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Interneurons and initial axon collaterals in the feline gracile nucleus demonstrated with the rapid Golgi technique
Brain Research, 111 (1976) 407-410
407
~ Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
Interneurons and initial axon collaterals in the feline gracile nucleus demonstrated with the rapid Golgi technique
ANDERS BLOMQV1ST and JAN WESTMAN Department of Anatomy, Biomedical Centre, University ~f Uppsala, Uppsala (Sweden)
(Accepted April 12th, 1976)
The feline gracile nucleus can no longer be regarded as a simple relay station in the dorsal column pathway. Neuroanatomical studies have revealed rostrocaudal differences regarding both the cytoarchitecture 15 and the termination of the different afferents 14,1~,2°. Furthermore, efferent fibers terminate in other places besides the thalamus. There are well documented projections both to the inferior olive 7 and to the cerebellum 8. The results of experiments employing neurophysiological methods suggest a complex pattern of impulse modulation in the nucleus. Thus a large part of the neurons projecting into the medial lemniscus is subjected both to surround inhibition and supraspinal controP°, 11. Another interesting feature is presynaptic inhibition 2. The possible morphological correlate, axo-axonal contacts 21, is also found in the gracile nucleus.5, iv. The results of the present study are of special interest in relation to the physiological findings discussed above. They were made during analysis of Golgi material in order to identify the different kinds of projection neurons recently demonstrated 3, 5,8 by use of retrograde axonal transport of HRP. The gracile nuclei of 19 cats, ranging in age from newborn to adult, were investigated. Staining was performed according to the rapid Golgi method 18. After exsanguination of the animal, the lower brain stem and upper spinal cord were dissected out and cut into smaller pieces. The specimens were immersed in a fixation solution of 2 ~ potassium dichromate and 0.2 ~,, osmium tetroxide and left for 2-6 days in darkness. They were then transferred to a solution of 0.75 °/o silver nitrate and kept in it for 1-2 days. The specimens were dehydrated in acetone and alcohol, embedded in celloidin and cut on a sliding microtome in 100-150 # m thick sections. Most specimens were cut in transverse sections, but longitudinal sections were also made. Drawings were made with the help of a binocular drawing apparatus. In the gracile nucleus, beside cells projecting into the medial lemniscus, neurons were found whose axons ramify after a short distance and end with fine branches showing boutons of passage and clusters of boutons (Fig. 1). These cells were classified as interneurons. So far, 10 such cells have been identified with certainty. They are small to medium sized, with a perikaryal diameter of 11-17/~m (mean 14). The number
408
\
100 pm
Fig. 1. Rapid Golgi impregnation. Kitten, I I days old. lnterneuron situated in the rostral part of the gracile nucleus.
of primary dendrites varies between 2 and 4 (mean 3). Mostly the dendrites are situated at each pole of the perikaryon. In 7 neurons the dendritic field occupies a narrow strip in the transverse plane, but three of the neurons have dendrites which spread widely in all directions. The lengths of individual dendrites are up to 200/,m. The axon emerges from a dendrite in 5 of the l0 neurons. Axons of projection neurons in the gracile nucleus frequently show initial collaterals. These collaterals leave the stem axon at varying distances from the perikaryon, the most proximal being found after 15 #m, and terminate within the nucleus with boutons of passage and clusters of boutons (Fig. 2). Up to 3 collaterals per stem axon have been observed. Neither interneurons nor axon collaterals have earlier been demonstrated anatomically in the feline gracile nucleus. However, the finding of surround inhibition ~z implied the presence of interneurons. The existence of axon collaterals was suggested by Gordon and Jukes n, who found transsynaptic inhibition of gracile neurons following stimulation of the medial lemniscus. In our study it has only been possible to identify a small number of cells as interneurons. However, most cells escape classification, as their axons are unimpregnated or leave the plane of section after a short distance. The fact that many small neurons in the gracile nucleus remain unlabeled following injection of H R P into gracile projection areas 4 suggests that interneurons are quite frequent. Although no final conclusion can be drawn from the present small material, it is interesting to note that interneurons with different dendritic organizations have been found. Further studies will reveal whether this or other features may permit a division of the interneurons into distinct morphological types. In the cuneate nucleus different types of interneurons are thought to mediate pre- and postsynaptic inhibition, respectively 1. Also the cells that receive pyramidal tract fibers may constitute a separate group of interneurons 1. The physiological importance of the initial collaterals has not been thoroughly investigated. As they seem to be rather frequent, it is tempting to suggest that the collaterals play a significant role in impulse modulation. In the rat cuneate nucleus
409
/
100 p m
Fig. 2. Rapid Golgi impregnation. Kitten, 2 days old. Projection neuron situated in the middle part of the gracile nucleus. Note the initial collateral which ramifies and terminates in the vicinity of the neuron.
