320
Brain Research, 84 (1975) 320 324 ~('~ Elsevier Scientific Publishing Company, Amsterdam
-
Printed in The Netherlands
Retrograde axonal transport of horseradish peroxidase in sensory nerves and ganglion cells of the rat
L A W R E N C E F U R S T M A N , S A M U E L SAPORTA AND L A W R E N C E K R U G E R
Departments of Orthodontics and Anatomy, UCLA Center for Health Sciences, Los Angeles, Calif. 90024 (U.S.A.) (Accepted October 29th, 1974)
Retrograde axonal transport of horseradish peroxidase (HRP) has recently been reported for a variety of central nervous system neurons including spinal and cranial motor neuronsS, 6, retinal ganglion cells and the isthmo-optic nucleus of chicks a, thalamic neurons projecting upon somatosensory cortical fields3,4,11, superior olivary neurons projecting to the cochlea 15 and a variety of central connections now under study in several laboratories2,7,9,10. The present report is an account of preliminary attempts to trace first-order afferent fibers into the central nervous system. The development of a method of labeling specific afferent nerves and end-organs might allow the tracing of functionally categorized nerves and would serve to follow connections that can only be traced from dorsal roots with degeneration methods. In the first group of experiments, the pulp cavity of one or more teeth in 12 rats was injected on one side of the mouth in various combinations ranging from a single tooth to all upper and lower teeth that could successfully be opened with a fine dental drill. A 5 #l volume of 0.9 N NaC1 containing 1 mg Sigma type VI horseradish peroxidase was injected into each tooth and the rats were sacrificed after 1 or 2 days by cardiac perfusion with a solution of l ~ glutaraldehyde-l ~ paraformaldehyde buffered with sodium cacodylate (pH 7.5). Both trigeminal ganglia were dissected free and immersed in the same perfusate for 24 h. The tissue was then immersed for an equal period of time in 5 ~ sucrose buffered with 0.1 M sodium cacodylate (pH 7.5). Serial frozen sections were cut at 60 p m and incubated in 3,3'-diaminobenzidine tetrahydrochloride and H202 for H R P 1. The contralateral trigeminal ganglion served as a control. Four ganglia (two ipsilateral to the injection and two contralateral controls) were processed as whole tissue blocks to reveal the presence of HRP. The tissue was treated in the same manner as the frozen section material with the exception that it was incubated en bloc for 3 h at room temperature. The tissue was then washed through 3 changes of distilled water, dehydrated and embedded in paraffin for serial sectioning, and Nissl stained.
321 Tracing axonal pathways proved more complex than anticipated after initial observations of occasional linear arrays of granules in peripheral nerve. Because only a small percentage of axons and perikarya are labeled for a given local injection of tooth, an attempt was made to label a large nerve trunk in 2 rats with HRP. For this purpose the rat sciatic nerve was cut above the knee and immersed in HRP-Ringer solution (0.05 mg HRP/ml Ringer) for one hour. One animal was sacrificed at 24 h and the other at 48 h. Lumbar dorsal root ganglia, sectors of peripheral nerve and dorsal root were processed either by incubation of free-floating frozen sections or by en b l o c incubation for peroxidase activity. The contralateral nerve and dorsal root ganglia were used as controls. In the first experiment where tooth pulps were injected, peroxidase reaction product was present in a limited number of ganglion cells in the ipsilateral trigeminal ganglion (Figs. 1 and 2). There were no labeled (HRP-containing) cells in the contralateral trigeminal ganglion. Following immersion of the sciatic nerve in HRP, labeled neurons were found throughout the extent of the ipsilateral dorsal root ganglion after 24 and 48 h (Fig. 3). The distribution of labeled neurons for single incisor injections was limited to a compact zone containing a maximum of 5 labeled neurons on the periphery of the ganglion. Neurons labeled by multiple tooth injections, however, were not distributed in separate discrete zones and determination of topographical relationships will require extensive