Brain Research, 118 (1976) 115-118 © Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
115
Short Communications
Axonal projections of spinal interneurones excited by group I afferents in the cat, revealed by intracellular staining with horseradish peroxidase
J. CZARKOWSKA*, E. JANKOWSKA and E. SYBIRSKA* Department of Physiology, University of GOteborg, Gi~teborg (Sweden) (Accepted September 9th, 1976)
Most conclusions on th~ neuronal organization of spinal reflexes, including the number of interneurones and the pattern of convergence in various reflex paths, are based on indirect evidence (see ref. 9). So far, even detailed studies of various types of interneurones a-5 did not, with only a few exceptions 6, resolve whether these interneurones are or are not interposed in reflex paths to motoneurones and, if so, whether they are the last order interneurones. This is becoming possible with the progress of techniques of intracellular staining of individual cells. With horseradish peroxidase, recently introduced for this purpose~,10, it is possible to visualize not only the soma and dendrites of the cells but also the trajectory of their axons, even to their terminals1,10 in well-stained cells. Using this technique we aimed now at finding out which of the interneurones located in laminae V and VI of Rexed in lower lumbar segments of the cat spinal cord project to motor nuclei and may terminate on motoneurones, and which of these interneurones could influence motoneurones only via some polysynaptic pathways. The sample of interneurones selected for staining included interneurones monosynaptically excited from group la muscle spindle afferents and/or from group Ib tendon organ afferents, whether or not they were affected by other fibre systems as well4, 5. The interneurones were penetrated with a glass micropipette filled with a 1015 ~o solution of horseradish peroxidase (HRP, Sigma type VI) in 0.5 M NaCI, which allowed both intracellular recording from the cells and the iontophoretic application o f the enzyme, as described previously 7. Of 55 cells kept for at least 5-10 min, 44 were sufficiently well stained to allow tracing of their axons; the remaining cells were either too poorly stained or, for unknown reasons, not stained at all. More than 75 ~o of the interneurones turned out to be funicular cells and to send their stem axons to either the ipsilateral lateral funiculus (21 cells) or to the ipsilateral (9 cells) or contralateral (5 cells) ventral funiculus where they could be followed for 1-2 mm. None of the cells projected, however, to thoracic or to more rostral levels of * On leave of absence from Nencki Institute of Experimental Biology, Warsaw, Poland.
116
Fig. 1. Schematic drawings of 6 pattenls of axonal projections of interneurones in laminae V-VII which are monosynaptically excited from group I afferents. Filled triangles are for cell bodies, while thick and thin lines represent stem axons and axon collaterals respectively. Further explanations in the text. the neuraxis as indicated by lack of antidromic activation on stimulation of either the ipsi- or contralateral spinal half at Th 13. Six main patterns of axonal projections have been revealed, These are schematically illustrated in Fig. 1, with examples of full reconstructions of the 1st and 5th patterns in Fig. 2. Photo reconstructions of cells with the 2nd and 4th patterns of projections are shown in Fig. 1 of the following reportL The cells were ascribed to one of the above groups depending on whether their stem axons projected to one of the funiculi or remained in the grey matter, and taking into account the trajectory of the axons and the area of terminal branching. The last feature was considered as being most essential from the point of view of the function of the cells. Even if without electronmicroscopic studies we cannot resolve whether the stained interneurones made synaptic contacts on cells with somata located within the areas of their terminal branching, it seems m o r e likely that their target cells were among these cells than among cells located further away and extending only their dendrites towards the interneurones. We assume, there-
117
