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The ultrastructural morphology of the subthalamic-nigral axon terminals intracellularly labeled with horseradish peroxidase
182
Brain Research, 299 (1984) 182-185 Elsevier
BRE 20171
The ultrastructural morphology of the subthalamic-nigral axon terminals intracellularly labeled with horseradish peroxidase H. T. CHANG, H. KITA and S. T. KITAI Department of Anatomy, College of Medicine, The University of Tennessee Center for the Health Sciences, 875 Monroe A venue, Memphis, TN38163 (U.S.A.) (Accepted January 3rd, 1984) Key words: subthalamic-nigral projection - - intracellular HRP injection - - electron microscopy
The labeled axons of neurons intraceUularly injected with horseradish peroxidase (HRP) in the rat subthalamic nucleus (STH) were studied with electron microscopy. The main axons and the efferent daughter branches were all myelinated. The morphology of the intrinsic axon terminals within STH was obscured by the dark HRP reaction products, but the labeled efferent STH terminals in the substantia nigra (SN) were revealed to contain small oval vesicles and formed asymmetrical synapses with dendrites of SN neurons. Although the size of the subthalamic nucleus (STH) is relatively small when c o m p a r e d to other nuclei in the basal ganglia, severe m o t o r disorders can result from pathological as well as experimental lesions of STH 1.9,13,18. Its functional importance is also reflected by its anatomical connections to the main output nuclei of the basla ganglia, including both segments of the globus pallidus ( G P ) , and the substantia nigra (SN) 2. Recent electrophysiological5 and anatomicaW studies have shown that most STH neurons send branching projections to both G P and SN. A n analysis of such branching projections at the individual neuronal level was recently conducted in our laboratory using a c o m b i n e d technique of intracellular recording and labeling with horseradish peroxidase ( H R P ) lo-12. However, the ultrastructural morphology of these STH efferent pathways has remained unknown. We report here results of our electron microscopic (EM) analysis of some of the H R P labeled STH axons. The detailed methods of material p r e p a r a t i o n have been r e p o r t e d previously3,n,12. Following light microscopic analyses 12, the thick sections containing H R P - l a b e l e d STH axons were r e - e m b e d d e d on blank plastic blocks and processed for electron mi-
croscopy. In order to avoid processes which were sometimes stained inadvertently by the occasional leakage of the HRP-containing electrodes, all H R P labeled elements examined in this study were previously identified at the light microscopic level and were traced back to H R P - l a b e l e d STH neurons. Since the concentration of H R P is usually very low at distal axonai processes, the H R P reaction products at these distal sites were often too faint to be traced via light microscopy on sections r e - e m b e d d e d on top of a blank plastic block. Therefore, the ultrastructural morphology of the STH efferent terminals in this study included only the most visible and the darkest terminals in SN. Efferent terminals in G P and entopeduncular nucleus (EP) were either too faint to be traced through r e - e m b e d d e d plastic blocks, or had to be trimmed off in the process of thin-sectioning for the terminals in SN. The results presented here included sections from 3 STH neurons intracellularly labeled with H R P ; the light microscopic m o r p h o l o g y of these cell are illustrated in Figs. 1 A , G and 5 of Kita et al. 12 The intrinsic collaterals of a H R P - l a b e l e d STH neuron consist of thin fibers with both boutons enpassage and boutons terminaux (Fig. 1). A t E M lev-
Correspondence: H. T. Chang, Department of Anatomy, College of Medicine. The University of Tennessee Center for the Health Sciences, 875 Monroe Avenue, Memphis, TN 38163, U.S.A.