recurrent collaterals are t h o u g h t to f o r m a p a t h w a y , including a n u m b e r o f intern e u r o n s t h a t p r o d u c e p r e s y n a p t i c inhibition o f c u n e o t h a l a m i c relay cells 9. Electron m i c r o s c o p i c a l investigations o f the gracile nucleus have revealed t h a t a large n u m b e r o f b o u t o n s r e m a i n u n c h a n g e d following t r a n s e c t i o n o f different afferentsS, 17. I t now seems evident t h a t m a n y o f these b o u t o n s are s u p p l i e d by interneurons and initial collaterals. The results o f the present G o l g i s t u d y have thus given new d a t a c o n c e r n i n g the s y n a p t o l o g y in the feline gracile nucleus. T h e y are in g o o d a g r e e m e n t with findings in investigations using o t h e r m e t h o d s a n d also indicate t h a t the gracile nucleus in the cat has an o r g a n i z a t i o n similar to t h a t in r o d e n t s 13,19. The a u t h o r s are i n d e b t e d to Dr. Bo W i k s t e n , who kindly put p a r t o f the investigated material at o u r disposal. The skilful technical assistance o f Mrs. Kfirstin F l i n k is gratefully a c k n o w l e d g e d . This s t u d y was s u p p o r t e d by grants from the Swedish Medical Research Council (Project No. B76-12X-02710-08).
I Andersen, P., Eccles, J. C., Schmidt, R. F. and Yokota, T., Identification of relay cells and interneurons in the cuneate nucleus, J. Neurophysiol., 27 (1964) 1080-1095. 2 Andersen, P., Etholm, B. and Gordon, G., Presynaptic and post-synaptic inhibition elicited in the cat's dorsal column nuclei by mechanical stimulation of skin, J. Physiol. (Lond.), 210 (1970) 433-455. 3 Berkley, K. J., Different targets of different neurons in nucleus gracilis of the cat, d. comp. Neurol., 163 (1975) 285 304. 4 Blomqvist, A., In preparation. 5 Blomqvist, A. and Westman, J., An electron microscopical study of the gracile nucleus in the cat Acta Soc. Med. upsalien., 75 (1970) 241-252.
