injections of TB in the rostral cervical cord and NY in the anterior lobe of the cerebellum. Reticular neurons labelled by TB were located within the nucleus reticularis gigantocellularis; pars ventralis, the nucleus reticularis lateralis, the nucleus reticularis gigantocellularis, the nuclei medullae oblongatae ventralis and dorsalis and along the dorsal and medial borders of the nucleus lateralis reticularis. Fig. 1. Plot of neurons in the medulla (rostral, A, to caudal, E) labelled by the spinal injection of True Blue (TB) shown at the lower right and the cerebellar injection of Nuclear Yellow (NY) illustrated at the upper left. The symbols for neurons labelled by TB and NY alone, as well as by both markers, are indicated on the illustration. The following structures are labelled after Oswaldo-Cruz and Rocha-Miranda [15]: cr, restiform body; Cu, nucleus cuneatus; CuL, nucleus cuneatus lateralis; Hg, nucleus hypoglossi; LR, nucleus reticularis lateralis; OI, nucleus olivaris inferior; RaM, nucleus magnus raphae; RaO, nucleus obscurus raphae; RGc, nucleus reticularis gigantocellularis; RGcv, nucleus reticularis gigantocellularis; pars ventralis; RV, nucleus medulla oblongatae ventralis; Vstl, nucleus vestibularis inferior.
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188 The latter c o r r e s p o n d s to the classical lateral reticular nucleus [9]. As can be seen f r o m Fig. 1 n e u r o n s labelled by NY were f o u n d in m a n y o f the same areas, b u t they were p a r t i c u l a r l y n u m e r o u s within the nucleus reticularis gigantocellularis (RGc) a n d the nucleus lateralis reticularis (LR). Labelled n e u r o n s in the RGc were often f o u n d in clusters medial to the hypoglossal nerve p r o b a b l y c o m p a r a b l e to the p a r a m e d i a n cell groups described in the cat [2]. As expected, NY-labelled n e u r o n s were also f o u n d in the inferior olive, the lateral c u n e a t e nucleus a n d the vestibular complex. Based o n the olivary labelling, it appeared that zones A - C of the anterior lobe were included in the injections [8, 14]. N e u r o n s labelled by T B or NY a l o n e were i n t e r m i n g l e d in the RGc (arrows in Fig. 1B, C; Fig. 2A). Very few n e u r o n s were double-labelled, however. In n o case did the n u m b e r o f d o u b l e - l a b e l l e d n e u r o n s exceed 2.0°7o of those labelled by either TB or NY alone a n d in one of them, n o reticular n e u r o n s were d o u b l e labelled. N e u r o n s c o n t a i n i n g TB or NY were also i n t e r m i n g l e d a l o n g the dorsal a n d medial borders of the nucleus lateralis reticularis, but no double-labelled n e u r o n s were f o u n d .
Fig. 2. Photomicrographs of True Blue (TB) and Nuclear Yellow (NY) labelled neurons in the nucleus reticularis gigantocellularis (RGc) (A) and the nucleus lateralis reticularis (LR), (B). The insert in A illustrates double-labelled (DL) neurons in the RGc. The bar at the lower left of A indicates 27/~m and can be used for A and B. The bar at the lower left of the insert indicates the same measurement. Dorsal and ventral are indicated for both A and B in B. The photomicrograph in A was taken from an animal in which TB was injected into the spinal cord and NY was injected into the anterior lobe of the cerebellum. 2B was taken from a case in which the markers were reversed.