183
Fig. 1. Light micrograph of an intrinsic terminal (arrow) in STH. Fig. 2A,B: stereo-pair electron micrographs of an intrinsic terminal labeled by HRP. Arrow points to the synapse formed by the terminal and a small dendrite. Due to the presence of dense HRP reaction products, it is difficult to discern the synaptic vesicles within the labeled terminal. Figs. 3-7. Electron micrographs of HRP-labeled STH axons. All at the same magnification. Fig. 3. The labeled main axon before branching in STH. Figs. 4 and 5. Thin sections near the branch point, note that the labeled axons are unmyelinated here. Fig. 6. The SN-projecting branch became myelinated in this section (lower labeled profile) while the GP-projecting branch remained unmyelinated (upper labeled profile). Fig. 7. The GP-projecting branch also became myelinated. Fig. 8. Electron micrograph of an HRP-labeled STH efferent terminal in SN. Note that the labeled terminal formed an asymmetrical synapse (arrow) and contained smaller vesicles than the neighboring unlabeled terminals. The postsynaptic dendrite appeared to be degenerating possibly due to traumatic injuries caused by the stimulating electrodes which were placed in SN for antidromically activating the STH neurons H.
184 el, the collateral fibers were thin (D largest diameter) = 0.35/tm), and the small terminals (D = 1/~m) formed synapses with dendritic shafts (Fig. 2A,B). Since these terminals were relatively close to the site of HRP injection (i.e. the soma), they were densely filled with H R P reaction products, often obscuring the organelles within them. .However, by tilting the EM specimen stage one could obtain a stereoscopic image of the labeled bouton and these were observed to contain oval vesicles (D = 40 nm). The type of these synaptic contacts, however, could not be determined clearly. The main axon branched into two projection daughter processes, one going toward SN, the other toward GP. At the EM level, the myelinated parent main axon (D = 1.23 x 0.48/~m for inner diameters of the axolemma, D = 1.74 × 0.91 p m at the outermost layer of myelin) (Fig. 3) shed its myelin at the branch point (Fig. 4) and gave rise to two unmyelinated daughter axons (Fig. 5) which became myelinated immediately (Figs. 6 and 7). Since the axons were of-tea tangentially sectioned, it was not immediately clear that the GP-projecting branch was thicker than the SN-projecting branch near the site of branching as was indicated by light microscopy t2. The efferent arborization of STH axons in SN consisted of thin fibers with both boutons en-passage and terminaux in the pars reticulata of SN 12. At the EM level, the HRP-labeled terminal collateral fibers were very thin (D up to 0.2/~m). The terminal boutons (D up to 2/~m) contained small oval vesicles (D = 40 nm) and formed asymmetrical synapses with dendritic shafts of SN neurons which were also ensheathed with unlabeled boutons containing large vesicles (D = 50 nm) (Fig. 8). In this study we have demonstrated for the first
This study was supported by National Research Service Award F32NS06951 to H.T.C. and NIH Grant NS14866 to S.T.K.
1 Carpenter, M. B., Whittier, J. R. and Mettler, F, A., Analysis of choreoid hyperkinesia in the rhesus monkey. Surgical and pharmacological analysis of hyperkinesia resulting from lesions in the subthalamic nucleus of Luys, J. cornp. Nearol., 92 (1950) 293-332. 2 Carpenter, M. B., Carleton, S. C., Keller, J. T. and Conte, P., Connections of the subthalamic nucleus in the monkey, Brain Research, 224 (1981) 1-29. 3 Chang, H. T., Wilson, C. J. and Kitai, S. T., Single neostriatal efferent axons in the gtobus pallidus: a light and electron microscopicstudy, Science, 213 (1981) 915-918. 4 Chang, H. T., Kita, H. and Kitai, S. T., The fine structure of the rat subthalamic nucleus: an electron microscopic study, J. comp. Neurol., 221 (1983) 113-123.
