Brain Research, 105 (1976) 557-562 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
557
Intracellular application of horseradish peroxidase and its light and electron microscopical appearance in spinocervical tract cells*
ELZBIETA JANKOWSKA, JONAS RASTAD AND JAN WESTMAN (E.J.) Department of Physiology, University of G6teborg, GOteborg, and Department of Anatomy, Biomedical Centre, University of Uppsala, Uppsala (Sweden)
(Accepted December 22nd, 1975)
Present techniques for the intracellular staining of physiologically identified neurons are not quite satisfactory for ultrastructural investigations. Iontophoretic administration of cobalt chloride for subsequent reaction with ammonium sulphide 12 does not allow fixation before incubation, a disadvantage as regards the ultrastructural preservation. If the injected cobalt chloride is allowed to react with diaminobenzidine 4 the fine structure of the cytoplasm appears to be damaged. This holds true also for iontophoretically injected Procion brown 2. Procion yellow 1~ is not electron dense and ultrastructural identification has to depend on changes of cell organelles9, la. Radioautographic techniques 5 are laborious because ultrastructural interpretation needs statistical analysis since the silver grains are situated at a distance from the source of activity 14. In this study the enzyme horseradish peroxidase (HRP) has been used for intracellular staining. H R P has recently found extensive use as a neuronal marker (for references, see ref. 10) in light and electron microscopical studies. The histochemical staining procedure employed seems not to damage the ultrastructure and the product of enzyme reaction is found in lysosome-like organelles 1°. In the present investigation the enzyme was administered intracellularly in spinocervical tract cells a, neurons previously studied with intracellular injections of Procion yellow 1,s. Cells were identified by monosynaptic excitation from the cutaneous nerves and by antidromic invasion from the dorsolateral funiculus in segment C3 (Fig. 1A-G). An additional criterion was the absence of antidromic invasion from C1 except at a high threshold. A total of 23 spinocervical tract cells were impaled with single barrelled micropipettes with tip diameters of 2-3/~m. The electrodes were filled by pressure with a 15-20 ~o (w/v) solution of H R P (Sigma, type II). In contrast to previous studies, where extracellular iontophoresis of H R P was performed 7,11, solutions in distilled water were initially used. In order to decrease the high electrode resistance by increasing ionic strength, both acid and basic solutions were tested. The best results were obtained * Reprints are available from: Department of Anatomy, Biomedical Centre, Box 571, S-751 23 Uppsala, Sweden.
558
Before injection
,~>
C3
After injection
~IOmV(D,E,G)
A
C
B
--
, ....
,1+5ms
_2 J C D
F
E
G
{ Hj
~
Fig. 1. A-G: upper records in A-C and D-G are from two spinocervical tract cells; lower records are from the surface of the spinal cord close to the site of microelectrode penetration. A and D show antidromic invasions from C3, with blocked action potential in A. Remaining records show monosynaptic and polysynaptic potentials from the superficial peroneal nerve (SP). Pictures were taken immediately before (B and E), immediately after (C and F) and a few minutes after (G) enzyme administration. Note recovery of postsynaptic potential in G. These cells were stained by passing 10 nA for 16 rain (A-C) and 13 min (D-G), respectively. H: schematic drawing of the same cell as in A-C and its localization in the lumbar dorsal horn. m, axon; ac, axon collaterals.
