132
BraiJ1 Research, 118 (1976)132 136 ~i Elsevier/North-Holland Biomedical Press, Amsterdam Printed ill The Netherlands
Intracellular staining of Purkinje cells and their axons with horseradish peroxidase
R. A. McCREA, G. A. BISHOP and S. T. KITAI
Morin Memorial Laboratory, Wayne State University School of Medicine, Department of Anatomy, Detroit, Mich. 48201 (U.S.A.) (Accepted September 3rd, 1976)
Electrophoretic injections of horseradish peroxidase (HRP) into single neurons with recording microelectrodes have recently been shown to be a useful method for staining neurons that have been studied electrophysiologically5,6,11. When the enzyme was then reacted with diaminobenzidine (DAB) and H~O2, both the dendrites and axonal processes of these cells could be visualized. We would like to report the results of applying this new staining technique to an area of the central nervous system where the morphology is already fairly well understood, the cerebellar cortex2, 9. Purkinje cells (Pk cells) in the anterior lobe and paramedian lobule of cats were stained by injecting HRP intracellularly through recording microelectrodes. The best results were obtained when the tip of a microelectrode was broken to a diameter of 1.5-1.0/am. The DC resistance of an electrode filled with a 4 ~ solution of HRP (Sigma Type VI in Tris buffer (pH 8.6) and 0.2 M KC1) was usually between 50 and 100 Mr2. The contralateral inferior olive was stimulated to aid in searching for and identifying Pk cells. The amplitude of the climbing fiber response in cells penetrated by these electrodes was usually quite low (10-40 mV), although the resting potential was sometimes higher. The low amplitude of the spikes was probably due to injury by penetration with electrodes with relatively large tips. Penetrated cells with resting potentials less than 20 mV were usually not injected. The HRP was injected electrophoretically with positive DC pulses (3.3 Hz, 200 msec duration, 15-40 nA for 12-45 min). The best staining was obtained when the soma was injected with 20-30 nA for over 20 min. Following survival periods of 6-27 h, the animal was perfused with a fixative of 1 ~ glutaraldehyde and 2 ~o paraformaldehyde in phosphate buffer (pH 7.4). Serial sagittal sections, 100/am thick, were cut on a freezing microtome, incubated with DAB and H~O2, mounted and counterstained with cresyI violet. The injected Pk cells were easily located, since usually they could be seen with the naked eye. The soma, dendrites and dendritic spines were typically filled with HRP (Fig. 1A and B). This diffuse brown staining usually appeared to be agranular at the light microscopic level; however, occasionally dark granules could be seen in the soma and large dendrites in addition to the diffuse staining.
133 Fig. 1A is a photomicrograph of a Pk cell in lobule III of the pars intermedia of the anterior lobe that was injected with 30 nA of HRP for 20 min. Note that the dendritic tree appears to be completely stained. At higher magnifications, the spines are clearly visible (Fig. 1B). In 22 of the 34 Pk cells stained so far, the injected cell was the only neural element stained by the injection. However, in almost every case a few nearby glial elements contained brown granules, which would indicate that some HRP was leaked by the microelectrode in its approach toward the cell or that there was a small amount of leakage from the punctured cell. In 12 cases, adjacent neural elements were stained. In these instances, the element most frequently stained was a nearby basket cell. Since the stained basket cell participated in the axonal plexus around the soma of the injected Pk cell, it is very possible that its axon was injured during penetration. We have found that cells whose axons are in the region of an electrophoretic injection of HRP may be stained in a manner similar to intracellularly injected neurons (unpublished observation). The selective staining of only one basket cell in many cases may reflect the fact that this was the only cell whose axon was injured in close proximity to the injection. One of the more promising characteristics of the technique of intracellularly staining neurons with HRP is that the enzyme appears to be transported anterogradely in the axon 11. Since the injected neuron is the only stained element in the tissue, the course of the axon can be followed in serial sections to its termination. The course of the axon of the cell in Fig. 1A is shown in the drawings of Fig. 2. The climbing fiber response of this cell is shown in Fig. 2D. The trajectory of the axon was reconstructed from seven 100 #m sections. It followed a rather uncomplicated course, tapering in diameter fi'om 1.5/~m in the granular layer to 1.0/~m just before entering the nucleus interpositus anterior (NIA). Arising from a Pk cell midway along the dorsal folium of lobule III, it issued a single local collateral and entered the white matter. After leaving the folium, it coursed dorsally for about 1 mm before assuming a rather direct