5 Deniau, J. M., Hammond, C., Chevalier, G. and Feger, J., Evidence for branched subthalamic nucleus projections to substantia nigra, entopeduncular nucleus and globus pallidus, Neurosci, Len., 9 (1978) 117-121. 6 Grofov~, I. and Rinvik, E., An experimental electron microscopic study on the striatonigral projection in the cat, Exp. Brain Res., 11 (1970) 249-262. 7 Hajdu, F., Hassler, R. and Bak, I. J., Electron microscopic study of the substantia nigra and the strio-nigral projection in the rat, Z. Zellforsch., 146 (1973) 207-221. 8 Hammond, C., Deniau, J. M., Rizsk, A. and Feger, J., Electrophysiological demonstration of an excitatory subthalamo-nigral pathway in the rat, Brain Research, 148 (1978) 235-244.
time the ultrastructural morphology of the intrinsic collaterals of STH neurons, their efferent axons, and the terminals in SN. Based on the size of the boutons and their vesicles, and the postsynaptic targets, the intrinsic collateral terminals of STH neurons are clearly different from the type 2 terminals which are characterized by their large size and preferential distribution on the somata and proximal large dendrites as described in normal materials previously 4. Although the intrinsic terminals resemble in size and shape the type 1 terminals in normal materials 4, the obscuring effect of the dense H R P reaction products made it impossible to determine whether they were a subpopulation of the type 1 terminals, many of which originated in the cerebral cortexl6. On the other hand, the density of the H R P reaction products in some of the efferent terminals in SN was optimal for both tracing at the light microscopic level and analysis at EM level. The morphology of the labeled boutons resembled the type III terminals in the normal rat SN as described by Hajdu et al. 7. These labeled boutons containing small vesicles and forming asymmetrical synapses contrasted sharply with the neighboring unlabeled boutons containing large vesicles and forming symmetrical synapses, most of which probably originated in the neostriatum6,7. Since the major striato-nigral pathway has been demonstrated to be inhibitory and probably GABAergicl4,15, the morphological differences of the STH efferent terminals and the striatal terminals suggest functional8 and perhaps chemical differences between these two pathways.
185 9 Hammond, C., Feger, J., Bioulac, B. and Souteyrand, J. P., Experimental hemiballism in the monkey produced by unilateral kainic acid lesion in corpus Luysii, Brain Research, 171 (1979) 577-580. 10 Hammond, C. and Yelnik, J., Intracellular labelling of rat subthalamic neurons with horseradish peroxidase: computer analysis of dendrites and characterization of axon arborization, Neuroscience, 8 (1983) 781-790. 11 Kita, H., Chang, H. T. and Kitai, S. T., Pallidal inputs to subthalamus: intracellular analysis, Brain Research, 264 (1983) 255--265. 12 Kita, H., Chang, H. T. and Kitai, S. T., The morphology of intracellularly labeled rat subthalamic neurons: a light microscopic analysis, J. comp. Neurol., 215 (1983) 245-257. 13 Martin, J. P., Hemichorea resulting from a local lesion of the brain (syndrome of body of Luys), Brain, 5 (1927) 637-651. 14 Ribak, C. E., Vaughn, J. E., Saito, K., Barber, R. and Roberts, E., Immunocytochemical localization of gluta-
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16
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mate decarboxylase in rat substantia nigra, Brain Research, 116 (1976) 287-298. Ribak, C. E., Vaughn, J. E. and Roberts, E., GABAergic nerve terminals decrease in the substantia nigra following hemitransections of the striatonigral and pallidonigral pathways, Brain Research, 192 (1980) 413--420. Romansky, K. V., Usunoff, K. G., Ivanov, D. P. and Galabov, G. P., Corticosubthalamic projection in the cat: an electron microscopic study, Brain Research, 163 (1979) 319-322. Van der Kooy, D. and Hattori, T., Single subthalamic nucleus neurons project to both the globus pallidus and substantia nigra in rat, J. comp. Neurol., 192 (1980) 751-768. Whittier, J. R. and Mettler, F. A., Studies on the subthalamus of the rhesus monkey. II. Hyperkinesia and other physiologic effects of subthalamic lesions with special reference to the subthalamic nucleus of Luys, J. comp. Neurol., 90 (1949) 319-472.