189 In light of the small number of double-labelled neurons, it seemed appropriate to ask if reticular neurons can be double labelled in large numbers. In a previous communication [12] we showed that 50% or more of RGc neurons labelled by injections o f TB in the cervical cord could also be labelled by NY injections o f the lumbar cord. In order to assure ourselves that cerebellar projecting neurons of the reticular formation could be double labelled, we made injections o f TB and NY into the same area o f the anterior lobe. Spread o f TB was much less than that o f NY as evidenced by direct examination and the labelling in the inferior olive. Olivary neurons containing TB were found in restricted areas o f the dorsal and medial accessory nuclei, but most, if not all of them contained NY. Neurons containing only NY were widespread in the same nuclei. In some sections through the RGc virtually every neuron was double labelled (insert, Fig. 2A). The use of fluorescent markers in double-labelling paradigms is one of the few methods available for determining if single neurons project to widely separate areas o f the brain. It appears that the method is sensitive, but it is not likely to reveal all o f the neurons within a given nucleus which provide collateral innervation to injected areas. We tried to make injections which included both terminal areas and fibers o f passage, but we cannot claim to have involved all o f the axons in question. It is also possible that small amounts o f one marker within a neuron were obscured by large amounts o f the other a n d / o r that double-labelled neurons were recorded as labelled by TB alone if their nucleus was not included in the section. In spite of such problems, double-labelled neurons are numerous after other injection combinations of the same markers [5, 12]. In the opossum (present study) and rat [20] overlap exists in the location of reticular neurons which innervate the spinal cord and cerebellum. Comparison of the published plots o f neurons labelled after spinal [3, 4, 6] and cerebellar [17, 19] injections of horseradish peroxidase suggests that similar overlap exists in the cat. Our results also indicate that a few reticular neurons in the areas of overlap innervate both the spinal cord and cerebellum, presumably via axonal collaterals. The Golgi studies of Scheibel and Scheibel [16] suggested that reticular neurons innervate widely separate areas of the neural axis although they did not demonstrate reticular neurons with collaterals in the spinal cord and cerebellum. In light o f the fact that reticular neurons are characterized by the presence of axonal collaterals [16], we were surprised by the small number o f neurons which project to both the spinal cord and cerebellum. The Golgi studies of the Scheibels were performed on newborn animals and it is possible that reticular neurons which provide collateral innervation to distant targets are more numerous during development than in the adult animal. Because o f its unique embryology [10], the opossum may be a good model for testing that hypothesis. The authors wish to thank Ms. Mary Ann Jarrell for the excellence of her technical help. We are also grateful to Mrs. Nina J. Anspaugh for typing the
190 manuscript
i n f i n a l f o r m a n d M r . K a r l B. R u b i n f o r p h o t o g r a p h i c
vestigation was supported
by U.S.P.H.S.
help. This in-
Grant BNS-8309245.
1 Brodal, A., Reticulo-cerebellar connections in the cat. An experimental study, J. comp. Neurol., 98 (1953) 113-154. 2 Brodal, A. and Torvik, A., Cerebellar projection of paramedian reticular nucleus of the medulla oblongata in cat, J. Neurophysiol., 17 (1954) 484-495. 3 Hayes, N.L. and Rustioni, A., Descending projections from the hralnstem and sensorimotor cortex to spinal enlargements in the cat. Single and double retrograde tracer studies, Exp. Brain Res., 41 (1981) 89-107. 4 Holstege, G. and Kuypers, H.G.J.M., The anatomy of brain stem pathways to the spinal cord in cat. In H.G.J.M. Kuypers and G.F. Martin (Eds.), Descending Pathways to the Spinal Cord, Progress in Brain Research, Vol. 57, Elsevier, Amsterdam, 1982, pp. 145-175. 5 Huisman, A.M., Kuypers, H.G.J.M. and Verburgh, C.A., Differences in collateralization of the descending spinal pathways from the red nucleus and other brain stem cell groups in cat and monkey. In H.G.J.M. Kuypers and G.F. Martin (Eds.), Descending Pathways to the Spinal Cord, Progress in Brain Research, Vol. 57, Elsevier, Amsterdam, 1982, pp. 186-217. 6 Kuypers, H.G.J.M. and Maisky, V.A., Retrograde axonal transport of horseradish peroxidase from spinal cord to brain stem cell groups in the cat, Neurosci. Lett., 1 (1975) 9-14. 