410 6 Blomqvist, A. and Westman. J., Combined HRP and Fink Heimer s|ainmg applied ~n the gracile nucleus in the cat, Brain ResearcM 99 (1975) 339 342. 7 Boesten, A. J. P. and Voogd, J., Projections o[" the dorsal column nuclei and the spinal cord on the inferior olive in the cqt. J. eon*p. Neto-ol., 161 (1975) 215-,238. 8 Cheek, M. D., Rustioni, A. and Trevino, D. L., Dorsal column nuclei projections to the cerebellar cortex in cats as revealed by the use of the retrograde transport of horseradish peroxidase, ,I. eomp. Neurol., 164 (1975) 31 46. 9 Davidson, N. and Smith, ( . A., A recurrent collateral pathway for presynaptic inhibition m the rat cuneate nucleus, Brain Research, 44 (I 972) 63- 71. 10 Gordon, G. and Jukes, M. G. M., Dual organization of the exteroceptive components of the cat's gracile nucleus, J. I-'hysiol. ( L o n d . , 173 (1964) 263--290. 11 Gordon, G. and Jukes, M. G. M,, Descending influences on the exteroceptive organizations of the cat's gracile nucleus, J. Phy,~iol. florid. ), 173 (I 964) 29 I-319. 12 Gordon, G. and Paine, ('. H., Functional organization in nucleus gracilis of the cat..I. IJhysiol. (Lond.), 153 (1960) 331 349. 13 Gulley, R. L., Golgi studies of the nucleus gracilis in the rat, Anat. Rec., 177 (1973) 325442. 14 Hand, P. J., Lumbosacral dorsal root terminations in the nucleus gracilis of the cat. Some observations on terminal degeneration in other medullary sensory nuclei, J. eomp. NeuroL, 126 (19661 137 156. 15 Kuypers, H. G. J. M. and Tuerk, J. D., The distribution of the cortical fibres within the nuclei cuneatus and gracilis in the cat, J. AHat. (Lond.), 98 (1964) 143-162. 16 Rustioni, A., Non-primary afferents to the nucleus gracilis from the lumbar cord of the cat, Brai, Research, 51 (1973) 81 95. 17 Rustioni, A. and Sotelo, ( . , Synaptic organization of the nucleus gracilis of the cat. Experimental identification of dorsal root fibers and cortical afferents, J. comp. Neurol., 155 (1974) 441 468. 18 Strong, O., Review of the Golgi method, J. eomp. Neurol., 6 (1896) 101-127. 19 Valverde, F.. The pyramidal tract in rodents. A study of its relations with the posterior column nuclei, dorsolateral reticular formation of the medulla oblongata, and cervical spinal cord, Z. ZellJbrsch., 71 (1966) 297-363. 20 Walberg, F., Corticofugal fibres to the nuclei of the dorsal columns, Brait~, 80 (19571 273 287. 21 Walberg, F., Axoaxonic contacts in the cuneate nucleus, probable basis for presynaptic depolarization, E.vp. Neurol., 13 (1965) 218 231.
407
~ Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
Interneurons and initial axon collaterals in the feline gracile nucleus demonstrated with the rapid Golgi technique
ANDERS BLOMQV1ST and JAN WESTMAN Department of Anatomy, Biomedical Centre, University ~f Uppsala, Uppsala (Sweden)
(Accepted April 12th, 1976)
The feline gracile nucleus can no longer be regarded as a simple relay station in the dorsal column pathway. Neuroanatomical studies have revealed rostrocaudal differences regarding both the cytoarchitecture 15 and the termination of the different afferents 14,1~,2°. Furthermore, efferent fibers terminate in other places besides the thalamus. There are well documented projections both to the inferior olive 7 and to the cerebellum 8. The results of experiments employing neurophysiological methods suggest a complex pattern of impulse modulation in the nucleus. Thus a large part of the neurons projecting into the medial lemniscus is subjected both to surround inhibition and supraspinal controP°, 11. Another interesting feature is presynaptic inhibition 2. The possible morphological correlate, axo-axonal contacts 21, is also found in the gracile nucleus.5, iv. The results of the present study are of special interest in relation to the physiological findings discussed above. They were made during analysis of Golgi material in order to identify the different kinds of projection neurons recently demonstrated 3, 5,8 by use of retrograde axonal transport of HRP. The gracile nuclei of 19 cats, ranging in age from newborn to adult, were investigated. Staining was performed according to the rapid Golgi method 18. After exsanguination of the animal, the lower brain stem and upper spinal cord were dissected out and cut into smaller pieces. The specimens were immersed in a fixation solution of 2 ~ potassium dichromate and 0.2 ~,, osmium tetroxide and left for 2-6 days in darkness. They were then transferred to a solution of 0.75 °/o silver nitrate and kept in it for 1-2 days. The specimens were dehydrated in acetone and alcohol, embedded in celloidin and cut on a sliding microtome in 100-150 # m thick sections. Most specimens were cut in transverse sections, but longitudinal sections were also made. Drawings were made with the help of a binocular drawing apparatus. In the gracile nucleus, beside cells projecting into the medial lemniscus, neurons were found whose axons ramify after a short distance and end with fine branches showing boutons of passage and clusters of boutons (Fig. 1). These cells were classified as interneurons. So far, 10 such cells have been identified with certainty. They are small to medium sized, with a perikaryal diameter of 11-17/~m (mean 14). The number
408
\
100 pm
Fig. 1. Rapid Golgi impregnation. Kitten, I I days old. lnterneuron situated in the rostral part of the gracile nucleus.