by dissolving H R P in 0.2 M N a O H giving a final p H o f a b o u t 8.5 and an electrode resistance o f 25-35 M ~ . In all cases H R P appeared to be ejected only by positive current. The a m o u n t o f enzyme administered was estimated by multiplying the time (in min) by the current (in hA). Continuous currents o f 5-10 nA for 5-25 min were passed through the electrodes while recording from the cells. Fig. l shows that during this period the cellular responses somewhat deteriorated (Fig. I C and F). This m a y have been due primarily to depolarization o f the cells rather than to an effect o f the injected enzyme, since several cells greatly recovered after the injection (compare Fig. l E and Fig. 1G). After diffusion o f H R P for 20-430 min the cats were perfused transcardially with a 4 l mixture o f 2 ~ (v/v) glutaraldehyde and l ~ (v/v) formaldehyde in 0.05 M sodium cacodylate buffer. The dorsal horn from the injected side o f segments L6-7 was dissected out, kept in fixative for 12 h and then cut transversely with a Sorvall TC-2 tissue sectioner. Sections about 120 # m thick were incubated for 30 min in a solution o f 0.05 ~ (w/v) diaminobenzidine (DAB) followed by a mixture o f 0.05 D A B and 0.01 ~ (v/v) hydrogen peroxide 6 for 30 min. Cells were processed for light and electron microscopy according to Rastad and Westman (to be published). This
559
Fig. 2. A: light micrograph of the same cell as in Fig. 1A-C. The arrow points dorsally, t, electrode track; ia, initial axon segment; m, myelinized axon. x 200. B: montage of light micrographs from one section of the same cell as in Fig. IA-C. Two axon collaterals (ac) leave the parent axon (m) at the same place (arrow). x 680. technique allowed light microscopical observation on the thick plastic embedded sections stained by osmium tetroxide and re-embedding for ultrathin serial sectioning. Electron microscopy was performed on 11 cells. Ultrathin sections were not contrasted. During the course of the study attempts were made to establish the maximal amount of enzyme, which could be administered without serious damage to the ultrastructural appearance of the cells. F o r light microscopical identification, more than 40 nA.min were needed. On the other hand applications of more than 170 nA.min seemed to increase the incidence of cell damage. N o correlation was observed between the ultrastructural preservation and the functional state of the cells after enzyme administration or between the preservation and the diffusion time. When cell damage was severe it was evident already in the light microscope. The contours of the cells then appeared irregular and shrunken. The dendrites emerged from the soma with abnormally thick proximal segments. The soma membrane, between primary dendrites, often showed a very concave shape thus demonstrating a shrunken perikaryal profile. At the ultrastructural level an increased cytoplasmic vacuolization occurred. A more advanced change was the cell shrinkage with a very condensed cytoplasm. The cell then showed densely packed mitochondria and lysosomes. No Golgi apparatus or endoplasmic reticulum were seen. The nucleus appeared disproportionally large with a diffuse outline of the nuclear membrane. These gravely damaged cells are not included in the description below.
560
Fig. 3. A: electron micrographs of the same cell as in Fig. 1A-C. The cell was stained 310 rain before perfusion. The arrow points dorsally, d, proximal dendrite; c, capillary with endothelial cell containing reaction product; i, filamentous inclusion body reaching the cell membrane at the approximate place of electrode penetration, x 1450. B: electron micrograph of the same cell as in Fig. 1A-C. The finely granular deposits are seen inside the stained cell. x 16,000.
In the light microscope the reaction p r o d u c t seemed evenly distributed (Fig. 2A) giving the injected cells a dark b r o w n appearance. One cell was exceptional, in that the reaction p r o d u c t was accumulated in the periphery of the soma. I n lighter stained cells, less t h a n a b o u t 80 n A . m i n , only few dendrites were seen a n d the nucleus
561 was darker than the cytoplasm. In heavier stained cells, more than about 80 nA.min, the nucleus was invisible. In general the soma was round to oval with mostly dorsally directed dendrites (Figs. 1H and 2A), which could be followed up to 450 ~m. The axons emerged from the ventral or lateral aspects of the cell bodies. The initial axon segments were easily identified. Four cells showed dorsomedially directed axon collaterals leaving the parent axon within the dorsal horn (Fig. 2B). Neither axonal projections in the white matter nor axon collaterals at more rostral levels (as found by Brown, Rose and Snow, personal communication, by HRP staining) were studied. In the electron microscope finely granular deposits were found in the soma, dendrites and axon. Lysosomes containing deposits were seen. Mitochondria and Golgi apparatus appeared normal. There was a sparse amount of granular endoplasmic reticulum. The cell membrane and, to a lesser extent, the nuclear envelope were outlined with deposits (Fig. 3A and B). Grains were also found in somaspines. Occasionally the outline of the stained cells showed invaginations containing adjacent neuronal and glial elements. The deposits were also seen in the nucleus giving it a darker appearance. The nucleolonema appeared slightly condensed. Small amounts of precipitate were seen extracellularly in the light microscope. To what extent this was due to diffusion of H R P from the electrode tip or to cleaning of the electrodes, by passing current, could not be clarified. Ultrastructurally minor amounts of reaction product were also seen in membrane bound organelles of nonpenetrated cells, mainly in glial profiles and endothelial cells (Fig. 3A). Electrode tracks could be followed as they were filled with stained blood cells (Fig. 2A). The localization of spinocervical cells was in agreement with earlier investigations 1,s. The present study also confirmed the general appearance of spinocervical tract cells seen after Procion yellow injections1, s. The major advantage of intracellular staining with H R P seems to be that amounts of enzyme large enough for an extensive light microscopical investigation of dendritic and proximal axonal aborization allow a reasonably good ultrastructural preservation. The electron dense deposits are easily identified. Stained profiles down to 0.35 ~m have been seen. The enzyme distributes quickly within the neuronal processes. The reaction product has a low osmium sensitivity and appropriate postfixation can be performed. The ultrastructural preservation is not as dependent on a short period between injection and perfusion as with other staining proceduresL The enzyme-filled micropipettes also have acceptable recording properties. It is yet not possible to state how complete the staining of cell processes is. Several facts suggest a rather extensive staining. Thus, no unstained dendrites of properly stained perikarya were seen, nor was any decreased density of reaction product observed in the first 150 # m of the processes. Although most of the perikarya and processes in the present investigation were preserved satisfactorily, some were seriously damaged. The reasons for this remain to be clarified. An estimate of possible loss of boutons 9 due to penetration and enzyme administration will have to await studies of non-injected cells. This paper will be followed by an extensive report of spinocervical tract cells labelled by both intracellular and extracellular techniques.