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Fig. 2. Reconstruction of axonal projections of 2 interneurones in a transverse plane. A: interneurone located in rostral L5, excited from Ib afferents from quadriceps, stained by ejecting HRP with 106 nA- min. Note that the stem axon bifurcated before entering the lateral funiculus. The dorsal and the ventral branches could be followed in the white matter over distances 1 mm rostrally and 3.5 mm caudally respectively. The ventral branch gave off axon collaterals which re-entered the ventral horn at distances 300 #m and 900/~m caudal from the soma. Axon collaterals given off from the stem axon branched in the ventral horn and in laminae V and VI, within 100 #m rostral and 300/zm caudal to the soma. B: interneurone located in rostral L7, excited by low and high threshold group I afferents from plantaris and flexor digitorum and hallucis longus, stained by ejecting HRP with 311 nA • min. Note that the axon of the cell divided into 3 branches only some 200 #m from the soma (at the first node of Ranvier) and that none of the branches entered the white matter. Only main axonal branches (thicker) and axon collaterals (thinner) were drawn in order to avoid mixing up the latter with the dendrites. Note that the two cells are reproduced in different scales. fore, t h a t t e r m i n a l b r a n c h i n g within m o t o r nuclei indicates direct c o n t a c t s with m o t o n e u r o n e s o r o t h e r cells with the s a m e l o c a t i o n (e.g. cells o f origin o f the ventral spinocerebellar tract), a n d t h a t t e r m i n a l b r a n c h i n g in R e x e d l a m i n a e V - V I I indicates s y n a p t i c c o n t a c t s on o t h e r i n t e r n e u r o n e s o r on cells o f origin o f some long ascending o r p r o p r i o spinal p a t h w a y s , a l t h o u g h t e r m i n a t i o n on distal p a r t s o f m o t o n e u r o n e dendrites c a n n o t be excluded in this case. O f the 6 p a t t e r n s o f p r o j e c t i o n s o f the g r o u p I excited i n t e r n e u r o n e s l o c a t e d in l a m i n a e V a n d VI the first two r e p r e s e n t p r o j e c t i o n s to the m o t o r nuclei, the second one being c o m b i n e d with projections to the a r e a s l o c a t e d d o r s a l l y a n d m e d i a l l y to them. The 3rd, 4th a n d 5th p a t t e r n s represent p r o j e c t i o n s within l a m i n a e V - V I I , s o m e w h a t m o r e laterally (groups 3 a n d 5) o r medially ( g r o u p 4), p r o b a b l y o v e r a g r e a t e r length o f the spinal c o r d in g r o u p s 3 a n d 4 t h a n in g r o u p 5 as j u d g e d f r o m p r o j e c t i o n s o f the stem axons to the white m a t t e r , o r their absence respectively. T h e 6th p a t t e r n o f p r o j e c t i o n s w o u l d subserve crossed spinal connexions. The stem axons o f all 5 cells b e l o n g i n g to this g r o u p crossed the midline t h r o u g h the ventral c o m m i s s u r e b u t n o t h i n g c a n be so far said a b o u t the d e s t i n a t i o n o f these axons because they c o u l d n o t be t r a c e d far enough.
118 F r o m the p o i n t o f view o f their i n p u t none o f the g r o u p s with the a b o v e described projection patterns constituted a uniform p o p u l a t i o n . I n t e r n e u r o n e s excited m o n o synaptically by a p p a r e n t l y b o t h l a a n d Ib afferents, f r o m one or several nerves, constituted a m a j o r i t y in all the groups. The Ia, but n o t Ib excited cells h a d m o s t often the 1st or the 4th p r o j e c t i o n p a t t e r n , a n d the Ib, but not l a excited cells had the 1st o r the 3rd projection p a t t e r n ; a m o n g the latter were also cells d i s y n a p t i c a l l y excited from I b afferents. In view o f a very extensive convergence on cells in the i n t e r m e d i a t e nucleus 4,'5 and o f difficulties in differentiating between Ia a n d lb effects from m o s t o f the electrically s t i m u l a t e d nerves (see ref. 8), except h a m s t r i n g a n d quadriceps, the discussion o f correlation between the p a t t e r n s o f projections o f the interneurones a n d their i n p u t will have to a w a i t the full report. This w o r k was s u p p o r t e d by the Swedish Medical Research Council (Project No. 00094).