of primary dendrites varies between 2 and 4 (mean 3). Mostly the dendrites are situated at each pole of the perikaryon. In 7 neurons the dendritic field occupies a narrow strip in the transverse plane, but three of the neurons have dendrites which spread widely in all directions. The lengths of individual dendrites are up to 200/,m. The axon emerges from a dendrite in 5 of the l0 neurons. Axons of projection neurons in the gracile nucleus frequently show initial collaterals. These collaterals leave the stem axon at varying distances from the perikaryon, the most proximal being found after 15 #m, and terminate within the nucleus with boutons of passage and clusters of boutons (Fig. 2). Up to 3 collaterals per stem axon have been observed. Neither interneurons nor axon collaterals have earlier been demonstrated anatomically in the feline gracile nucleus. However, the finding of surround inhibition ~z implied the presence of interneurons. The existence of axon collaterals was suggested by Gordon and Jukes n, who found transsynaptic inhibition of gracile neurons following stimulation of the medial lemniscus. In our study it has only been possible to identify a small number of cells as interneurons. However, most cells escape classification, as their axons are unimpregnated or leave the plane of section after a short distance. The fact that many small neurons in the gracile nucleus remain unlabeled following injection of H R P into gracile projection areas 4 suggests that interneurons are quite frequent. Although no final conclusion can be drawn from the present small material, it is interesting to note that interneurons with different dendritic organizations have been found. Further studies will reveal whether this or other features may permit a division of the interneurons into distinct morphological types. In the cuneate nucleus different types of interneurons are thought to mediate pre- and postsynaptic inhibition, respectively 1. Also the cells that receive pyramidal tract fibers may constitute a separate group of interneurons 1. The physiological importance of the initial collaterals has not been thoroughly investigated. As they seem to be rather frequent, it is tempting to suggest that the collaterals play a significant role in impulse modulation. In the rat cuneate nucleus
409
/
100 p m
Fig. 2. Rapid Golgi impregnation. Kitten, 2 days old. Projection neuron situated in the middle part of the gracile nucleus. Note the initial collateral which ramifies and terminates in the vicinity of the neuron.
recurrent collaterals are t h o u g h t to f o r m a p a t h w a y , including a n u m b e r o f intern e u r o n s t h a t p r o d u c e p r e s y n a p t i c inhibition o f c u n e o t h a l a m i c relay cells 9. Electron m i c r o s c o p i c a l investigations o f the gracile nucleus have revealed t h a t a large n u m b e r o f b o u t o n s r e m a i n u n c h a n g e d following t r a n s e c t i o n o f different afferentsS, 17. I t now seems evident t h a t m a n y o f these b o u t o n s are s u p p l i e d by interneurons and initial collaterals. The results o f the present G o l g i s t u d y have thus given new d a t a c o n c e r n i n g the s y n a p t o l o g y in the feline gracile nucleus. T h e y are in g o o d a g r e e m e n t with findings in investigations using o t h e r m e t h o d s a n d also indicate t h a t the gracile nucleus in the cat has an o r g a n i z a t i o n similar to t h a t in r o d e n t s 13,19. The a u t h o r s are i n d e b t e d to Dr. Bo W i k s t e n , who kindly put p a r t o f the investigated material at o u r disposal. The skilful technical assistance o f Mrs. Kfirstin F l i n k is gratefully a c k n o w l e d g e d . This s t u d y was s u p p o r t e d by grants from the Swedish Medical Research Council (Project No. B76-12X-02710-08).