562 NOTE ADDED IN PROOF A f t e r this p a p e r was accepted for publication, the light microscopical results o f intracellular application o f H R P especially for tracing axonal projections o f spinal n eu ro n s were published. (SNow, P. J., RosE, P. K., AND BROWN, A., Tr aci n g axons and a x o n collaterals o f spinal neurones using intracellular injection o f horseradish peroxidase, Science, 191 (1976) 312-313.) We wish to th a n k M r A l f J o h a n s s o n an d Mrs. Lau r i Larsson for skilful technical assistance. This study was s u p p o r t e d by the Swedish Med i cal R e s e a r c h Council Projects B76-12X-02710-08 a n d B76-14X-00094-12B.
1 BROWN,A. G., HousE, C. R., AND HUME,R. B., Physiology and morphology of identified spinal cord neurones, J. Physiol. (Lond.J, 244 (1974) 10-12. 2 CHRISTENSEN,B. N., Procion Brown: an intracellular dye for light and electron microscopy, Science, 182 (1973) 1255-1256. 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.J, 154 (1960) 89-114. 4 GILETTE, R., AND POMERANZ,B., Neuron geometry and circuitry via the electron microscope: intracellular staining with osmiophilic polymer, Science, 182 (1973) 1256-1258. 5 GLOaUS, A., Lux, H. D., AND SCHUBERT, P., Somadendritic spread of intracellularly injected tritiated glycine in cat spinal motoneurons, Brain Research, I I (1968) 440-445. 6 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. 7 GRAYBIEL,A. M., AND DEVOR, M., A microelectrophoretic delivery technique for use with horseradish peroxidase, Brain Research, 68 (1974) 167-173. 8 JANKOWSKA,E., Identification of interneurons interposed in different spinal reflex pathways. In M. SANT1NI(Ed.), Proc. Golgi Cent. Syrup., Raven Press, New York, 1975, pp. 235-246. 9 KELLERTH,J.-O., lntracellular staining of cat spinal motoneurons with Procion Yellow for ultrastructural studies, Brain Research, 50 (1973) 415J, l 8. 10 LA VAIL,J. H., AND LA VAIL, 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) 303-357. 11 LYNCH,G., DEADWYLER,S., ANDGALL,C., Labeling of central nervous system neurons with extracellular recording microelectrodes, Brain Research, 66 (1974) 337-341. 12 PITMAN,R. M., TWEEDLE,C. O., AND COHEN, M. J., Branching of central neurons: intracellular cobalt injection for light and electron microscopy, Science, 176 (1972) 412-414. 13 PURVES,D., AND MCMAHAN, U. J., The distribution of synapses on a physiologically identified motor neuron in the central nervous system of the leech. An electron microscope study after the injection of the fluorescent dye Procion Yellow, J. Cell Biol., 55 (1972) 205-220. 14 SALPETER, M. M., BACHMANN,L., AND SALPETER, E. E., Resolution in electron microscope radioautography, J. Cell Biol., 41 (1969)1-20. 15 STRETTON,A. O. W., AND KRAVITZ,E. A., Neuronal geometry: determination with a technique of intracellular dye injection, Science, 162 (1968) 132-134.
