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Combined light and electron microscopic tracing of neurons, including axons and synaptic terminals, after intracellular injection of horseradish peroxidase
Neuroseience Letters, 2 (1976) 307--313
307
© Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
COMBINED LIGHT AND E L E C T R O N MICROSCOPIC T R A C I N G OF NEURONS, INCLUDING AXONS AND SYNAPTIC TERMINALS, A F T E R I N T R A C E L L U L A R INJECTION OF H O R S E R A D I S H PEROXIDASE
STAFFAN CULLHEIM and JAN-OLOF KELLERTH Department of Anatomy, Karolinska Instituter, S-104 01 Stockholm 60 (Sweden)
(Received May 10th, 1976) (Accepted May 10th, 1976)
SUMMARY By using intracellular deposition of horseradish peroxidase (HRP), a method has been designed for combined light and electron microscopic tracing of single fibers and synaptic terminals, belonging to physiologically characterized neurons. This technique has been successfully tested on the axon collateral systems of cat a-motoneurons, b u t should also be applicable to other types of neurons.
One major aim in morphological studies of the central nervous system is to define the axonal pathways and synaptic projections of various populations of neurons. A number of methods have been developed for this purpose, most of them utilizing lesions with subsequent degeneration or loss of axons and terminals [1,5]. Recently, methods using the anterograde transport of extracellularly deposited tritiated amino acids [ 2] and horseradish peroxidase (HRP) [9,11] have been developed. Successful intracellular deposition of H R P has been obtained in spinocervical neurons [ 8,12], thus allowing light microscopic demonstration of cell bodies, axons and axon collaterals [12]. Since the reaction product of HRP is electron-opaque, this substance also seems to be well suited for electron microscopic tracing of stained neuronal profiles [8,9,11]. Thus, utilizing the active intraneuronal transport of H R P we have developed a technique for the combined light microscopic (LM) and electron microscopic (EM) tracing of single, physiologically defined neurons, including their dendrites, axons and synaptic terminals. Adult cats were anesthetized with Nembutal (40 mg/kg) and a lumbosacral laminectomy was performed. The dorsal roots Ls--S, were cut on the left side. Various muscle nerves were dissected free in the left hind limb and m o u n t e d on stimulating electrodes. The animals were paralyzed with Flaxedil (gallamine triethiodide) and artificially ventilated. A pneumothorax was usually performed.
308 Single-barrelled glass micropipettes were used to impale motoneurons which were identified b y antidromic stimulation of the muscle nerves. The microelectrodes were filled with HRP b y first boiling them in distilled water, then sucking away as much water as possible from above with a thin catheter and instead inserting 5 pl of a 25% solution of HRP (type VI; Sigma) in 0.1 M NaOH. This H R P solution was allowed to diffuse down into the tips for 2--3 days before use. On the day of the experiment the filling of the electrodes was completed with a solution of 25% HRP (type II; Sigma) in 0.1 M NaOH. The electrode resistances ranged between 30 and 60 M~2 (tip diameters 1.5--2.5 pm). HRP was injected intracellularly b y passing a 10--25 nA constant positive current through the electrode during 15--40 min. In order to facilitate the later tracing and reconstruction of the HRP-stained neurons (see below), the
/
/
J
1 mm
Fig. 1. Reconstruction of an HRP-stained (330 n A . min) lateral gastrocnemius a-motoneuron located in the ventral horn o f the L~ segment. The reconstruction is based on 40 transverse LM sections covering a total longitudinal length o f 1.2 m m . Three axon collaterals, illustrated as dotted lines, leave the m o t o r axon at places indicated by small arrows. T h e dashed arrow shows the direction of the microelectrode insertion.