7 Kuypers, H.G.J.M., Bentivoglio, M., Catsman-Berrevoets, C.E. and Bharos, T.B., Double retrograde neuronal labelling through divergent axon collaterals, using two fluorescent tracers with the same excitation wavelength which label different features of the cell, Exp. Brain Res., 40 (1980) 383-392. 8 Linauts, M. and Martin, G.F., The organization of olivo-cerebellar projections in the opossum, Didelphis virginiana, as revealed by the retrograde transport of horseradish peroxidase, J. comp. Neurol., 179 (1978) 355-382. 9 Martin, G.F., Andrezik, J.A., Crutcher, K., Lilaauts, M. and Panneton, M., The lateral reticular nucleus of the opossum (Didelphis virginiana). II. Connections, J. comp. Neurol., 17 (1977) 151-186. 10 Martin, G.F., Beals, J.K., Culherson, J.L., Dom, R., Goode, G. and Humhertson, A.O., Observations on the development of brainstem-spinal systems in the North American opossum, J. comp. Neurol., 181 (1978) 271-289. 11 Martin, G.F., Humhertson, A.O., Laxson, L.C., Panneton, W.M. and Tschismadia, I., Spinal projections from the mesencephalic and pontine reticular formation in the North American opossum. A study using axonal transport techniques, J. comp. Neurol., 187 (1979) 373-400. 12 Martin, G.F., Cabana, T. and Humhertson, A.O., Evidence for collateral innervation of the cervical and lumbar enlargements of the spinal cord by single reticular and raphe neurons. Studies using fluorescent markers in double-labelling experiments on the North American opossum, Neurosci. Lett., 24 (1981) 1-6. 13 Martin, G.F., Cabana, T., Humbertson, A.O., Laxson, L.C. and Panneton, W.M., Spinal projections from the medullary reticular formation of the North American opossum: evidence for connectional heterogeneity, J. comp. Neurol., 196 (1981) 663-682. 14 Martin, G.F., Bishop, G.A., Culberson, J.L., King, J.S., Laxson, C., Linauts, M. and Panneton, M., Spino-olivocerehellar circuits in the North American opossum with notes on their development. In S.L. Palay and V. Chan-Palay (Eds.), The Cerebellum, New Vistas, Experimental Brain Research, Supplementum 6, Springer-Verlag, Heidelberg, 1982, pp. 162-188. 15 Oswaldo-Cruz, E. and Rocha-Miranda, C.E., The Brain of the Opossum (Didelphis virginiana). A Cytoarchitectural Atlas in Stereotaxic Coordinates, Instituto de Biofisica, Universidade Federal de Rio de Janeiro, 1968.
191 16 Scheibel, M.E. and Scheibel, A.B., Structural substrates for integrative patterns in the brainstem reticular core. In H.H. Jasper, L.D. Proctor, R.S. Knighton, W.S. Noshay and R.T. Costello (Eds.), Reticular Formation of the Brain (Henry Ford Hospital Symposium), Little Brown, Boston, 1958, pp. 31-55. 17 Somana, R. and Walberg, F., Cerebellar afferents from the paramedian reticular nucleus studied with retrograde transport of horseradish peroxidase, Anat. Embryol., 154 (1978) 353-368. 18 Torvik, A. and Brodal, A., The origin of reticulospinal fibers in the cat. An experimental study, Anat. Rec., 128 (1957) 113-137. 19 Walberg, F., The vestibulo- and reticulocerebellar projections in the cat as studied with horseradish peroxidase as a retrograde tracer. In S.L. Palay and V. Chan-Palay (Eds.), The Cerebellum, New Vistas, Experimental Brain Research Supplementum 9, Springer-Verlag, Heidelberg, 1982, pp. 477-500. 20 Waltzer, R.P. and Martin, G.F., A double labelling study demonstrating that most cells in the nucleus reticularis gigantocellularis and adjacent raphe project to either the anterior lobe of the cerebellum or the spinal cord in the rat, Neurosci. Abstr., 8 (1982) 250:2.
185
Elsevier Scientific Publishers Ireland Ltd. NSL 02742 A DOUBLE-LABELLING STUDY OF RETICULAR COLLATERALS TO THE SPINAL CORD AND CEREBELLUM OF THE NORTH AMERICAN OPOSSUM
G.F. MARTIN and R.P. WALTZER Department of Anatomy, College of Medicine, The Ohio State University, 1645 Neil A venue, Columbus, OH 43210 (U.S.A.)
(Received July 12th, 1983; Revised version received January 23rd, 1984; Accepted March 13th, 1984)
Key words: reticular formation - spinal cord - cerebellum - collaterals
Injections of horseradish peroxidase into either the spinal cord or cerebellum label neurons in the gigantocellular and lateral reticular nuclei of the North American opossum. In order to determine if neurons which project to the spinal cord and cerebellum are intermingled in these two nuclei and if single neurons provide collaterals to both areas, we have employed fluorescent markers in double-labelling experiments. Our results show that reticular neurons innervating either the spinal cord or cerebellum are often close together and that a few provide collaterals to both areas. Neurons providing such collaterals are rare, however, comprising 2% or less of those innervating either target alone.