557
Intracellular application of horseradish peroxidase and its light and electron microscopical appearance in spinocervical tract cells*
ELZBIETA JANKOWSKA, JONAS RASTAD AND JAN WESTMAN (E.J.) Department of Physiology, University of G6teborg, GOteborg, and Department of Anatomy, Biomedical Centre, University of Uppsala, Uppsala (Sweden)
(Accepted December 22nd, 1975)
Present techniques for the intracellular staining of physiologically identified neurons are not quite satisfactory for ultrastructural investigations. Iontophoretic administration of cobalt chloride for subsequent reaction with ammonium sulphide 12 does not allow fixation before incubation, a disadvantage as regards the ultrastructural preservation. If the injected cobalt chloride is allowed to react with diaminobenzidine 4 the fine structure of the cytoplasm appears to be damaged. This holds true also for iontophoretically injected Procion brown 2. Procion yellow 1~ is not electron dense and ultrastructural identification has to depend on changes of cell organelles9, la. Radioautographic techniques 5 are laborious because ultrastructural interpretation needs statistical analysis since the silver grains are situated at a distance from the source of activity 14. In this study the enzyme horseradish peroxidase (HRP) has been used for intracellular staining. H R P has recently found extensive use as a neuronal marker (for references, see ref. 10) in light and electron microscopical studies. The histochemical staining procedure employed seems not to damage the ultrastructure and the product of enzyme reaction is found in lysosome-like organelles 1°. In the present investigation the enzyme was administered intracellularly in spinocervical tract cells a, neurons previously studied with intracellular injections of Procion yellow 1,s. Cells were identified by monosynaptic excitation from the cutaneous nerves and by antidromic invasion from the dorsolateral funiculus in segment C3 (Fig. 1A-G). An additional criterion was the absence of antidromic invasion from C1 except at a high threshold. A total of 23 spinocervical tract cells were impaled with single barrelled micropipettes with tip diameters of 2-3/~m. The electrodes were filled by pressure with a 15-20 ~o (w/v) solution of H R P (Sigma, type II). In contrast to previous studies, where extracellular iontophoresis of H R P was performed 7,11, solutions in distilled water were initially used. In order to decrease the high electrode resistance by increasing ionic strength, both acid and basic solutions were tested. The best results were obtained * Reprints are available from: Department of Anatomy, Biomedical Centre, Box 571, S-751 23 Uppsala, Sweden.
558
Before injection
,~>
C3
After injection
~IOmV(D,E,G)
A
C
B
--
, ....
,1+5ms
_2 J C D
F
E
G
{ Hj
~
Fig. 1. A-G: upper records in A-C and D-G are from two spinocervical tract cells; lower records are from the surface of the spinal cord close to the site of microelectrode penetration. A and D show antidromic invasions from C3, with blocked action potential in A. Remaining records show monosynaptic and polysynaptic potentials from the superficial peroneal nerve (SP). Pictures were taken immediately before (B and E), immediately after (C and F) and a few minutes after (G) enzyme administration. Note recovery of postsynaptic potential in G. These cells were stained by passing 10 nA for 16 rain (A-C) and 13 min (D-G), respectively. H: schematic drawing of the same cell as in A-C and its localization in the lumbar dorsal horn. m, axon; ac, axon collaterals.