1 Cullheim, S. and Kellerth, J. O., Combined light and electron microscopical tracing of neurones, including axons and synaptic terminals, after intracellular injection of horseradish peroxidase, Neurosci. Left., 2 (1976) 307-313. 2 Czarkowska, J., Jankowska, E. and Sybirska, E., Diameter and internodal length of stem axons of spinal interneurones excited by group I afferents in the cat, Brain Research, 118 (1976) 119-122. 3 Eccles, J. C., Eccles, R. M. and Lundberg, A., Types of neurone in and around the intermediate nucleus of the lumbosacral cord, J. Physiol. (Lond.), 154 (1960) 89-114. 4 Hongo, T., Jankowska, E. and Lundberg, A., Convergence of excitatory and inhibitory action on interneurones in the lumbosacral cord, Exp. Brain Res., 1 (1966) 338-358. 5 Hongo, T., Jankowska, E. and Lundberg, A., The rubrospinal tract. IV. Effects on interneurones, Exp. Brain Res., 15 (1972) 54-78. 6 Jankowska, E., Identification of interneurons interposed in different spinal reflex pathways. In M. Santini (Ed.), Proc. Golgi Cent. Syrup., Raven Press, New York, 1975, pp. 235-246. 7 Jankowska, E., Rastad, J. and Westman, J., Intracellular application of horseradish peroxidase and its light and electron microscopical appearance in spinocervical tract cells, Brain Research, 105 (1976) 557-562. 8 Lucas, M. E. and Willis, W. D., Identification of muscle afferents which activate interneurons in the intermediate nucleus, J. Neurophysiol., 37 (1974) 282-293. 9 Lundberg, A., The control of spinal mechanisms from the brain. In D. B. Tower (Ed.), The Nervous System, Vol. 1, Raven Press, New York, 1975, pp. 253-265. 10 Snow, P. J., Rose, P. K. and Brown, A., Tracing axons and axon collaterals of spinal neurones using intracellular injection of horseradish peroxidase, Science, 191 (1976) 312-313.
115
Short Communications
Axonal projections of spinal interneurones excited by group I afferents in the cat, revealed by intracellular staining with horseradish peroxidase
J. CZARKOWSKA*, E. JANKOWSKA and E. SYBIRSKA* Department of Physiology, University of GOteborg, Gi~teborg (Sweden) (Accepted September 9th, 1976)
Most conclusions on th~ neuronal organization of spinal reflexes, including the number of interneurones and the pattern of convergence in various reflex paths, are based on indirect evidence (see ref. 9). So far, even detailed studies of various types of interneurones a-5 did not, with only a few exceptions 6, resolve whether these interneurones are or are not interposed in reflex paths to motoneurones and, if so, whether they are the last order interneurones. This is becoming possible with the progress of techniques of intracellular staining of individual cells. With horseradish peroxidase, recently introduced for this purpose~,10, it is possible to visualize not only the soma and dendrites of the cells but also the trajectory of their axons, even to their terminals1,10 in well-stained cells. Using this technique we aimed now at finding out which of the interneurones located in laminae V and VI of Rexed in lower lumbar segments of the cat spinal cord project to motor nuclei and may terminate on motoneurones, and which of these interneurones could influence motoneurones only via some polysynaptic pathways. The sample of interneurones selected for staining included interneurones monosynaptically excited from group la muscle spindle afferents and/or from group Ib tendon organ afferents, whether or not they were affected by other fibre systems as well4, 5. The interneurones were penetrated with a glass micropipette filled with a 1015 ~o solution of horseradish peroxidase (HRP, Sigma type VI) in 0.5 M NaCI, which allowed both intracellular recording from the cells and the iontophoretic application o f the enzyme, as described previously 7. Of 55 cells kept for at least 5-10 min, 44 were sufficiently well stained to allow tracing of their axons; the remaining cells were either too poorly stained or, for unknown reasons, not stained at all. More than 75 ~o of the interneurones turned out to be funicular cells and to send their stem axons to either the ipsilateral lateral funiculus (21 cells) or to the ipsilateral (9 cells) or contralateral (5 cells) ventral funiculus where they could be followed for 1-2 mm. None of the cells projected, however, to thoracic or to more rostral levels of * On leave of absence from Nencki Institute of Experimental Biology, Warsaw, Poland.