T h e results o f retrograde degeneration studies in kittens [1, 18] have suggested that little overlap exists in the location o f reticular neurons which innervate the spinal c o r d and cerebellum. In the course o f studies using the N o r t h A m e r i c a n o p o s s u m , however, we have m a d e injections o f horseradish peroxidase into the spinal cord [11, 13] and cerebellum [8, 9] and have been impressed by the degree to which neurons are labelled in some o f the same reticular nuclei. This observation has led us to investigate the question o f overlap m o r e directly using the doublelabelling technique described in Kuypers et al. [7]. This technique has also allowed us to determine whether single reticular neurons innervate b o t h the spinal cord and cerebellum, p r e s u m a b l y via axonal collaterals. Eight adult o p o s s u m s were used for o u r experiments. Five o f t h e m received injections o f True Blue (TB) in the rostral cervical cord, followed 6-7 days later by injections o f Nuclear Yellow (NY) in the cerebellum. In two cases the markers were reversed. In one control experiment, b o t h TB and NY were injected into the cerebellum. All o f the injections were intentionally large and those in the spinal cord o f t e n involved reticular axons in the white matter as well as terminal fields [11, 13]. Since H R P studies showed that injections o f the anterior lobe and posterior lobe vermis were particularly effective for labelling reticular neurons, m o s t o f the 0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.
186
cerebellar injections were made into those areas (6 animals). In one animal injections were made into the lobus simplex, crus I and II as well as the paramedian lobule. All of the cerebellar injections were limited to the cortex. The markers were injected at different times because of their different rates of transport [7] and in all cases the second injection was made ipsilateral to the first on the basis of the predominant laterality of the connections in question. Neurons are labelled contralateral to both spinal or cerebellar injection, but we wished to maximize the potential for overlap in the double-labelling experiments. All animals were anesthetized with sodium pentobarbitol (40 mg/kg body wt.) for the surgical exposures and injections. After stabilization in either a spinal frame or head holder the injections were made using a micropipette attached to a Hamilton syringe. Both markers were dissolved in distilled water (2-10o70 solutions) and delivered in volumes varying from 1.0 to 4.5 #1. Twenty-four hours or less after the NY injection(s) the animals were reanesthetized and sacrificed. The survival time for NY transport was adjusted so as to minimize glial labelling. In two cases the animals were unperfused. Their brains and spinal cords were removed under deep anesthesia, frozen immediately with dry ice and sectioned at 32/zm on a cryostat. The remaining 5 animals were perfused with 30°70 formalin buffered to a pH of 7.2 with a cacodylate buffer after Huisman et al. [5]. In the perfused cases the removed brain and injected segment of the spinal cord were sectioned at 40/~m on a freezing microtome. All sections were mounted immediately, coverslipped using a nonfluorescent mounting medium and examined using a Leitz microscope equipped for epifluorescence. Both markers were viewed using an excitation wavelength of 360 nm and the positions of neurons labelled by TB or NY alone or by both markers were recorded using an x - y plotter interfaced with the microscope stage by potentiometers. The terminology used for the opossum's brainstem was taken from Oswaldo-Cruz and Rocha-Miranda [15]. Counts of labelled neurons were taken from evenly spaced (1 out of 4) sections. Fig. 1 shows the medullary labelling present in a representative case subjected to injections of TB in the rostral cervical cord and NY in the anterior lobe of the cerebellum. Reticular neurons labelled by TB were located within the nucleus reticularis gigantocellularis; pars ventralis, the nucleus reticularis lateralis, the nucleus reticularis gigantocellularis, the nuclei medullae oblongatae ventralis and dorsalis and along the dorsal and medial borders of the nucleus lateralis reticularis. Fig. 1. Plot of neurons in the medulla (rostral, A, to caudal, E) labelled by the spinal injection of True Blue (TB) shown at the lower right and the cerebellar injection of Nuclear Yellow (NY) illustrated at the upper left. The symbols for neurons labelled by TB and NY alone, as well as by both markers, are indicated on the illustration. The following structures are labelled after Oswaldo-Cruz and Rocha-Miranda [15]: cr, restiform body; Cu, nucleus cuneatus; CuL, nucleus cuneatus lateralis; Hg, nucleus hypoglossi; LR, nucleus reticularis lateralis; OI, nucleus olivaris inferior; RaM, nucleus magnus raphae; RaO, nucleus obscurus raphae; RGc, nucleus reticularis gigantocellularis; RGcv, nucleus reticularis gigantocellularis; pars ventralis; RV, nucleus medulla oblongatae ventralis; Vstl, nucleus vestibularis inferior.