trajectory toward NIA. The axon made an abrupt turn just before entering the substance of this nucleus [,Figs. 1D and 2), a common characteristic of the Pk cell axons we have followed. Other Pk axons have been observed to take a very complicated course after exiting the folium in which they originated. In fact, on 4 occasions they have actually reversed in direction, coursing several hundred micrometers away from the deep nuclei before resuming a nuclear bound trajectory. The point at which axons were most likely to become twisted and contorted was at the mouth ofa folium (Fig. 2). At this point many axons were observed to course laterally for a millimeter or more before resuming a nuclear bound trajectory. Pk axons always gave rise to one or more recurrent collaterals while passing through the granular layer or immediately after entering the white matter. These fine collaterals terminated by forming a beaded plexus in and just beneath the Pk cell layer. This plexus, which corresponds to the infraganglionic and supraganglionic plexuses of Jakob 4, extended 250-500/~m in the sagittal plane (perpendicular to the axis of the folium) and 200-300/~m in the frontal plane. Figs. 1C and 2B show some of the recurrent collateral plexus formed by the cell in Fig. 1A. Occasionally one or more collaterals would terminate in the granular layer, and in 3 cells beaded collaterals
\ Q
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135
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Fig. 2. A: the course of the axon of the Pk cell shown in Fig. 1. Reconstructed from seven 100/~m thick sections. NIA, nucleus interpositus anterior; NIP, nucleus interpositus posterior. B: drawing of the recurrent collateral plexus of this neuron. C: terminal arborization of the axon; reconstructed from three 100/~m sections. D: climbing fiber response of the illustrated neuron. arose from the plexus in the Pk cell layer and climbed into the molecular layer. The morphology and areas of termination of these recurrent collaterals were similar to the descriptions of investigators using the Golgi techniquea, 8-1°. It is interesting that none of the 16 Pk axons we have followed to the deep nuclei have branched outside of the folium in which they originated until their terminal arborization in the deep nuclei. Fig. 1D is a photomicrograph of the axon of the cell in Fig. 1A as it enters NIA. At this point the axon is considerably less stained than it is closer to the soma. Presumably this fading was due to a lower concentration of H R P and was consistently observed. Some of the most significant factors which appeared to affect the stainability of the terminal arborization were the length of the axon, the period of time between the injection and perfusion (survival time), and the amount of current injected. This would indicate that anterograde axonal transport was important in determining the amount of H R P in the terminal arborization. The survival time of Pk cells with visible terminal arborizations ranged from 16 to 27 h and their length ranged from 6.3 to 10.5 mm. The best results were obtained from Pk cells in the deep folia of the cortex, presumably
Fig. 1. A: photomicrograph of the soma and dendrites of a Pk cell in lobule III injected with HRP. higher power photomicrograph of the proximal and spine laden distal dendrites of the same cell. C: axon and singlerecurrent collateral from samecell; upward arrow points to endothelial cell, downward arrow points to red blood cell. D: same axon as it enters NIA. B:
136 because the distance these axons traveled before terminating was considerably shorter. Fig. 2C is a drawing o f the terminal arborization o f the Pk cell shown in Fig. 1. After entering N I A , the axon branched repeatedly, arborizing anteriorly and ventrally. The form o f this arborization was similar to the terminal arborization of Pk cells described by investigators using the Golgi techniqueS,7,10. The exact dimensions o f the termination of this axon were difficult to determine, since the smaller branches were quite faint. The largest terminal arborization we have observed extended 700 # m vertically, 250 # m anterior-posteriorly, and was contained in two 100/~m thick sections. To our knowledge, this is the first time that the axon of a single identified projection neuron has been traced all the way to its site o f termination. This is a preliminary report on our systematic investigation of the axons of Pk cells by use o f intracellutar injections o f HRP. Application o f the Golgi technique has revealed many details of organization of the cerebellar cortex and the cerebellar nuclei, lntraceltular injections o f H R P in selected Pk cells will help elucidate some o f the details of the organization of the cortico-nuclear projection. The authors wish to express their appreciation to Dr. C. Fox for the use of his photographic facilities, and to Dr. J. Rafols for the use o f his microscope and drawing tube. This work was supported by N I H Grants NS 00405 and R R 5384.
