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The nigrotectal projection in the cat: An electron microscope autoradiographic study
0306-4522/87 $3.00 + 0.00 Pergamon Journals Ltd
Neuroscience Vol. 21, No. 2, pp. 529-539, 1987 Printed in Great Britain
0 1987 IBRO
THE NIGROTECTAL PROJECTION IN THE CAT: AN ELECTRON MICROSCOPE AUTORADIOGRAPHIC STUDY M. BEHAN,* C.-S. LJNt and W. C. HALLt *Neuroscience Training Program and School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706, U.S.A. and tDuke University Medical Center, Durham, NC 27710, U.S.A. Abstract-Recent evidence indicates that the nigrotectal tract plays an important role in regulating the premotor responses of cells in the in the intermediate gray layer of the superior colliculus. The purpose of the present study was to characterize the ultrastructure of nigrotectal terminals and of their postsynaptic targets in the intermediate gray layer. Nigrotectal terminals were identified in the electron microscope by labeling them autoradiographically, following injections of tritiated proline into the substantia nigra pars reticulata. The majority of nigrotectal terminals contain a high proportion of pleomorphic vesicles and form symmetrical synaptic contacts. Most of these terminals synapse with small dendritic profiles (2.00 pm + 0.83 SD), which may be the distal dendrites of neurons in the intermediate gray layer. Less than 10% of the labeled contacts are made with cell bodies or initial axonal segments.
Several lines of evidence support the view that the nigra. Some of these results have been presented in substantia nigra pars reticulata influences the eye abstract form4 movement-related responses of neurons in the interEXPERIMENTAL PROCEDURES mediate gray layer of the superior colliculus.22~2*~40~41 The substantia nigra pars reticulata projects to the Six normal adult cats were used. The cats were anesthedeeper layers of the superior colliculus,‘3~23~36 and tized with pentobarbital administered intravenously, and then received two 0.2 ~1 injections of a mixture of anatomical studies have shown a close correspon[‘Hlproline and [3H]leucine (25pCi/pl each) into each dence between the terminal distribution of the nigrosubstantia nigra pars reticulata. The syringe needle was tectal tract, and the location of cells or origin of the guided stereotaxically into the substantia nigra by an predorsal bundle, the pathway which projects to approach that was 20” from vertical. The needle track penetrated the lateral geniculate nucleus and did not encontralateral brainstem gaze centers.28 Moreover, croach on the superior colliculus. Following a 24 h survival physiological studies suggest that the nigrotectal tract period, the animal was reanesthetized, given 1 ml of sodium may serve to gate the eye movement related bursts of heparin intravenously and perfused intracardially. The perimpulses in cells of the intermediate gray layer. In fusate consisted of 1.5 1 of 1% glutaraldehyde and 1% particular, Hikosaka and Wurtz,“-” have reported paraformaldehyde in 0.1 M Sorensen’s phosphate buffer, pH 7.4, with 0.002% CaCl, at 37°C. This solution was that many nigrotectal cells have a high level of followed by 21 of buffered 2% glutaraldehyde and 2% spontaneous activity which decreases before saccades paraformaldehyde at 37°C. After the perfusion, the brain to a visual or auditory stimulus. Since the nigrotectal was refrigerated at 4”C, undisturbed in the skull. Two hours projection has an inhibitory influence on cells in later the brain was exposed and stored in the more dilute the deeper layers of the superior colliculus,6s7*24this fixative overnight at 4°C. The following day the brain was removed from the skull, and the superior colliculus and decrease in nigral activity may serve to disinhibit the caudal diencephalon were dissected out and cut into sections cells of the intermediate gray layer. There is also 1OOpm thick with an Oxford Vibratome. The tissue was evidence that the inhibitory influence of the nigrosubsequently postfixed in 2% 0~0, in phosphate buffer tectal tract is mediated by gamma aminobutyric acid with 5% sucrose, washed, dehydrated and flat-embedded in Epon-Araldite. (GABA). GABAergic neurons are present in the Selected sections containing the injection sites in animals substantia nigra pars reticulata,33s3?and at least some 82-106, 82-109, 82-137 and 82-138 were mounted on gelaof the nigrotectal terminals are GABAergic.*,4’ Furtinized glass slides and dipped in Kodak NTB-2 photothermore, the activity of deep tectal neurons and graphic emulsion. The tissue was exposed for 6 weeks after saccadic eye movements are strongly influenced by which the sections were developed in D19 at 15°C counter stained with Cresyl Violet and cover slipped. For electron GABA agonists and antagonists.16 In the present study, we extend these findings by microscopic autoradiography, squares from the embedded 100 pm sections were cut from the intermediate gray layer describing the ultrastructure of nigrotectal terminals of the superior colliculus in the middle of its rostrocaudal and their postsynaptic targets, after the terminals extent, and remounted on resin blocks. Thin sections from these squares were cut and placed on celloidin-coated glass have been labeled autoradiographically following slides, and then coated with a monolayer of Ilford L4 injections of tritiated proline into the substantia *Author to whom correspondence should be addressed. Abbreoiurion:
GABA, gamma aminobutyric acid.