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188 The latter c o r r e s p o n d s to the classical lateral reticular nucleus [9]. As can be seen f r o m Fig. 1 n e u r o n s labelled by NY were f o u n d in m a n y o f the same areas, b u t they were p a r t i c u l a r l y n u m e r o u s within the nucleus reticularis gigantocellularis (RGc) a n d the nucleus lateralis reticularis (LR). Labelled n e u r o n s in the RGc were often f o u n d in clusters medial to the hypoglossal nerve p r o b a b l y c o m p a r a b l e to the p a r a m e d i a n cell groups described in the cat [2]. As expected, NY-labelled n e u r o n s were also f o u n d in the inferior olive, the lateral c u n e a t e nucleus a n d the vestibular complex. Based o n the olivary labelling, it appeared that zones A - C of the anterior lobe were included in the injections [8, 14]. N e u r o n s labelled by T B or NY a l o n e were i n t e r m i n g l e d in the RGc (arrows in Fig. 1B, C; Fig. 2A). Very few n e u r o n s were double-labelled, however. In n o case did the n u m b e r o f d o u b l e - l a b e l l e d n e u r o n s exceed 2.0°7o of those labelled by either TB or NY alone a n d in one of them, n o reticular n e u r o n s were d o u b l e labelled. N e u r o n s c o n t a i n i n g TB or NY were also i n t e r m i n g l e d a l o n g the dorsal a n d medial borders of the nucleus lateralis reticularis, but no double-labelled n e u r o n s were f o u n d .
Fig. 2. Photomicrographs of True Blue (TB) and Nuclear Yellow (NY) labelled neurons in the nucleus reticularis gigantocellularis (RGc) (A) and the nucleus lateralis reticularis (LR), (B). The insert in A illustrates double-labelled (DL) neurons in the RGc. The bar at the lower left of A indicates 27/~m and can be used for A and B. The bar at the lower left of the insert indicates the same measurement. Dorsal and ventral are indicated for both A and B in B. The photomicrograph in A was taken from an animal in which TB was injected into the spinal cord and NY was injected into the anterior lobe of the cerebellum. 2B was taken from a case in which the markers were reversed.
189 In light of the small number of double-labelled neurons, it seemed appropriate to ask if reticular neurons can be double labelled in large numbers. In a previous communication [12] we showed that 50% or more of RGc neurons labelled by injections o f TB in the cervical cord could also be labelled by NY injections o f the lumbar cord. In order to assure ourselves that cerebellar projecting neurons of the reticular formation could be double labelled, we made injections o f TB and NY into the same area o f the anterior lobe. Spread o f TB was much less than that o f NY as evidenced by direct examination and the labelling in the inferior olive. Olivary neurons containing TB were found in restricted areas o f the dorsal and medial accessory nuclei, but most, if not all of them contained NY. Neurons containing only NY were widespread in the same nuclei. In some sections through the RGc virtually every neuron was double labelled (insert, Fig. 2A). The use of fluorescent markers in double-labelling paradigms is one of the few methods available for determining if single neurons project to widely separate areas o f the brain. It appears that the method is sensitive, but it is not likely to reveal all o f the neurons within a given nucleus which provide collateral innervation to injected areas. We tried to make injections which included both terminal areas and fibers o f passage, but we cannot claim to have involved all o f the axons in question. It is also possible that small amounts o f one marker within a neuron were obscured by large amounts o f the other a n d / o r that double-labelled neurons were recorded as labelled by TB alone if their nucleus was not included in the section. In spite of such problems, double-labelled neurons are numerous after other injection combinations of the same markers [5, 12]. In the opossum (present study) and rat [20] overlap exists in the location of reticular neurons which innervate the spinal cord and cerebellum. Comparison of the published plots o f neurons labelled after spinal [3, 4, 6] and cerebellar [17, 19] injections of horseradish peroxidase suggests that similar overlap exists in the cat. Our results also indicate that a few reticular neurons in the areas of overlap innervate both the spinal cord and cerebellum, presumably via axonal collaterals. The Golgi studies of Scheibel and Scheibel [16] suggested that reticular neurons innervate widely separate areas of the neural axis although they did not demonstrate reticular neurons with collaterals in the spinal cord and cerebellum. In light o f the fact that reticular neurons are characterized by the presence of axonal collaterals [16], we were surprised by the small number o f neurons which project to both the spinal cord and cerebellum. The Golgi studies of the Scheibels were performed on newborn animals and it is possible that reticular neurons which provide collateral innervation to distant targets are more numerous during development than in the adult animal. Because o f its unique embryology [10], the opossum may be a good model for testing that hypothesis. The authors wish to thank Ms. Mary Ann Jarrell for the excellence of her technical help. We are also grateful to Mrs. Nina J. Anspaugh for typing the
190 manuscript
i n f i n a l f o r m a n d M r . K a r l B. R u b i n f o r p h o t o g r a p h i c
vestigation was supported
by U.S.P.H.S.
help. This in-
Grant BNS-8309245.
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