Chan-Palay, V., Afferent axons and their relations with neurons in the nucleus lateralis of the cerebellum: A light microscopic study, Z. Anat. Entwickl.-Gesch., 142 (1973) 1-21. 2 Eccles, J., lto, M. and SzentS.gothai, J., The Cerebellum as a Neuronal Machine, Springer, Berlin, 1967. 3 Fox, C. A., HiUman, D. E., Siegesmund, K. A. and Dutta, C. R., The primate cerebellar cortex: a Golgi and electron microscopic study. In C. A. Fox and R. S. Snider (Eds0, The Cerebellum, Progr. Brain Res., Vol. 25, Elsevier, Amsterdam, 1967, pp. 174-225. 4 Jakob, A., Das Kleinhirn. In yon M61tendorff's Handbueh tier microskopischen Anatomic des Menschen, Nervensystem, Vol. 4, Springer, Berlin, 1928, pp. 674--916. 5 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. 6 Kitai, S. T., Kocsis, J. D., Preston, R. J. and Sugimori, M., Monosynaptic inputs to caudate neurones identified by intracellular injection of horseradish peroxidase, Brain Research, 109 (1976) 601-606. 7 Matsushita, M. and Iwahori, N., Structural organization of the interpositus and the dentate nuclei, Brain Research, 35 (1971) 17-36. 80'Leary, J. L., Petty, J., Smith, J. M., O'Leary, M. and Inukai, J., Cerebellar cortex of rat and other animals. A structural and ultrastructural study, J. comp. Neurol., 134 (1968) 401-432. 9 Palay, S. L. and Chan-Palay, V., Cerebellar Cortex Cytology and Organization, Springer, New York, 1974. 10 Ram6n y Cajal, S., Histologie du Systdme Nerveux de l'Homme et des Vertdbrds, Maloine, Paris, 1911, pp. 12-15. 1l Snow, P. J., Rose, P. K. and Brown, A. G., Tracing axons and axon collaterals of spinal neurons using intracetlular injection of horseradish peroxidase, Science, 191 (1976) 312-313. 1
BraiJ1 Research, 118 (1976)132 136 ~i Elsevier/North-Holland Biomedical Press, Amsterdam Printed ill The Netherlands
Intracellular staining of Purkinje cells and their axons with horseradish peroxidase
R. A. McCREA, G. A. BISHOP and S. T. KITAI
Morin Memorial Laboratory, Wayne State University School of Medicine, Department of Anatomy, Detroit, Mich. 48201 (U.S.A.) (Accepted September 3rd, 1976)
Electrophoretic injections of horseradish peroxidase (HRP) into single neurons with recording microelectrodes have recently been shown to be a useful method for staining neurons that have been studied electrophysiologically5,6,11. When the enzyme was then reacted with diaminobenzidine (DAB) and H~O2, both the dendrites and axonal processes of these cells could be visualized. We would like to report the results of applying this new staining technique to an area of the central nervous system where the morphology is already fairly well understood, the cerebellar cortex2, 9. Purkinje cells (Pk cells) in the anterior lobe and paramedian lobule of cats were stained by injecting HRP intracellularly through recording microelectrodes. The best results were obtained when the tip of a microelectrode was broken to a diameter of 1.5-1.0/am. The DC resistance of an electrode filled with a 4 ~ solution of HRP (Sigma Type VI in Tris buffer (pH 8.6) and 0.2 M KC1) was usually between 50 and 100 Mr2. The contralateral inferior olive was stimulated to aid in searching for and identifying Pk cells. The amplitude of the climbing fiber response in cells penetrated by these electrodes was usually quite low (10-40 mV), although the resting potential was sometimes higher. The low amplitude of the spikes was probably due to injury by penetration with electrodes with relatively large tips. Penetrated cells with resting potentials less than 20 mV were usually not injected. The HRP was injected electrophoretically with positive DC pulses (3.3 Hz, 200 msec duration, 15-40 nA for 12-45 min). The best staining was obtained when the soma was injected with 20-30 nA for over 20 min. Following survival periods of 6-27 h, the animal was perfused with a fixative of 1 ~ glutaraldehyde and 2 ~o paraformaldehyde in phosphate buffer (pH 7.4). Serial sagittal sections, 100/am thick, were cut on a freezing microtome, incubated with DAB and H~O2, mounted and counterstained with cresyI violet. The injected Pk cells were easily located, since usually they could be seen with the naked eye. The soma, dendrites and dendritic spines were typically filled with HRP (Fig. 1A and B). This diffuse brown staining usually appeared to be agranular at the light microscopic level; however, occasionally dark granules could be seen in the soma and large dendrites in addition to the diffuse staining.