309
A
j,
/
'
1ram
" I i
B
100 pm
Fig. 2. A: reconstruction of the axon and collateral system of an HRP-stained (600 nA. rain) sciatic a-motoneuron. B: reconstruction of the same collateral system at a higher magnification. The branches of this collateral tree seem to give rise to swellings of both 'en passant' and terminal types. The arrow points at the position of the synaptic terminal illustrated in Fig. 5. number of injected cells was usually restricted to 8--10 in each experiment, and, furthermore, the cells were separated by a distance of at least 400 p m in the rostrocaudal direction. One to six hours after the HRP-injection the cats were sacrificed by perfusion with 5% glutaraldehyde in Millonig's buffer (300 mOsm/1) through the descending aorta. The appropriate regions of the spinal cord were cut out and immersed in the perfusion fixative over night. Transverse serial sections (30 pm) were cut with a Vibratome (Oxford Lab.) and processed for HRP [4]. After a quick light microscopic (LM) screening the sections containing stained neuronal profiles were osmificated according to a modification [6] of the Marchi m e t h o d [10] by putting t h e m in a mixture containing 1 part 1% OsO4 and 3 parts 1% KC103, both dissolved in Millonig's buffer (300 mOsm/1). After 20 h in this medium, the sections were dehydrated with acetone and embedded in Vestopal according to the 'section embedding' procedure described by Holl~inder [7]. In contrast to conventional osmification procedures the modified Marchi m e t h o d [6] produces only a moderate background staining of the myelin and allows LM tracing also of very small HRP-filled neuronal processes. The use of osmificated sections is of course benificial for later ultrastructural analysis of the stained profiles located within these sections. To obtain LM reconstructions of the stained neurons (Figs. 1 and 2) the embedded sections were studied in a Leitz microscope equipped with a camera lucida arrangement for simultaneous viewing and drawing. Light micrographs (Fig. 3) were obtained by the use of a Leitz
310
Fig. 3. Light micrographs of the motoneuron reconstructed in Fig. 2. Sections (30 ~m thick) osmificated according to the modified [6] method of Marchi [10]. A: the cell body and parts of the proximal dendrites. B: the motor axon and the origin (arrow) of the axon collateral. C: branches of the axon collateral system. D: a terminal swelling originating from a branch of the axon collateral system. The major part of this branch was located in another section. p h o t o m i c r o s c o p e ( O r t o l u x ) . D e t a i l e d m a p s w e r e also d r a w n at large magnificat i o n s t o a l l o w an e x a c t (+ 1 p m ) d e t e r m i n a t i o n o f t h e t h r e e - d i m e n s i o n a l posit i o n o f t h e H R P - s t a i n e d p r o f i l e s in r e l a t i o n t o l a n d m a r k s like b l o o d vessels a n d n e u r o n a l s t r u c t u r e s w i t h i n e a c h section. T h e s e m a p s w e r e u s e d f o r t h e i d e n t i f i c a t i o n o f stained n e u r o n a l processes in t h e e l e c t r o n m i c r o s c o p e (EM). T h e a t t e m p t s t o inject H R P i n t o m o t o n e u r o n a l cell b o d i e s was successful in r o u g h l y 50% o f t h e cases. T h e cell b o d i e s o f H R P - s t a i n e d n e u r o n s o f t e n s h o w e d signs o f d a m a g e or f r a g m e n t a t i o n , b u t usually t h e staining o f t h e a x o n s and, in p a r t i c u l a r , t h e d e n d r i t e s was still s u f f i c i e n t t o p e r m i t tracing. E x t e n s i v e tracing o f d e n d r i t e s , a x o n s a n d s y n a p t i c t e r m i n a l s c o u l d t h e r e f o r e b e p e r f o r m e d in a b o u t 80% o f t h e stained n e u r o n s . A f t e r t h e LM studies had been completed the sections were mounted on blocks of Vestopal to permit u l t r a t h i n sectioning. E a c h s e c t i o n was t r i m m e d d o w n w i t h a P y r a m i t o m e
311 (LKB) leaving only a small region containing those stained structures which had previously been selected in the LM for ultrastructural analysis. Ultrathin serial sections were cut transversly with an ultramicrotome (LKB 4801), stained with uranyl acetate and lead citrate and examined in a Philips 301 electron microscope. Electron microscopically, neuronal profiles exhibiting the same ultrastructural characteristics as those shown in Figs. 4 and 5 were always present at the exact location predicted from the previous LM maps. Thus, the HRPstained dendrites and axons were characterized b y the presence of a large number of electron-dense granules scattered in a cytoplasm of low electron density (Fig. 4). This finding is consistent with earlier reports on the appearance of the peroxidase reaction products in neuronal cell bodies after intracellular injection [8]. In the synaptic boutons, an electron-dense material adhered to t h e membranes of vesicles and mitochondria and partly also to the inside of the outer membrane of the bouton. Profiles showing these characteristics were never observed in other places than those predicted from the LM studies, nor in control material from this region. Fig. 5 shows a stained synaptic b o u t o n which belongs to the collateral system described in Figs. 2--4. The position of this terminal within the section matches exactly that of the collateral swelling indicated b y the arrow in Fig. 2B. Electron microscopically a direct continuity could also be demonstrated between this b o u t o n and one of the stained collateral branches. According to
Fig. 4. Electron micrograph of a transverse section through two myelinated fibers. The fiber indicated by the asterisk contains electron-dense granules and is a branch of the HRP-stained axon collateral system shown in Figs. 2 and 3.