116
Fig. 1. Schematic drawings of 6 pattenls of axonal projections of interneurones in laminae V-VII which are monosynaptically excited from group I afferents. Filled triangles are for cell bodies, while thick and thin lines represent stem axons and axon collaterals respectively. Further explanations in the text. the neuraxis as indicated by lack of antidromic activation on stimulation of either the ipsi- or contralateral spinal half at Th 13. Six main patterns of axonal projections have been revealed, These are schematically illustrated in Fig. 1, with examples of full reconstructions of the 1st and 5th patterns in Fig. 2. Photo reconstructions of cells with the 2nd and 4th patterns of projections are shown in Fig. 1 of the following reportL The cells were ascribed to one of the above groups depending on whether their stem axons projected to one of the funiculi or remained in the grey matter, and taking into account the trajectory of the axons and the area of terminal branching. The last feature was considered as being most essential from the point of view of the function of the cells. Even if without electronmicroscopic studies we cannot resolve whether the stained interneurones made synaptic contacts on cells with somata located within the areas of their terminal branching, it seems m o r e likely that their target cells were among these cells than among cells located further away and extending only their dendrites towards the interneurones. We assume, there-
117
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Fig. 2. Reconstruction of axonal projections of 2 interneurones in a transverse plane. A: interneurone located in rostral L5, excited from Ib afferents from quadriceps, stained by ejecting HRP with 106 nA- min. Note that the stem axon bifurcated before entering the lateral funiculus. The dorsal and the ventral branches could be followed in the white matter over distances 1 mm rostrally and 3.5 mm caudally respectively. The ventral branch gave off axon collaterals which re-entered the ventral horn at distances 300 #m and 900/~m caudal from the soma. Axon collaterals given off from the stem axon branched in the ventral horn and in laminae V and VI, within 100 #m rostral and 300/zm caudal to the soma. B: interneurone located in rostral L7, excited by low and high threshold group I afferents from plantaris and flexor digitorum and hallucis longus, stained by ejecting HRP with 311 nA • min. Note that the axon of the cell divided into 3 branches only some 200 #m from the soma (at the first node of Ranvier) and that none of the branches entered the white matter. Only main axonal branches (thicker) and axon collaterals (thinner) were drawn in order to avoid mixing up the latter with the dendrites. Note that the two cells are reproduced in different scales. fore, t h a t t e r m i n a l b r a n c h i n g within m o t o r nuclei indicates direct c o n t a c t s with m o t o n e u r o n e s o r o t h e r cells with the s a m e l o c a t i o n (e.g. cells o f origin o f the ventral spinocerebellar tract), a n d t h a t t e r m i n a l b r a n c h i n g in R e x e d l a m i n a e V - V I I indicates s y n a p t i c c o n t a c t s on o t h e r i n t e r n e u r o n e s o r on cells o f origin o f some long ascending o r p r o p r i o spinal p a t h w a y s , a l t h o u g h t e r m i n a t i o n on distal p a r t s o f m o t o n e u r o n e dendrites c a n n o t be excluded in this case. O f the 6 p a t t e r n s o f p r o j e c t i o n s o f the g r o u p I excited i n t e r n e u r o n e s l o c a t e d in l a m i n a e V a n d VI the first two r e p r e s e n t p r o j e c t i o n s to the m o t o r nuclei, the second one being c o m b i n e d with projections to the a r e a s l o c a t e d d o r s a l l y a n d m e d i a l l y to them. The 3rd, 4th a n d 5th p a t t e r n s represent p r o j e c t i o n s within l a m i n a e V - V I I , s o m e w h a t m o r e laterally (groups 3 a n d 5) o r medially ( g r o u p 4), p r o b a b l y o v e r a g r e a t e r length o f the spinal c o r d in g r o u p s 3 a n d 4 t h a n in g r o u p 5 as j u d g e d f r o m p r o j e c t i o n s o f the stem axons to the white m a t t e r , o r their absence respectively. T h e 6th p a t t e r n o f p r o j e c t i o n s w o u l d subserve crossed spinal connexions. The stem axons o f all 5 cells b e l o n g i n g to this g r o u p crossed the midline t h r o u g h the ventral c o m m i s s u r e b u t n o t h i n g c a n be so far said a b o u t the d e s t i n a t i o n o f these axons because they c o u l d n o t be t r a c e d far enough.