by dissolving H R P in 0.2 M N a O H giving a final p H o f a b o u t 8.5 and an electrode resistance o f 25-35 M ~ . In all cases H R P appeared to be ejected only by positive current. The a m o u n t o f enzyme administered was estimated by multiplying the time (in min) by the current (in hA). Continuous currents o f 5-10 nA for 5-25 min were passed through the electrodes while recording from the cells. Fig. l shows that during this period the cellular responses somewhat deteriorated (Fig. I C and F). This m a y have been due primarily to depolarization o f the cells rather than to an effect o f the injected enzyme, since several cells greatly recovered after the injection (compare Fig. l E and Fig. 1G). After diffusion o f H R P for 20-430 min the cats were perfused transcardially with a 4 l mixture o f 2 ~ (v/v) glutaraldehyde and l ~ (v/v) formaldehyde in 0.05 M sodium cacodylate buffer. The dorsal horn from the injected side o f segments L6-7 was dissected out, kept in fixative for 12 h and then cut transversely with a Sorvall TC-2 tissue sectioner. Sections about 120 # m thick were incubated for 30 min in a solution o f 0.05 ~ (w/v) diaminobenzidine (DAB) followed by a mixture o f 0.05 D A B and 0.01 ~ (v/v) hydrogen peroxide 6 for 30 min. Cells were processed for light and electron microscopy according to Rastad and Westman (to be published). This
559
Fig. 2. A: light micrograph of the same cell as in Fig. 1A-C. The arrow points dorsally, t, electrode track; ia, initial axon segment; m, myelinized axon. x 200. B: montage of light micrographs from one section of the same cell as in Fig. IA-C. Two axon collaterals (ac) leave the parent axon (m) at the same place (arrow). x 680. technique allowed light microscopical observation on the thick plastic embedded sections stained by osmium tetroxide and re-embedding for ultrathin serial sectioning. Electron microscopy was performed on 11 cells. Ultrathin sections were not contrasted. During the course of the study attempts were made to establish the maximal amount of enzyme, which could be administered without serious damage to the ultrastructural appearance of the cells. F o r light microscopical identification, more than 40 nA.min were needed. On the other hand applications of more than 170 nA.min seemed to increase the incidence of cell damage. N o correlation was observed between the ultrastructural preservation and the functional state of the cells after enzyme administration or between the preservation and the diffusion time. When cell damage was severe it was evident already in the light microscope. The contours of the cells then appeared irregular and shrunken. The dendrites emerged from the soma with abnormally thick proximal segments. The soma membrane, between primary dendrites, often showed a very concave shape thus demonstrating a shrunken perikaryal profile. At the ultrastructural level an increased cytoplasmic vacuolization occurred. A more advanced change was the cell shrinkage with a very condensed cytoplasm. The cell then showed densely packed mitochondria and lysosomes. No Golgi apparatus or endoplasmic reticulum were seen. The nucleus appeared disproportionally large with a diffuse outline of the nuclear membrane. These gravely damaged cells are not included in the description below.
560
Fig. 3. A: electron micrographs of the same cell as in Fig. 1A-C. The cell was stained 310 rain before perfusion. The arrow points dorsally, d, proximal dendrite; c, capillary with endothelial cell containing reaction product; i, filamentous inclusion body reaching the cell membrane at the approximate place of electrode penetration, x 1450. B: electron micrograph of the same cell as in Fig. 1A-C. The finely granular deposits are seen inside the stained cell. x 16,000.
In the light microscope the reaction p r o d u c t seemed evenly distributed (Fig. 2A) giving the injected cells a dark b r o w n appearance. One cell was exceptional, in that the reaction p r o d u c t was accumulated in the periphery of the soma. I n lighter stained cells, less t h a n a b o u t 80 n A . m i n , only few dendrites were seen a n d the nucleus
561 was darker than the cytoplasm. In heavier stained cells, more than about 80 nA.min, the nucleus was invisible. In general the soma was round to oval with mostly dorsally directed dendrites (Figs. 1H and 2A), which could be followed up to 450 ~m. The axons emerged from the ventral or lateral aspects of the cell bodies. The initial axon segments were easily identified. Four cells showed dorsomedially directed axon collaterals leaving the parent axon within the dorsal horn (Fig. 2B). Neither axonal projections in the white matter nor axon collaterals at more rostral levels (as found by Brown, Rose and Snow, personal communication, by HRP staining) were studied. In the electron microscope finely granular deposits were found in the soma, dendrites and axon. Lysosomes containing deposits were seen. Mitochondria and Golgi apparatus appeared normal. There was a sparse amount of granular endoplasmic reticulum. The cell membrane and, to a lesser extent, the nuclear envelope were outlined with deposits (Fig. 3A and B). Grains were also found in somaspines. Occasionally the outline of the stained cells showed invaginations containing adjacent neuronal and glial elements. The deposits were also seen in the nucleus giving it a darker appearance. The nucleolonema appeared slightly condensed. Small amounts of precipitate were seen extracellularly in the light microscope. To what extent this was due to diffusion of H R P from the electrode tip or to cleaning of the electrodes, by passing current, could not be clarified. Ultrastructurally minor amounts of reaction product were also seen in membrane bound organelles of nonpenetrated cells, mainly in glial profiles and endothelial cells (Fig. 3A). Electrode tracks could be followed as they were filled with stained blood cells (Fig. 2A). The localization of spinocervical cells was in agreement with earlier investigations 1,s. The present study also confirmed the general appearance of spinocervical tract cells seen after Procion yellow injections1, s. The major advantage of intracellular staining with H R P seems to be that amounts of enzyme large enough for an extensive light microscopical investigation of dendritic and proximal axonal aborization allow a reasonably good ultrastructural preservation. The electron dense deposits are easily identified. Stained profiles down to 0.35 ~m have been seen. The enzyme distributes quickly within the neuronal processes. The reaction product has a low osmium sensitivity and appropriate postfixation can be performed. The ultrastructural preservation is not as dependent on a short period between injection and perfusion as with other staining proceduresL The enzyme-filled micropipettes also have acceptable recording properties. It is yet not possible to state how complete the staining of cell processes is. Several facts suggest a rather extensive staining. Thus, no unstained dendrites of properly stained perikarya were seen, nor was any decreased density of reaction product observed in the first 150 # m of the processes. Although most of the perikarya and processes in the present investigation were preserved satisfactorily, some were seriously damaged. The reasons for this remain to be clarified. An estimate of possible loss of boutons 9 due to penetration and enzyme administration will have to await studies of non-injected cells. This paper will be followed by an extensive report of spinocervical tract cells labelled by both intracellular and extracellular techniques.