312
Fig. 5. Electron micrograph of the same terminal (p) as indicated by the arrow in Fig. 2B. Note that an electron-dense material adheres to the vesicle membranes and also to the interior of the cell membrane, thus giving the terminal an appearance which contrasts markedly to that of normal unstained terminals (n). The mitochondria of the stained boutons were sometimes ruptured, d = postsynaptic dendrite.
physiological evidence [3], this b o u t o n and its postsynaptic element should thus represent the cholinergic synapse between a m o t o r axon collateral and a Renshaw cell. A more detailed morphological description of this t y p e of synapse will be published later. ACKNOWLEDGEMENTS
This study was supported by grants from the Swedish Medical Research Council (Project 2886) and the Karolinska Institutet. The electron microscopic evaluations by Drs. S. Conmdi and L.-O. Ronnevi, and the skillfultechnical assistance of Miss M. Berghman, Miss K. Carlsson and Mrs. L. Stuart are gratefully acknowledged.
313
REFERENCES i Alksne, J.F., Blackstad, Th.W., Walberg, F. and White Jr., L.E., Electron microscopy of axon degeneration: a valuable tool in experimental neuroanatomy, Ergebn. Anat., 39 (1966) 1--32. 2 Cowan, W.M., Gottlieb, D.I., Hendrickson, A.E., Price, J.L. and Woolsey, T.A., The autoradiographic demonstration of axonal connections in the central nervous system, Brain Res., 37 (1972) 21--51. 3 Eccles, J.C., Fatt, P. and Koketsu, K., Cholinergic and inhibitory synapses in a pathway from motor-axon collaterals to motoneurons, J. Physiol. (Lond.), 126 (1954) 524--562. 4 Graham, R.C. and Karnovsky, M.J., The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem., 14 (1966) 291--302. 5 Grant, G. and Walberg, F., The light and electron microscopical appearance of anterograde and retrograde neuronal degeneration. In K. Fuxe, L. Olson and Y. Zotterman (Eds.), Dynamics of Degeneration and Growth in Neurons, Pergamon Press, Oxford, 1974, pp. 5--18. 6 Hildebrand, C. and Aldskogius, H., Electron microscopic identification of Marchipositive bodies and argyrophilic granules in the spinal cord white matter of the guinea pig. Submitted to J. comp. Neurol. 7 Holl~/nder, H., The section embedding (SE) technique. A new method for the combined light and electron microscopic examination of central nervous tissue, Brain Res., 20 (1970) 39--47. 8 Jankowska, E., Rastad, J. and Westman, J., Intracellular application of horseradish peroxidase and its light and electron microscopical appearance in spinocervical tract cells, Brain Res., 105 (1976) 557--562. 9 LaVail, J.H. and LaVail, M.M., The retrograde intraaxonal transport of horseradish peroxidase in the chick visual system: a light and electron microscopic study, J. comp. Neurol., 157 (1974) 303--358. 10 Marchi, V., Sulle degenerazioni consecutive all' estirpazione totale e parziale del cervelletto, Riv. Sper. Freniat., 12 (1886) 50--56. 11 Nauta, H.J.W., Kaiserman-Abramof, I.R. and Lasek, R.J., Electron microscopic observations of horseradish peroxidase transported from the caudoputamen to the substantia nigra in the rat: possible involvement of the agranular reticulum, Brain Res., 85 (1975) 373--384. 12 Snow, P.J., Rose, P.K. and Brown, A.G., Tracing axons and axon collaterals of spinal neurons using intracellular injection of horseradish peroxidase, Science, 191 (1976) 312--313.