reconstruction. One or two days after cut nerve immersion, linear arrays of label can be seen at the level of the ganglion, in peripheral nerve and in spinal root fibers (Fig. 4). Aggregation is also seen around Schwann cell nuclei and oligodendrocyte nuclei in the trigeminal root. Distal portions of the nerve reveal the presence of HRP label primarily in a periaxonal distribution (Figs. 5 and 6) within myelin but a small quantity of reaction product also appears to remain in the axoplasm. The HRP reaction product for the 2 incubation procedures was slightly different. Free-floating frozen section incubation disclosed large dark granules of H R P (Fig. 1). Incubation en b l o c produced a fine granular reaction product which made recognition of HRP-containing cells more difficult than recognition of the large granule reaction product present in cells following incubation of sections (Fig. 2). However, the reaction product in both procedures is essentially similar and ganglion cells or peripheral nerves containing HRP are easily recognized. The HRP reaction product in sensory ganglion cells after en b l o c or section incubation does not differ significantly in our experience from HRP labeling of neurons within the central nervous system. The pulp cavity was selected as an example of a sensory receptor of particular interest because of its unique role in pain in man. The use of HRP for labeling of spinal and trigeminal ganglion neurons established the generality of the method first applied to tracing axons from motor end-plates to motoneurons 6. The feasibility of tracing first order afferents by the same method has now been demonstrated. The distribution of HRP in the zone of myelin and the presence of some diagonal streaks of granules suggest, in addition, that there may be significant passage of HRP within
322
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323 the S c h m i d t - L a n t e r m a n incisures as suggested b y Singer a n d his c o - w o r k e r s 12-14. T h e l i m i t e d q u a n t i t y o f H R P r e a c t i o n p r o d u c t within a x o p l a s m after 24 h suggests t h a t the p e r o x i d a s e molecule is n o t sequestered locally to a great extent, b u t is transp o r t e d to the p e r i k a r y o n a n d b e y o n d into the d o r s a l r o o t ( a l t h o u g h in small a m o u n t s ) within a relatively s h o r t p e r i o d o f time. G r e a t e r u p t a k e o f H R P within sensory g a n g l i o n cells can be seen after 48 h t h a n in g a n g l i o n cells o f a n i m a l s sacrificed after 24 h. Conversely, there is m o r e H R P r e a c t i o n p r o d u c t in p e r i p h e r a l nerve after 24 h t h a n is p r e s e n t after 48 h. O n the basis o f these o b s e r v a t i o h s it m a y be expected t h a t an a p p r o p r i a t e survival t i m e c a n be f o u n d where H R P is p r e s e n t only in p e r i p h e r a l nerve while n e u r o n s o f the d o r s a l r o o t g a n g l i o n o r t r i g e m i n a l ganglion are d e v o i d o f reaction product. These initial o b s e r v a t i o n s raise a n u m b e r o f questions. E l u c i d a t i o n o f the site o f u p t a k e , a n d the t i m e course a n d features o f t r a n s p o r t are crucial to further interp r e t a t i o n o f this m e t h o d . H o w e v e r , the p r e l i m i n a r y findings suggest t h a t r e t r o g r a d e t r a n s p o r t o f H R P m a y p r o v i d e a m e a n s for tracing first-order sensory axons in a m a n n e r t h a t is n o t feasible with d e g e n e r a t i o n m e t h o d s because o f the feeble r e a c t i o n in d o r s a l r o o t s after section o f p e r i p h e r a l nerve fibers. The d e p e n d e n c e o f p r o t e i n t r a n s p o r t on m i c r o t u b u l e s m a y a c c o u n t for the l i m i t e d q u a n t i t y o f H R P in d o r s a l r o o t fibers as c o m p a r e d to ganglion cells, a n d m a y be a l i m i t a t i o n in t r a c i n g afferents centrally. This research was s u p p o r t e d b y the U.S. Public H e a l t h Service t h r o u g h N I H G r a n t NS-5685. Excellent technical assistance was p r o v i d e d by Ms. S h a r o n S a m p o g n a a n d Mr. E v a n R e a s o r .