118 F r o m the p o i n t o f view o f their i n p u t none o f the g r o u p s with the a b o v e described projection patterns constituted a uniform p o p u l a t i o n . I n t e r n e u r o n e s excited m o n o synaptically by a p p a r e n t l y b o t h l a a n d Ib afferents, f r o m one or several nerves, constituted a m a j o r i t y in all the groups. The Ia, but n o t Ib excited cells h a d m o s t often the 1st or the 4th p r o j e c t i o n p a t t e r n , a n d the Ib, but not l a excited cells had the 1st o r the 3rd projection p a t t e r n ; a m o n g the latter were also cells d i s y n a p t i c a l l y excited from I b afferents. In view o f a very extensive convergence on cells in the i n t e r m e d i a t e nucleus 4,'5 and o f difficulties in differentiating between Ia a n d lb effects from m o s t o f the electrically s t i m u l a t e d nerves (see ref. 8), except h a m s t r i n g a n d quadriceps, the discussion o f correlation between the p a t t e r n s o f projections o f the interneurones a n d their i n p u t will have to a w a i t the full report. This w o r k was s u p p o r t e d by the Swedish Medical Research Council (Project No. 00094).
1 Cullheim, S. and Kellerth, J. O., Combined light and electron microscopical tracing of neurones, including axons and synaptic terminals, after intracellular injection of horseradish peroxidase, Neurosci. Left., 2 (1976) 307-313. 2 Czarkowska, J., Jankowska, E. and Sybirska, E., Diameter and internodal length of stem axons of spinal interneurones excited by group I afferents in the cat, Brain Research, 118 (1976) 119-122. 3 Eccles, J. C., Eccles, R. M. and Lundberg, A., Types of neurone in and around the intermediate nucleus of the lumbosacral cord, J. Physiol. (Lond.), 154 (1960) 89-114. 4 Hongo, T., Jankowska, E. and Lundberg, A., Convergence of excitatory and inhibitory action on interneurones in the lumbosacral cord, Exp. Brain Res., 1 (1966) 338-358. 5 Hongo, T., Jankowska, E. and Lundberg, A., The rubrospinal tract. IV. Effects on interneurones, Exp. Brain Res., 15 (1972) 54-78. 6 Jankowska, E., Identification of interneurons interposed in different spinal reflex pathways. In M. Santini (Ed.), Proc. Golgi Cent. Syrup., Raven Press, New York, 1975, pp. 235-246. 7 Jankowska, E., Rastad, J. and Westman, J., Intracellular application of horseradish peroxidase and its light and electron microscopical appearance in spinocervical tract cells, Brain Research, 105 (1976) 557-562. 8 Lucas, M. E. and Willis, W. D., Identification of muscle afferents which activate interneurons in the intermediate nucleus, J. Neurophysiol., 37 (1974) 282-293. 9 Lundberg, A., The control of spinal mechanisms from the brain. In D. B. Tower (Ed.), The Nervous System, Vol. 1, Raven Press, New York, 1975, pp. 253-265. 10 Snow, P. J., Rose, P. K. and Brown, A., Tracing axons and axon collaterals of spinal neurones using intracellular injection of horseradish peroxidase, Science, 191 (1976) 312-313.