307
© Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
COMBINED LIGHT AND E L E C T R O N MICROSCOPIC T R A C I N G OF NEURONS, INCLUDING AXONS AND SYNAPTIC TERMINALS, A F T E R I N T R A C E L L U L A R INJECTION OF H O R S E R A D I S H PEROXIDASE
STAFFAN CULLHEIM and JAN-OLOF KELLERTH Department of Anatomy, Karolinska Instituter, S-104 01 Stockholm 60 (Sweden)
(Received May 10th, 1976) (Accepted May 10th, 1976)
SUMMARY By using intracellular deposition of horseradish peroxidase (HRP), a method has been designed for combined light and electron microscopic tracing of single fibers and synaptic terminals, belonging to physiologically characterized neurons. This technique has been successfully tested on the axon collateral systems of cat a-motoneurons, b u t should also be applicable to other types of neurons.
One major aim in morphological studies of the central nervous system is to define the axonal pathways and synaptic projections of various populations of neurons. A number of methods have been developed for this purpose, most of them utilizing lesions with subsequent degeneration or loss of axons and terminals [1,5]. Recently, methods using the anterograde transport of extracellularly deposited tritiated amino acids [ 2] and horseradish peroxidase (HRP) [9,11] have been developed. Successful intracellular deposition of H R P has been obtained in spinocervical neurons [ 8,12], thus allowing light microscopic demonstration of cell bodies, axons and axon collaterals [12]. Since the reaction product of HRP is electron-opaque, this substance also seems to be well suited for electron microscopic tracing of stained neuronal profiles [8,9,11]. Thus, utilizing the active intraneuronal transport of H R P we have developed a technique for the combined light microscopic (LM) and electron microscopic (EM) tracing of single, physiologically defined neurons, including their dendrites, axons and synaptic terminals. Adult cats were anesthetized with Nembutal (40 mg/kg) and a lumbosacral laminectomy was performed. The dorsal roots Ls--S, were cut on the left side. Various muscle nerves were dissected free in the left hind limb and m o u n t e d on stimulating electrodes. The animals were paralyzed with Flaxedil (gallamine triethiodide) and artificially ventilated. A pneumothorax was usually performed.
308 Single-barrelled glass micropipettes were used to impale motoneurons which were identified b y antidromic stimulation of the muscle nerves. The microelectrodes were filled with HRP b y first boiling them in distilled water, then sucking away as much water as possible from above with a thin catheter and instead inserting 5 pl of a 25% solution of HRP (type VI; Sigma) in 0.1 M NaOH. This H R P solution was allowed to diffuse down into the tips for 2--3 days before use. On the day of the experiment the filling of the electrodes was completed with a solution of 25% HRP (type II; Sigma) in 0.1 M NaOH. The electrode resistances ranged between 30 and 60 M~2 (tip diameters 1.5--2.5 pm). HRP was injected intracellularly b y passing a 10--25 nA constant positive current through the electrode during 15--40 min. In order to facilitate the later tracing and reconstruction of the HRP-stained neurons (see below), the
/
/
J
1 mm
Fig. 1. Reconstruction of an HRP-stained (330 n A . min) lateral gastrocnemius a-motoneuron located in the ventral horn o f the L~ segment. The reconstruction is based on 40 transverse LM sections covering a total longitudinal length o f 1.2 m m . Three axon collaterals, illustrated as dotted lines, leave the m o t o r axon at places indicated by small arrows. T h e dashed arrow shows the direction of the microelectrode insertion.