Figs. 1-6. Horseradish peroxidase (HRP) in peripheral nerve, sensory ganglion neurons and dorsal root fibers of rat following introduction of HRP in the periphery. The HRP reaction product is seen as small dark brown granules within cells or fibers. Frozen sections were counter-stained with cresylecht violet and paraffin sections with basic fuchsin. Fig. 1. Trigeminal ganglion 48 h after injection of the ipsilateral lower incisor with HRP. Incubated as a free-floating frozen section cut at 60 ffm. Granular reaction product is prominent within the perikaryon of a single sensory ganglion cell. Some granules of HRP reaction product overlay the edges of the nucleus but are localized within cytoplasm, x 650. Fig. 2. The same experiment illustrated in Fig. 1 with en bloc incubation and paraffin sectioning at 7/~m. Perikaryal reaction product is less pronounced than in frozen section due to the very fine reaction product produced by en bloc incubation, but the darker color produced by the HRP is still distinctive throughout the whole cell. x 650. Fig. 3. HRP labeled sensory ganglion cells from L7 dorsal root ganglion 48 h after immersion of cut sciatic nerve in HRP. Incubated free-floating frozen section, x 650. Fig. 4. Trigeminal sensory root within the glial dome 48 h after injection of ipsilateral incisors. Note the accumulation of HRP reaction product around oligodendrocyte nuclei and the presence of a linear array of reaction product along a root axon. x 650. Fig. 5. Cross section of sciatic nerve 5 cm from cut 24 h after immersion in HRP reveals most of the HRP is associated with the myelin sheath. Nerve was processed en bloc and sectioned transversely in paraffin. × 650. Fig. 6. Longitudinal section of same portion of nerve as shown in Fig. 5, 24 h after immersion in HRP. Reaction product is linearly aggregated and heaviest in the myelinated zone of each nerve fiber. Aggregation of HRP reaction product can be seen around Schwann cell nuclei (arrows). x 425,
324 1 GRAHAM,R. C., JR., AND KARNOVSKY, M.J., The early stages of" absorption of injected horse radish peroxidase in the proximal tubules of the mouse kidney: ultra-structural cytochemistr~ by a new technique, J. Histochem. Cytochem., 14 (19661 291-302. 2 GRAYBIEL, A. M., AND DEVOR, M., A microelectrophoretic delivery technique for use with horseradish peroxidase, Brain Research, 68 (1974) 167-173. 3 JONES, E. G., AND LEAVITT, R.Y., Demonstration of thalamo-cortical connectivity in the cat somatosensory system by retrograde axonal transport of horseradish peroxidase, Brain Research. 63 (1973) 414-418. 4 JONES, E. G., AND LEAV1TT, R. Y., Retrograde axonal transport and the demonstration of nonspecific projections to the cerebral cortex and striatum from thalamic intralaminar nuclei in the rat, cat, and monkey, J. comp. Neurol., 154 (1974) 349-378. 5 KRISTENSSON, K., OLSSON, Y., AND SJOSTRAND, J., Axonal uptake and retrograde transport of exogenous proteins in the hypoglossal nerve, Brain Research, 32 (1972) 399-406. 6 KRISTENSSON, K., AND OLSSON, Y., Retrograde axonal transport of protein, Brain Research, 29 (1971) 363-365. 7 KUYPERS, U. G. J. U., KIEVIT, J., AND GROEN-KLEVANT, A. C., Retrograde axonal transport of horseradish peroxidase in rat's forebrain, Brain Research, 67 (1974) 211-218. 8 LAVAIL, J. H., AND LAVAIL, M. M., Retrograde axonal transport in the central nervous system, Science, 176 (1972) 1416-1417. 9 LAVAIL, J. H., WINSTON, K . R . , AND TISH, A., A method based on retrograde intra-axonal transport of protein for identification of cell bodies of origin of axons terminating within the CNS, Brain Research, 58 (1973) 470-477. 10 NAUTA, H. J. W., PRITZ, M. B., AND LASEK, R. J., Afferents in the rat caudoputamen studied with horseradish peroxidase. An evaluation of a retrograde neuroanatomical research method, Brain Research, 67 (1974) 219-238. 11 RALSTON, H. J., AND SHARP, P. V., The identification of thalamocortical relay cells in the adult cat by means of retrograde axonal transport of horseradish peroxidase, Brain Research, 62 (1973) 273-278. 12 SINGER, M., ANO BRYANT, S. V., Movements in the myelin Schwann sheath of the vertebrate axon, Nature (Lond.), 221 (1969) 1148-1150. 13 SINGER, M., KRISHNAN, N., AND FYICE, D. S., Penetration of ruthenium red into peripheral nerve fibers, Anat. Rec., 173 (1972) 375-390. 14 KRISHNAN, N., AND SINGER, M., Penetration of peroxidase into peripheral nerve fibers, Amer. J. Anat., 136 (1973) 1 14. 15 WARR, W. B., Localization of olivocochlear neurons by means of retrograde transport of horseradish peroxidase, Anat. Ree., 174 (1973) 464.