Brain Research, 299 (1984) 182-185 Elsevier
BRE 20171
The ultrastructural morphology of the subthalamic-nigral axon terminals intracellularly labeled with horseradish peroxidase H. T. CHANG, H. KITA and S. T. KITAI Department of Anatomy, College of Medicine, The University of Tennessee Center for the Health Sciences, 875 Monroe A venue, Memphis, TN38163 (U.S.A.) (Accepted January 3rd, 1984) Key words: subthalamic-nigral projection - - intracellular HRP injection - - electron microscopy
The labeled axons of neurons intraceUularly injected with horseradish peroxidase (HRP) in the rat subthalamic nucleus (STH) were studied with electron microscopy. The main axons and the efferent daughter branches were all myelinated. The morphology of the intrinsic axon terminals within STH was obscured by the dark HRP reaction products, but the labeled efferent STH terminals in the substantia nigra (SN) were revealed to contain small oval vesicles and formed asymmetrical synapses with dendrites of SN neurons. Although the size of the subthalamic nucleus (STH) is relatively small when c o m p a r e d to other nuclei in the basal ganglia, severe m o t o r disorders can result from pathological as well as experimental lesions of STH 1.9,13,18. Its functional importance is also reflected by its anatomical connections to the main output nuclei of the basla ganglia, including both segments of the globus pallidus ( G P ) , and the substantia nigra (SN) 2. Recent electrophysiological5 and anatomicaW studies have shown that most STH neurons send branching projections to both G P and SN. A n analysis of such branching projections at the individual neuronal level was recently conducted in our laboratory using a c o m b i n e d technique of intracellular recording and labeling with horseradish peroxidase ( H R P ) lo-12. However, the ultrastructural morphology of these STH efferent pathways has remained unknown. We report here results of our electron microscopic (EM) analysis of some of the H R P labeled STH axons. The detailed methods of material p r e p a r a t i o n have been r e p o r t e d previously3,n,12. Following light microscopic analyses 12, the thick sections containing H R P - l a b e l e d STH axons were r e - e m b e d d e d on blank plastic blocks and processed for electron mi-
croscopy. In order to avoid processes which were sometimes stained inadvertently by the occasional leakage of the HRP-containing electrodes, all H R P labeled elements examined in this study were previously identified at the light microscopic level and were traced back to H R P - l a b e l e d STH neurons. Since the concentration of H R P is usually very low at distal axonai processes, the H R P reaction products at these distal sites were often too faint to be traced via light microscopy on sections r e - e m b e d d e d on top of a blank plastic block. Therefore, the ultrastructural morphology of the STH efferent terminals in this study included only the most visible and the darkest terminals in SN. Efferent terminals in G P and entopeduncular nucleus (EP) were either too faint to be traced through r e - e m b e d d e d plastic blocks, or had to be trimmed off in the process of thin-sectioning for the terminals in SN. The results presented here included sections from 3 STH neurons intracellularly labeled with H R P ; the light microscopic m o r p h o l o g y of these cell are illustrated in Figs. 1 A , G and 5 of Kita et al. 12 The intrinsic collaterals of a H R P - l a b e l e d STH neuron consist of thin fibers with both boutons enpassage and boutons terminaux (Fig. 1). A t E M lev-
Correspondence: H. T. Chang, Department of Anatomy, College of Medicine. The University of Tennessee Center for the Health Sciences, 875 Monroe Avenue, Memphis, TN 38163, U.S.A.