309
A
j,
/
'
1ram
" I i
B
100 pm
Fig. 2. A: reconstruction of the axon and collateral system of an HRP-stained (600 nA. rain) sciatic a-motoneuron. B: reconstruction of the same collateral system at a higher magnification. The branches of this collateral tree seem to give rise to swellings of both 'en passant' and terminal types. The arrow points at the position of the synaptic terminal illustrated in Fig. 5. number of injected cells was usually restricted to 8--10 in each experiment, and, furthermore, the cells were separated by a distance of at least 400 p m in the rostrocaudal direction. One to six hours after the HRP-injection the cats were sacrificed by perfusion with 5% glutaraldehyde in Millonig's buffer (300 mOsm/1) through the descending aorta. The appropriate regions of the spinal cord were cut out and immersed in the perfusion fixative over night. Transverse serial sections (30 pm) were cut with a Vibratome (Oxford Lab.) and processed for HRP [4]. After a quick light microscopic (LM) screening the sections containing stained neuronal profiles were osmificated according to a modification [6] of the Marchi m e t h o d [10] by putting t h e m in a mixture containing 1 part 1% OsO4 and 3 parts 1% KC103, both dissolved in Millonig's buffer (300 mOsm/1). After 20 h in this medium, the sections were dehydrated with acetone and embedded in Vestopal according to the 'section embedding' procedure described by Holl~inder [7]. In contrast to conventional osmification procedures the modified Marchi m e t h o d [6] produces only a moderate background staining of the myelin and allows LM tracing also of very small HRP-filled neuronal processes. The use of osmificated sections is of course benificial for later ultrastructural analysis of the stained profiles located within these sections. To obtain LM reconstructions of the stained neurons (Figs. 1 and 2) the embedded sections were studied in a Leitz microscope equipped with a camera lucida arrangement for simultaneous viewing and drawing. Light micrographs (Fig. 3) were obtained by the use of a Leitz
310
Fig. 3. Light micrographs of the motoneuron reconstructed in Fig. 2. Sections (30 ~m thick) osmificated according to the modified [6] method of Marchi [10]. A: the cell body and parts of the proximal dendrites. B: the motor axon and the origin (arrow) of the axon collateral. C: branches of the axon collateral system. D: a terminal swelling originating from a branch of the axon collateral system. The major part of this branch was located in another section. p h o t o m i c r o s c o p e ( O r t o l u x ) . D e t a i l e d m a p s w e r e also d r a w n at large magnificat i o n s t o a l l o w an e x a c t (+ 1 p m ) d e t e r m i n a t i o n o f t h e t h r e e - d i m e n s i o n a l posit i o n o f t h e H R P - s t a i n e d p r o f i l e s in r e l a t i o n t o l a n d m a r k s like b l o o d vessels a n d n e u r o n a l s t r u c t u r e s w i t h i n e a c h section. T h e s e m a p s w e r e u s e d f o r t h e i d e n t i f i c a t i o n o f stained n e u r o n a l processes in t h e e l e c t r o n m i c r o s c o p e (EM). T h e a t t e m p t s t o inject H R P i n t o m o t o n e u r o n a l cell b o d i e s was successful in r o u g h l y 50% o f t h e cases. T h e cell b o d i e s o f H R P - s t a i n e d n e u r o n s o f t e n s h o w e d signs o f d a m a g e or f r a g m e n t a t i o n , b u t usually t h e staining o f t h e a x o n s and, in p a r t i c u l a r , t h e d e n d r i t e s was still s u f f i c i e n t t o p e r m i t tracing. E x t e n s i v e tracing o f d e n d r i t e s , a x o n s a n d s y n a p t i c t e r m i n a l s c o u l d t h e r e f o r e b e p e r f o r m e d in a b o u t 80% o f t h e stained n e u r o n s . A f t e r t h e LM studies had been completed the sections were mounted on blocks of Vestopal to permit u l t r a t h i n sectioning. E a c h s e c t i o n was t r i m m e d d o w n w i t h a P y r a m i t o m e
311 (LKB) leaving only a small region containing those stained structures which had previously been selected in the LM for ultrastructural analysis. Ultrathin serial sections were cut transversly with an ultramicrotome (LKB 4801), stained with uranyl acetate and lead citrate and examined in a Philips 301 electron microscope. Electron microscopically, neuronal profiles exhibiting the same ultrastructural characteristics as those shown in Figs. 4 and 5 were always present at the exact location predicted from the previous LM maps. Thus, the HRPstained dendrites and axons were characterized b y the presence of a large number of electron-dense granules scattered in a cytoplasm of low electron density (Fig. 4). This finding is consistent with earlier reports on the appearance of the peroxidase reaction products in neuronal cell bodies after intracellular injection [8]. In the synaptic boutons, an electron-dense material adhered to t h e membranes of vesicles and mitochondria and partly also to the inside of the outer membrane of the bouton. Profiles showing these characteristics were never observed in other places than those predicted from the LM studies, nor in control material from this region. Fig. 5 shows a stained synaptic b o u t o n which belongs to the collateral system described in Figs. 2--4. The position of this terminal within the section matches exactly that of the collateral swelling indicated b y the arrow in Fig. 2B. Electron microscopically a direct continuity could also be demonstrated between this b o u t o n and one of the stained collateral branches. According to
Fig. 4. Electron micrograph of a transverse section through two myelinated fibers. The fiber indicated by the asterisk contains electron-dense granules and is a branch of the HRP-stained axon collateral system shown in Figs. 2 and 3.