Brain Research, 84 (1975) 320 324 ~('~ Elsevier Scientific Publishing Company, Amsterdam
-
Printed in The Netherlands
Retrograde axonal transport of horseradish peroxidase in sensory nerves and ganglion cells of the rat
L A W R E N C E F U R S T M A N , S A M U E L SAPORTA AND L A W R E N C E K R U G E R
Departments of Orthodontics and Anatomy, UCLA Center for Health Sciences, Los Angeles, Calif. 90024 (U.S.A.) (Accepted October 29th, 1974)
Retrograde axonal transport of horseradish peroxidase (HRP) has recently been reported for a variety of central nervous system neurons including spinal and cranial motor neuronsS, 6, retinal ganglion cells and the isthmo-optic nucleus of chicks a, thalamic neurons projecting upon somatosensory cortical fields3,4,11, superior olivary neurons projecting to the cochlea 15 and a variety of central connections now under study in several laboratories2,7,9,10. The present report is an account of preliminary attempts to trace first-order afferent fibers into the central nervous system. The development of a method of labeling specific afferent nerves and end-organs might allow the tracing of functionally categorized nerves and would serve to follow connections that can only be traced from dorsal roots with degeneration methods. In the first group of experiments, the pulp cavity of one or more teeth in 12 rats was injected on one side of the mouth in various combinations ranging from a single tooth to all upper and lower teeth that could successfully be opened with a fine dental drill. A 5 #l volume of 0.9 N NaC1 containing 1 mg Sigma type VI horseradish peroxidase was injected into each tooth and the rats were sacrificed after 1 or 2 days by cardiac perfusion with a solution of l ~ glutaraldehyde-l ~ paraformaldehyde buffered with sodium cacodylate (pH 7.5). Both trigeminal ganglia were dissected free and immersed in the same perfusate for 24 h. The tissue was then immersed for an equal period of time in 5 ~ sucrose buffered with 0.1 M sodium cacodylate (pH 7.5). Serial frozen sections were cut at 60 p m and incubated in 3,3'-diaminobenzidine tetrahydrochloride and H202 for H R P 1. The contralateral trigeminal ganglion served as a control. Four ganglia (two ipsilateral to the injection and two contralateral controls) were processed as whole tissue blocks to reveal the presence of HRP. The tissue was treated in the same manner as the frozen section material with the exception that it was incubated en bloc for 3 h at room temperature. The tissue was then washed through 3 changes of distilled water, dehydrated and embedded in paraffin for serial sectioning, and Nissl stained.
321 Tracing axonal pathways proved more complex than anticipated after initial observations of occasional linear arrays of granules in peripheral nerve. Because only a small percentage of axons and perikarya are labeled for a given local injection of tooth, an attempt was made to label a large nerve trunk in 2 rats with HRP. For this purpose the rat sciatic nerve was cut above the knee and immersed in HRP-Ringer solution (0.05 mg HRP/ml Ringer) for one hour. One animal was sacrificed at 24 h and the other at 48 h. Lumbar dorsal root ganglia, sectors of peripheral nerve and dorsal root were processed either by incubation of free-floating frozen sections or by en b l o c incubation for peroxidase activity. The contralateral nerve and dorsal root ganglia were used as controls. In the first experiment where tooth pulps were injected, peroxidase reaction product was present in a limited number of ganglion cells in the ipsilateral trigeminal ganglion (Figs. 1 and 2). There were no labeled (HRP-containing) cells in the contralateral trigeminal ganglion. Following immersion of the sciatic nerve in HRP, labeled neurons were found throughout the extent of the ipsilateral dorsal root ganglion after 24 and 48 h (Fig. 3). The distribution of labeled neurons for single incisor injections was limited to a compact zone containing a maximum of 5 labeled neurons on the periphery of the ganglion. Neurons labeled by multiple tooth injections, however, were not distributed in separate discrete zones and determination of topographical relationships will require extensive