183
Fig. 1. Light micrograph of an intrinsic terminal (arrow) in STH. Fig. 2A,B: stereo-pair electron micrographs of an intrinsic terminal labeled by HRP. Arrow points to the synapse formed by the terminal and a small dendrite. Due to the presence of dense HRP reaction products, it is difficult to discern the synaptic vesicles within the labeled terminal. Figs. 3-7. Electron micrographs of HRP-labeled STH axons. All at the same magnification. Fig. 3. The labeled main axon before branching in STH. Figs. 4 and 5. Thin sections near the branch point, note that the labeled axons are unmyelinated here. Fig. 6. The SN-projecting branch became myelinated in this section (lower labeled profile) while the GP-projecting branch remained unmyelinated (upper labeled profile). Fig. 7. The GP-projecting branch also became myelinated. Fig. 8. Electron micrograph of an HRP-labeled STH efferent terminal in SN. Note that the labeled terminal formed an asymmetrical synapse (arrow) and contained smaller vesicles than the neighboring unlabeled terminals. The postsynaptic dendrite appeared to be degenerating possibly due to traumatic injuries caused by the stimulating electrodes which were placed in SN for antidromically activating the STH neurons H.
184 el, the collateral fibers were thin (D largest diameter) = 0.35/tm), and the small terminals (D = 1/~m) formed synapses with dendritic shafts (Fig. 2A,B). Since these terminals were relatively close to the site of HRP injection (i.e. the soma), they were densely filled with H R P reaction products, often obscuring the organelles within them. .However, by tilting the EM specimen stage one could obtain a stereoscopic image of the labeled bouton and these were observed to contain oval vesicles (D = 40 nm). The type of these synaptic contacts, however, could not be determined clearly. The main axon branched into two projection daughter processes, one going toward SN, the other toward GP. At the EM level, the myelinated parent main axon (D = 1.23 x 0.48/~m for inner diameters of the axolemma, D = 1.74 × 0.91 p m at the outermost layer of myelin) (Fig. 3) shed its myelin at the branch point (Fig. 4) and gave rise to two unmyelinated daughter axons (Fig. 5) which became myelinated immediately (Figs. 6 and 7). Since the axons were of-tea tangentially sectioned, it was not immediately clear that the GP-projecting branch was thicker than the SN-projecting branch near the site of branching as was indicated by light microscopy t2. The efferent arborization of STH axons in SN consisted of thin fibers with both boutons en-passage and terminaux in the pars reticulata of SN 12. At the EM level, the HRP-labeled terminal collateral fibers were very thin (D up to 0.2/~m). The terminal boutons (D up to 2/~m) contained small oval vesicles (D = 40 nm) and formed asymmetrical synapses with dendritic shafts of SN neurons which were also ensheathed with unlabeled boutons containing large vesicles (D = 50 nm) (Fig. 8). In this study we have demonstrated for the first
This study was supported by National Research Service Award F32NS06951 to H.T.C. and NIH Grant NS14866 to S.T.K.
1 Carpenter, M. B., Whittier, J. R. and Mettler, F, A., Analysis of choreoid hyperkinesia in the rhesus monkey. Surgical and pharmacological analysis of hyperkinesia resulting from lesions in the subthalamic nucleus of Luys, J. cornp. Nearol., 92 (1950) 293-332. 2 Carpenter, M. B., Carleton, S. C., Keller, J. T. and Conte, P., Connections of the subthalamic nucleus in the monkey, Brain Research, 224 (1981) 1-29. 3 Chang, H. T., Wilson, C. J. and Kitai, S. T., Single neostriatal efferent axons in the gtobus pallidus: a light and electron microscopicstudy, Science, 213 (1981) 915-918. 4 Chang, H. T., Kita, H. and Kitai, S. T., The fine structure of the rat subthalamic nucleus: an electron microscopic study, J. comp. Neurol., 221 (1983) 113-123.