562 NOTE ADDED IN PROOF A f t e r this p a p e r was accepted for publication, the light microscopical results o f intracellular application o f H R P especially for tracing axonal projections o f spinal n eu ro n s were published. (SNow, P. J., RosE, P. K., AND BROWN, A., Tr aci n g axons and a x o n collaterals o f spinal neurones using intracellular injection o f horseradish peroxidase, Science, 191 (1976) 312-313.) We wish to th a n k M r A l f J o h a n s s o n an d Mrs. Lau r i Larsson for skilful technical assistance. This study was s u p p o r t e d by the Swedish Med i cal R e s e a r c h Council Projects B76-12X-02710-08 a n d B76-14X-00094-12B.
1 BROWN,A. G., HousE, C. R., AND HUME,R. B., Physiology and morphology of identified spinal cord neurones, J. Physiol. (Lond.J, 244 (1974) 10-12. 2 CHRISTENSEN,B. N., Procion Brown: an intracellular dye for light and electron microscopy, Science, 182 (1973) 1255-1256. 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.J, 154 (1960) 89-114. 4 GILETTE, R., AND POMERANZ,B., Neuron geometry and circuitry via the electron microscope: intracellular staining with osmiophilic polymer, Science, 182 (1973) 1256-1258. 5 GLOaUS, A., Lux, H. D., AND SCHUBERT, P., Somadendritic spread of intracellularly injected tritiated glycine in cat spinal motoneurons, Brain Research, I I (1968) 440-445. 6 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. 7 GRAYBIEL,A. M., AND DEVOR, M., A microelectrophoretic delivery technique for use with horseradish peroxidase, Brain Research, 68 (1974) 167-173. 8 JANKOWSKA,E., Identification of interneurons interposed in different spinal reflex pathways. In M. SANT1NI(Ed.), Proc. Golgi Cent. Syrup., Raven Press, New York, 1975, pp. 235-246. 9 KELLERTH,J.-O., lntracellular staining of cat spinal motoneurons with Procion Yellow for ultrastructural studies, Brain Research, 50 (1973) 415J, l 8. 10 LA VAIL,J. H., AND LA VAIL, 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) 303-357. 11 LYNCH,G., DEADWYLER,S., ANDGALL,C., Labeling of central nervous system neurons with extracellular recording microelectrodes, Brain Research, 66 (1974) 337-341. 12 PITMAN,R. M., TWEEDLE,C. O., AND COHEN, M. J., Branching of central neurons: intracellular cobalt injection for light and electron microscopy, Science, 176 (1972) 412-414. 13 PURVES,D., AND MCMAHAN, U. J., The distribution of synapses on a physiologically identified motor neuron in the central nervous system of the leech. An electron microscope study after the injection of the fluorescent dye Procion Yellow, J. Cell Biol., 55 (1972) 205-220. 14 SALPETER, M. M., BACHMANN,L., AND SALPETER, E. E., Resolution in electron microscope radioautography, J. Cell Biol., 41 (1969)1-20. 15 STRETTON,A. O. W., AND KRAVITZ,E. A., Neuronal geometry: determination with a technique of intracellular dye injection, Science, 162 (1968) 132-134.