reconstruction. One or two days after cut nerve immersion, linear arrays of label can be seen at the level of the ganglion, in peripheral nerve and in spinal root fibers (Fig. 4). Aggregation is also seen around Schwann cell nuclei and oligodendrocyte nuclei in the trigeminal root. Distal portions of the nerve reveal the presence of HRP label primarily in a periaxonal distribution (Figs. 5 and 6) within myelin but a small quantity of reaction product also appears to remain in the axoplasm. The HRP reaction product for the 2 incubation procedures was slightly different. Free-floating frozen section incubation disclosed large dark granules of H R P (Fig. 1). Incubation en b l o c produced a fine granular reaction product which made recognition of HRP-containing cells more difficult than recognition of the large granule reaction product present in cells following incubation of sections (Fig. 2). However, the reaction product in both procedures is essentially similar and ganglion cells or peripheral nerves containing HRP are easily recognized. The HRP reaction product in sensory ganglion cells after en b l o c or section incubation does not differ significantly in our experience from HRP labeling of neurons within the central nervous system. The pulp cavity was selected as an example of a sensory receptor of particular interest because of its unique role in pain in man. The use of HRP for labeling of spinal and trigeminal ganglion neurons established the generality of the method first applied to tracing axons from motor end-plates to motoneurons 6. The feasibility of tracing first order afferents by the same method has now been demonstrated. The distribution of HRP in the zone of myelin and the presence of some diagonal streaks of granules suggest, in addition, that there may be significant passage of HRP within
322
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323 the S c h m i d t - L a n t e r m a n incisures as suggested b y Singer a n d his c o - w o r k e r s 12-14. T h e l i m i t e d q u a n t i t y o f H R P r e a c t i o n p r o d u c t within a x o p l a s m after 24 h suggests t h a t the p e r o x i d a s e molecule is n o t sequestered locally to a great extent, b u t is transp o r t e d to the p e r i k a r y o n a n d b e y o n d into the d o r s a l r o o t ( a l t h o u g h in small a m o u n t s ) within a relatively s h o r t p e r i o d o f time. G r e a t e r u p t a k e o f H R P within sensory g a n g l i o n cells can be seen after 48 h t h a n in g a n g l i o n cells o f a n i m a l s sacrificed after 24 h. Conversely, there is m o r e H R P r e a c t i o n p r o d u c t in p e r i p h e r a l nerve after 24 h t h a n is p r e s e n t after 48 h. O n the basis o f these o b s e r v a t i o h s it m a y be expected t h a t an a p p r o p r i a t e survival t i m e c a n be f o u n d where H R P is p r e s e n t only in p e r i p h e r a l nerve while n e u r o n s o f the d o r s a l r o o t g a n g l i o n o r t r i g e m i n a l ganglion are d e v o i d o f reaction product. These initial o b s e r v a t i o n s raise a n u m b e r o f questions. E l u c i d a t i o n o f the site o f u p t a k e , a n d the t i m e course a n d features o f t r a n s p o r t are crucial to further interp r e t a t i o n o f this m e t h o d . H o w e v e r , the p r e l i m i n a r y findings suggest t h a t r e t r o g r a d e t r a n s p o r t o f H R P m a y p r o v i d e a m e a n s for tracing first-order sensory axons in a m a n n e r t h a t is n o t feasible with d e g e n e r a t i o n m e t h o d s because o f the feeble r e a c t i o n in d o r s a l r o o t s after section o f p e r i p h e r a l nerve fibers. The d e p e n d e n c e o f p r o t e i n t r a n s p o r t on m i c r o t u b u l e s m a y a c c o u n t for the l i m i t e d q u a n t i t y o f H R P in d o r s a l r o o t fibers as c o m p a r e d to ganglion cells, a n d m a y be a l i m i t a t i o n in t r a c i n g afferents centrally. This research was s u p p o r t e d b y the U.S. Public H e a l t h Service t h r o u g h N I H G r a n t NS-5685. Excellent technical assistance was p r o v i d e d by Ms. S h a r o n S a m p o g n a a n d Mr. E v a n R e a s o r .