312
Fig. 5. Electron micrograph of the same terminal (p) as indicated by the arrow in Fig. 2B. Note that an electron-dense material adheres to the vesicle membranes and also to the interior of the cell membrane, thus giving the terminal an appearance which contrasts markedly to that of normal unstained terminals (n). The mitochondria of the stained boutons were sometimes ruptured, d = postsynaptic dendrite.
physiological evidence [3], this b o u t o n and its postsynaptic element should thus represent the cholinergic synapse between a m o t o r axon collateral and a Renshaw cell. A more detailed morphological description of this t y p e of synapse will be published later. ACKNOWLEDGEMENTS
This study was supported by grants from the Swedish Medical Research Council (Project 2886) and the Karolinska Institutet. The electron microscopic evaluations by Drs. S. Conmdi and L.-O. Ronnevi, and the skillfultechnical assistance of Miss M. Berghman, Miss K. Carlsson and Mrs. L. Stuart are gratefully acknowledged.
313
REFERENCES i Alksne, J.F., Blackstad, Th.W., Walberg, F. and White Jr., L.E., Electron microscopy of axon degeneration: a valuable tool in experimental neuroanatomy, Ergebn. Anat., 39 (1966) 1--32. 2 Cowan, W.M., Gottlieb, D.I., Hendrickson, A.E., Price, J.L. and Woolsey, T.A., The autoradiographic demonstration of axonal connections in the central nervous system, Brain Res., 37 (1972) 21--51. 3 Eccles, J.C., Fatt, P. and Koketsu, K., Cholinergic and inhibitory synapses in a pathway from motor-axon collaterals to motoneurons, J. Physiol. (Lond.), 126 (1954) 524--562. 4 Graham, R.C. and Karnovsky, M.J., The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem., 14 (1966) 291--302. 5 Grant, G. and Walberg, F., The light and electron microscopical appearance of anterograde and retrograde neuronal degeneration. In K. Fuxe, L. Olson and Y. Zotterman (Eds.), Dynamics of Degeneration and Growth in Neurons, Pergamon Press, Oxford, 1974, pp. 5--18. 6 Hildebrand, C. and Aldskogius, H., Electron microscopic identification of Marchipositive bodies and argyrophilic granules in the spinal cord white matter of the guinea pig. Submitted to J. comp. Neurol. 7 Holl~/nder, H., The section embedding (SE) technique. A new method for the combined light and electron microscopic examination of central nervous tissue, Brain Res., 20 (1970) 39--47. 8 Jankowska, E., Rastad, J. and Westman, J., Intracellular application of horseradish peroxidase and its light and electron microscopical appearance in spinocervical tract cells, Brain Res., 105 (1976) 557--562. 9 LaVail, J.H. and LaVail, M.M., The retrograde intraaxonal transport of horseradish peroxidase in the chick visual system: a light and electron microscopic study, J. comp. Neurol., 157 (1974) 303--358. 10 Marchi, V., Sulle degenerazioni consecutive all' estirpazione totale e parziale del cervelletto, Riv. Sper. Freniat., 12 (1886) 50--56. 11 Nauta, H.J.W., Kaiserman-Abramof, I.R. and Lasek, R.J., Electron microscopic observations of horseradish peroxidase transported from the caudoputamen to the substantia nigra in the rat: possible involvement of the agranular reticulum, Brain Res., 85 (1975) 373--384. 12 Snow, P.J., Rose, P.K. and Brown, A.G., Tracing axons and axon collaterals of spinal neurons using intracellular injection of horseradish peroxidase, Science, 191 (1976) 312--313.