I Andersen, P., Eccles, J. C., Schmidt, R. F. and Yokota, T., Identification of relay cells and interneurons in the cuneate nucleus, J. Neurophysiol., 27 (1964) 1080-1095. 2 Andersen, P., Etholm, B. and Gordon, G., Presynaptic and post-synaptic inhibition elicited in the cat's dorsal column nuclei by mechanical stimulation of skin, J. Physiol. (Lond.), 210 (1970) 433-455. 3 Berkley, K. J., Different targets of different neurons in nucleus gracilis of the cat, d. comp. Neurol., 163 (1975) 285 304. 4 Blomqvist, A., In preparation. 5 Blomqvist, A. and Westman, J., An electron microscopical study of the gracile nucleus in the cat Acta Soc. Med. upsalien., 75 (1970) 241-252.
410 6 Blomqvist, A. and Westman. J., Combined HRP and Fink Heimer s|ainmg applied ~n the gracile nucleus in the cat, Brain ResearcM 99 (1975) 339 342. 7 Boesten, A. J. P. and Voogd, J., Projections o[" the dorsal column nuclei and the spinal cord on the inferior olive in the cqt. J. eon*p. Neto-ol., 161 (1975) 215-,238. 8 Cheek, M. D., Rustioni, A. and Trevino, D. L., Dorsal column nuclei projections to the cerebellar cortex in cats as revealed by the use of the retrograde transport of horseradish peroxidase, ,I. eomp. Neurol., 164 (1975) 31 46. 9 Davidson, N. and Smith, ( . A., A recurrent collateral pathway for presynaptic inhibition m the rat cuneate nucleus, Brain Research, 44 (I 972) 63- 71. 10 Gordon, G. and Jukes, M. G. M., Dual organization of the exteroceptive components of the cat's gracile nucleus, J. I-'hysiol. ( L o n d . , 173 (1964) 263--290. 11 Gordon, G. and Jukes, M. G. M,, Descending influences on the exteroceptive organizations of the cat's gracile nucleus, J. Phy,~iol. florid. ), 173 (I 964) 29 I-319. 12 Gordon, G. and Paine, ('. H., Functional organization in nucleus gracilis of the cat..I. IJhysiol. (Lond.), 153 (1960) 331 349. 13 Gulley, R. L., Golgi studies of the nucleus gracilis in the rat, Anat. Rec., 177 (1973) 325442. 14 Hand, P. J., Lumbosacral dorsal root terminations in the nucleus gracilis of the cat. Some observations on terminal degeneration in other medullary sensory nuclei, J. eomp. NeuroL, 126 (19661 137 156. 15 Kuypers, H. G. J. M. and Tuerk, J. D., The distribution of the cortical fibres within the nuclei cuneatus and gracilis in the cat, J. AHat. (Lond.), 98 (1964) 143-162. 16 Rustioni, A., Non-primary afferents to the nucleus gracilis from the lumbar cord of the cat, Brai, Research, 51 (1973) 81 95. 17 Rustioni, A. and Sotelo, ( . , Synaptic organization of the nucleus gracilis of the cat. Experimental identification of dorsal root fibers and cortical afferents, J. comp. Neurol., 155 (1974) 441 468. 18 Strong, O., Review of the Golgi method, J. eomp. Neurol., 6 (1896) 101-127. 19 Valverde, F.. The pyramidal tract in rodents. A study of its relations with the posterior column nuclei, dorsolateral reticular formation of the medulla oblongata, and cervical spinal cord, Z. ZellJbrsch., 71 (1966) 297-363. 20 Walberg, F., Corticofugal fibres to the nuclei of the dorsal columns, Brait~, 80 (19571 273 287. 21 Walberg, F., Axoaxonic contacts in the cuneate nucleus, probable basis for presynaptic depolarization, E.vp. Neurol., 13 (1965) 218 231.