133 Fig. 1A is a photomicrograph of a Pk cell in lobule III of the pars intermedia of the anterior lobe that was injected with 30 nA of HRP for 20 min. Note that the dendritic tree appears to be completely stained. At higher magnifications, the spines are clearly visible (Fig. 1B). In 22 of the 34 Pk cells stained so far, the injected cell was the only neural element stained by the injection. However, in almost every case a few nearby glial elements contained brown granules, which would indicate that some HRP was leaked by the microelectrode in its approach toward the cell or that there was a small amount of leakage from the punctured cell. In 12 cases, adjacent neural elements were stained. In these instances, the element most frequently stained was a nearby basket cell. Since the stained basket cell participated in the axonal plexus around the soma of the injected Pk cell, it is very possible that its axon was injured during penetration. We have found that cells whose axons are in the region of an electrophoretic injection of HRP may be stained in a manner similar to intracellularly injected neurons (unpublished observation). The selective staining of only one basket cell in many cases may reflect the fact that this was the only cell whose axon was injured in close proximity to the injection. One of the more promising characteristics of the technique of intracellularly staining neurons with HRP is that the enzyme appears to be transported anterogradely in the axon 11. Since the injected neuron is the only stained element in the tissue, the course of the axon can be followed in serial sections to its termination. The course of the axon of the cell in Fig. 1A is shown in the drawings of Fig. 2. The climbing fiber response of this cell is shown in Fig. 2D. The trajectory of the axon was reconstructed from seven 100 #m sections. It followed a rather uncomplicated course, tapering in diameter fi'om 1.5/~m in the granular layer to 1.0/~m just before entering the nucleus interpositus anterior (NIA). Arising from a Pk cell midway along the dorsal folium of lobule III, it issued a single local collateral and entered the white matter. After leaving the folium, it coursed dorsally for about 1 mm before assuming a rather direct trajectory toward NIA. The axon made an abrupt turn just before entering the substance of this nucleus [,Figs. 1D and 2), a common characteristic of the Pk cell axons we have followed. Other Pk axons have been observed to take a very complicated course after exiting the folium in which they originated. In fact, on 4 occasions they have actually reversed in direction, coursing several hundred micrometers away from the deep nuclei before resuming a nuclear bound trajectory. The point at which axons were most likely to become twisted and contorted was at the mouth ofa folium (Fig. 2). At this point many axons were observed to course laterally for a millimeter or more before resuming a nuclear bound trajectory. Pk axons always gave rise to one or more recurrent collaterals while passing through the granular layer or immediately after entering the white matter. These fine collaterals terminated by forming a beaded plexus in and just beneath the Pk cell layer. This plexus, which corresponds to the infraganglionic and supraganglionic plexuses of Jakob 4, extended 250-500/~m in the sagittal plane (perpendicular to the axis of the folium) and 200-300/~m in the frontal plane. Figs. 1C and 2B show some of the recurrent collateral plexus formed by the cell in Fig. 1A. Occasionally one or more collaterals would terminate in the granular layer, and in 3 cells beaded collaterals
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Fig. 2. A: the course of the axon of the Pk cell shown in Fig. 1. Reconstructed from seven 100/~m thick sections. NIA, nucleus interpositus anterior; NIP, nucleus interpositus posterior. B: drawing of the recurrent collateral plexus of this neuron. C: terminal arborization of the axon; reconstructed from three 100/~m sections. D: climbing fiber response of the illustrated neuron. arose from the plexus in the Pk cell layer and climbed into the molecular layer. The morphology and areas of termination of these recurrent collaterals were similar to the descriptions of investigators using the Golgi techniquea, 8-1°. It is interesting that none of the 16 Pk axons we have followed to the deep nuclei have branched outside of the folium in which they originated until their