Figs. 1-6. Horseradish peroxidase (HRP) in peripheral nerve, sensory ganglion neurons and dorsal root fibers of rat following introduction of HRP in the periphery. The HRP reaction product is seen as small dark brown granules within cells or fibers. Frozen sections were counter-stained with cresylecht violet and paraffin sections with basic fuchsin. Fig. 1. Trigeminal ganglion 48 h after injection of the ipsilateral lower incisor with HRP. Incubated as a free-floating frozen section cut at 60 ffm. Granular reaction product is prominent within the perikaryon of a single sensory ganglion cell. Some granules of HRP reaction product overlay the edges of the nucleus but are localized within cytoplasm, x 650. Fig. 2. The same experiment illustrated in Fig. 1 with en bloc incubation and paraffin sectioning at 7/~m. Perikaryal reaction product is less pronounced than in frozen section due to the very fine reaction product produced by en bloc incubation, but the darker color produced by the HRP is still distinctive throughout the whole cell. x 650. Fig. 3. HRP labeled sensory ganglion cells from L7 dorsal root ganglion 48 h after immersion of cut sciatic nerve in HRP. Incubated free-floating frozen section, x 650. Fig. 4. Trigeminal sensory root within the glial dome 48 h after injection of ipsilateral incisors. Note the accumulation of HRP reaction product around oligodendrocyte nuclei and the presence of a linear array of reaction product along a root axon. x 650. Fig. 5. Cross section of sciatic nerve 5 cm from cut 24 h after immersion in HRP reveals most of the HRP is associated with the myelin sheath. Nerve was processed en bloc and sectioned transversely in paraffin. × 650. Fig. 6. Longitudinal section of same portion of nerve as shown in Fig. 5, 24 h after immersion in HRP. Reaction product is linearly aggregated and heaviest in the myelinated zone of each nerve fiber. Aggregation of HRP reaction product can be seen around Schwann cell nuclei (arrows). x 425,
324 1 GRAHAM,R. C., JR., AND KARNOVSKY, M.J., The early stages of" absorption of injected horse radish peroxidase in the proximal tubules of the mouse kidney: ultra-structural cytochemistr~ by a new technique, J. Histochem. Cytochem., 14 (19661 291-302. 2 GRAYBIEL, A. M., AND DEVOR, M., A microelectrophoretic delivery technique for use with horseradish peroxidase, Brain Research, 68 (1974) 167-173. 3 JONES, E. G., AND LEAVITT, R.Y., Demonstration of thalamo-cortical connectivity in the cat somatosensory system by retrograde axonal transport of horseradish peroxidase, Brain Research. 63 (1973) 414-418. 4 JONES, E. G., AND LEAV1TT, R. Y., Retrograde axonal transport and the demonstration of nonspecific projections to the cerebral cortex and striatum from thalamic intralaminar nuclei in the rat, cat, and monkey, J. comp. Neurol., 154 (1974) 349-378. 5 KRISTENSSON, K., OLSSON, Y., AND SJOSTRAND, J., Axonal uptake and retrograde transport of exogenous proteins in the hypoglossal nerve, Brain Research, 32 (1972) 399-406. 6 KRISTENSSON, K., AND OLSSON, Y., Retrograde axonal transport of protein, Brain Research, 29 (1971) 363-365. 7 KUYPERS, U. G. J. U., KIEVIT, J., AND GROEN-KLEVANT, A. C., Retrograde axonal transport of horseradish peroxidase in rat's forebrain, Brain Research, 67 (1974) 211-218. 8 LAVAIL, J. H., AND LAVAIL, M. M., Retrograde axonal transport in the central nervous system, Science, 176 (1972) 1416-1417. 9 LAVAIL, J. H., WINSTON, K . R . , AND TISH, A., A method based on retrograde intra-axonal transport of protein for identification of cell bodies of origin of axons terminating within the CNS, Brain Research, 58 (1973) 470-477. 10 NAUTA, H. J. W., PRITZ, M. B., AND LASEK, R. J., Afferents in the rat caudoputamen studied with horseradish peroxidase. An evaluation of a retrograde neuroanatomical research method, Brain Research, 67 (1974) 219-238. 11 RALSTON, H. J., AND SHARP, P. V., The identification of thalamocortical relay cells in the adult cat by means of retrograde axonal transport of horseradish peroxidase, Brain Research, 62 (1973) 273-278. 12 SINGER, M., ANO BRYANT, S. V., Movements in the myelin Schwann sheath of the vertebrate axon, Nature (Lond.), 221 (1969) 1148-1150. 13 SINGER, M., KRISHNAN, N., AND FYICE, D. S., Penetration of ruthenium red into peripheral nerve fibers, Anat. Rec., 173 (1972) 375-390. 14 KRISHNAN, N., AND SINGER, M., Penetration of peroxidase into peripheral nerve fibers, Amer. J. Anat., 136 (1973) 1 14. 15 WARR, W. B., Localization of olivocochlear neurons by means of retrograde transport of horseradish peroxidase, Anat. Ree., 174 (1973) 464.