5 Deniau, J. M., Hammond, C., Chevalier, G. and Feger, J., Evidence for branched subthalamic nucleus projections to substantia nigra, entopeduncular nucleus and globus pallidus, Neurosci, Len., 9 (1978) 117-121. 6 Grofov~, I. and Rinvik, E., An experimental electron microscopic study on the striatonigral projection in the cat, Exp. Brain Res., 11 (1970) 249-262. 7 Hajdu, F., Hassler, R. and Bak, I. J., Electron microscopic study of the substantia nigra and the strio-nigral projection in the rat, Z. Zellforsch., 146 (1973) 207-221. 8 Hammond, C., Deniau, J. M., Rizsk, A. and Feger, J., Electrophysiological demonstration of an excitatory subthalamo-nigral pathway in the rat, Brain Research, 148 (1978) 235-244.
time the ultrastructural morphology of the intrinsic collaterals of STH neurons, their efferent axons, and the terminals in SN. Based on the size of the boutons and their vesicles, and the postsynaptic targets, the intrinsic collateral terminals of STH neurons are clearly different from the type 2 terminals which are characterized by their large size and preferential distribution on the somata and proximal large dendrites as described in normal materials previously 4. Although the intrinsic terminals resemble in size and shape the type 1 terminals in normal materials 4, the obscuring effect of the dense H R P reaction products made it impossible to determine whether they were a subpopulation of the type 1 terminals, many of which originated in the cerebral cortexl6. On the other hand, the density of the H R P reaction products in some of the efferent terminals in SN was optimal for both tracing at the light microscopic level and analysis at EM level. The morphology of the labeled boutons resembled the type III terminals in the normal rat SN as described by Hajdu et al. 7. These labeled boutons containing small vesicles and forming asymmetrical synapses contrasted sharply with the neighboring unlabeled boutons containing large vesicles and forming symmetrical synapses, most of which probably originated in the neostriatum6,7. Since the major striato-nigral pathway has been demonstrated to be inhibitory and probably GABAergicl4,15, the morphological differences of the STH efferent terminals and the striatal terminals suggest functional8 and perhaps chemical differences between these two pathways.
185 9 Hammond, C., Feger, J., Bioulac, B. and Souteyrand, J. P., Experimental hemiballism in the monkey produced by unilateral kainic acid lesion in corpus Luysii, Brain Research, 171 (1979) 577-580. 10 Hammond, C. and Yelnik, J., Intracellular labelling of rat subthalamic neurons with horseradish peroxidase: computer analysis of dendrites and characterization of axon arborization, Neuroscience, 8 (1983) 781-790. 11 Kita, H., Chang, H. T. and Kitai, S. T., Pallidal inputs to subthalamus: intracellular analysis, Brain Research, 264 (1983) 255--265. 12 Kita, H., Chang, H. T. and Kitai, S. T., The morphology of intracellularly labeled rat subthalamic neurons: a light microscopic analysis, J. comp. Neurol., 215 (1983) 245-257. 13 Martin, J. P., Hemichorea resulting from a local lesion of the brain (syndrome of body of Luys), Brain, 5 (1927) 637-651. 14 Ribak, C. E., Vaughn, J. E., Saito, K., Barber, R. and Roberts, E., Immunocytochemical localization of gluta-
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mate decarboxylase in rat substantia nigra, Brain Research, 116 (1976) 287-298. Ribak, C. E., Vaughn, J. E. and Roberts, E., GABAergic nerve terminals decrease in the substantia nigra following hemitransections of the striatonigral and pallidonigral pathways, Brain Research, 192 (1980) 413--420. Romansky, K. V., Usunoff, K. G., Ivanov, D. P. and Galabov, G. P., Corticosubthalamic projection in the cat: an electron microscopic study, Brain Research, 163 (1979) 319-322. Van der Kooy, D. and Hattori, T., Single subthalamic nucleus neurons project to both the globus pallidus and substantia nigra in rat, J. comp. Neurol., 192 (1980) 751-768. Whittier, J. R. and Mettler, F. A., Studies on the subthalamus of the rhesus monkey. II. Hyperkinesia and other physiologic effects of subthalamic lesions with special reference to the subthalamic nucleus of Luys, J. comp. Neurol., 90 (1949) 319-472.