terminal arborization in the deep nuclei. Fig. 1D is a photomicrograph of the axon of the cell in Fig. 1A as it enters NIA. At this point the axon is considerably less stained than it is closer to the soma. Presumably this fading was due to a lower concentration of H R P and was consistently observed. Some of the most significant factors which appeared to affect the stainability of the terminal arborization were the length of the axon, the period of time between the injection and perfusion (survival time), and the amount of current injected. This would indicate that anterograde axonal transport was important in determining the amount of H R P in the terminal arborization. The survival time of Pk cells with visible terminal arborizations ranged from 16 to 27 h and their length ranged from 6.3 to 10.5 mm. The best results were obtained from Pk cells in the deep folia of the cortex, presumably
Fig. 1. A: photomicrograph of the soma and dendrites of a Pk cell in lobule III injected with HRP. higher power photomicrograph of the proximal and spine laden distal dendrites of the same cell. C: axon and singlerecurrent collateral from samecell; upward arrow points to endothelial cell, downward arrow points to red blood cell. D: same axon as it enters NIA. B:
136 because the distance these axons traveled before terminating was considerably shorter. Fig. 2C is a drawing o f the terminal arborization o f the Pk cell shown in Fig. 1. After entering N I A , the axon branched repeatedly, arborizing anteriorly and ventrally. The form o f this arborization was similar to the terminal arborization of Pk cells described by investigators using the Golgi techniqueS,7,10. The exact dimensions o f the termination of this axon were difficult to determine, since the smaller branches were quite faint. The largest terminal arborization we have observed extended 700 # m vertically, 250 # m anterior-posteriorly, and was contained in two 100/~m thick sections. To our knowledge, this is the first time that the axon of a single identified projection neuron has been traced all the way to its site o f termination. This is a preliminary report on our systematic investigation of the axons of Pk cells by use o f intracellutar injections o f HRP. Application o f the Golgi technique has revealed many details of organization of the cerebellar cortex and the cerebellar nuclei, lntraceltular injections o f H R P in selected Pk cells will help elucidate some o f the details of the organization of the cortico-nuclear projection. The authors wish to express their appreciation to Dr. C. Fox for the use of his photographic facilities, and to Dr. J. Rafols for the use o f his microscope and drawing tube. This work was supported by N I H Grants NS 00405 and R R 5384.
Chan-Palay, V., Afferent axons and their relations with neurons in the nucleus lateralis of the cerebellum: A light microscopic study, Z. Anat. Entwickl.-Gesch., 142 (1973) 1-21. 2 Eccles, J., lto, M. and SzentS.gothai, J., The Cerebellum as a Neuronal Machine, Springer, Berlin, 1967. 3 Fox, C. A., HiUman, D. E., Siegesmund, K. A. and Dutta, C. R., The primate cerebellar cortex: a Golgi and electron microscopic study. In C. A. Fox and R. S. Snider (Eds0, The Cerebellum, Progr. Brain Res., Vol. 25, Elsevier, Amsterdam, 1967, pp. 174-225. 4 Jakob, A., Das Kleinhirn. In yon M61tendorff's Handbueh tier microskopischen Anatomic des Menschen, Nervensystem, Vol. 4, Springer, Berlin, 1928, pp. 674--916. 5 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. 6 Kitai, S. T., Kocsis, J. D., Preston, R. J. and Sugimori, M., Monosynaptic inputs to caudate neurones identified by intracellular injection of horseradish peroxidase, Brain Research, 109 (1976) 601-606. 7 Matsushita, M. and Iwahori, N., Structural organization of the interpositus and the dentate nuclei, Brain Research, 35 (1971) 17-36. 80'Leary, J. L., Petty, J., Smith, J. M., O'Leary, M. and Inukai, J., Cerebellar cortex of rat and other animals. A structural and ultrastructural study, J. comp. Neurol., 134 (1968) 401-432. 9 Palay, S. L. and Chan-Palay, V., Cerebellar Cortex Cytology and Organization, Springer, New York, 1974. 10 Ram6n y Cajal, S., Histologie du Systdme Nerveux de l'Homme et des Vertdbrds, Maloine, Paris, 1911, pp. 12-15. 1l Snow, P. J., Rose, P. K. and Brown, A. G., Tracing axons and axon collaterals of spinal neurons using intracetlular injection of horseradish peroxidase, Science, 191 (1976) 312-313. 1