emulsion.26 After 30-70 weeks exposure, the slides were developed in D19 at 15°C. The emulsion-sectioncelloidin sandwich was floated off on water and the sections were collected on mesh grids. Sections were stained in an LKB
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automatic stainer for 20 min each in uranyl acetate and lead citrate at 30°C. Sections were viewed and photographed on a Phillips 410 electron microscope. Injection sites in animals 82-106, 82-109, 82-137 and 82-138 were plotted with a microprojector in bright field ilIumination. Electron microscopic autoradiographs from animals 82-138 (left side) were analysed by the percentage density method described by Bachman and Salpeter.2,3sA circle with 50% probability of enclosing the site of the radioactive disintegration was drawn around the center of each silver grain, and the identity of profiles contained within the circle ascertained. Eight different categories of profiles were identified: profiles containing round vesicles, profiles containing pleomorphic vesicles, myelinated axons, dendrites, glia. cell bodies, blood vessels and unidentified. Often the circle drawn around each grain enclosed more than one profile type. When this occurred, the grain was divided among the profiles according to the relative area occupied by each protile category in the tissue, using the method of Wiiliams.43 Every silver grain in a 1mm2thin section was analysed in this fashion for the percentage distribution of grains. To calculate the percentage area occupied by each profile category, a montage of six micrographs was taken from the upper left hand corner of each of four randomly chosen grid squares, in the same thin section that was used for the silver grain analysis. The celluiar elements underlying 25 random points on a transparent overlay placed on each of the 24 micrographs were recorded. Percentage density was obtained by dividing percentage distribution by percentage area. If the distribution of grains over the entire tissue is random, then percentage density should have a value of 1. Thus, values greater than 1 indicate that the particular profile is selectively labeled. The size distribution of labeled terminals in all six animals was obtained by entering the outlines of terminals with synaptic specializations on a digitizing tablet interfaced with a Harris 6024/5 computer. The size distribution of postsynaptic dendrites was measured by entering the outlines of only those dendrites which were clearly cut in cross-section, and in which transverse sections of microtubules could be identified. RESULTS
Injection sites Injection sites from animals 82-106,82-109,82-137 and 82-138 were plotted after a six week exposure, with the maximum spread of labet or “halo” inch&d in each drawing. Sections were not available from cases 82- 116 and 82- 154 for plotting the injection sites as all the tissue was processed for electron microscopy. In 82-106, only one injection was made. This injection was we11 localized in the substantia nigra, deep to the rostra1 third of the superior colliculus (Figs I and 2). The injection sites in 82-109 were centered in the rostra1 pole of the tegmentum. Tissue was analysed from the right side of 82-109 only. There was some spread of iabel along the needle track on the right side which may have involved the su~uneifo~ nucleus (Fig. 2). The injection sites in 82-137 were concentrated at the diencephalic-mesencephalic boundary. Tissue was taken from the left side only, as the injection on the right side was too superficial to involve the substantia nigra (Fig. 3). However, the injection site on the right
side may have included a small part of the subcuneiform nucleus and the pardbigeminal nucleus.” Only the tissue from the left side of 82- I38 was used
in the analysis. While there exists the possibility that a few of the labeled terminals are from the subcuneiform and/or parabigeminal nucleus, the following points argue against this. First, the spread of label into these areas was very restricted, and never included labeled ceil bodies. Secondly, labeled terminals in each animal were examined separately, and there was no indication of more than one type of termina1 in any of the six animals. Finally, neither of these areas, the subcuneiform or the parabigeminal nucleus, projects heavily to the intermediate gray layer.9,‘3 Identl~cat~on of ~igrotecta~ terminals Percentage density analysis. Percentage density analysis of.the distribution of silver grains in 82-138 (left side) following an injection of tritiated amino acid in the substantia nigra, was carried out for the following categories: (1) profiles containing round synaptic vesicles, (2) profiles containing pleomorphic synaptic vesicles, (3) myelinated axons, (4) dendrites or dendritic appendages, (5) glia, (6) cell bodies, (7) blood vessels, and (8) unidentified, a category which probably includes unmyelinated axons and preterminals. The analysis summarized in Table 1, was carried out on 535 silver grains overlying tissue from the inte~ediate gray layer of 82-138 (left side). A total of 573 points were used to generate the data for percentage area. Profiles in category 2, those containing mostly pleomorphic vesicles, were most heavily labeled, with a percentage density value of 2.3. Terminals containing mostly round vesicles with a percentage density value of 0.8, were unlabeled. Dendrites had a percentage density value of 1.0. Based on the percentage density analysis, these results suggest that most of the nigrotectal terminals contain pleomorphic vesicles. Grain density analysis. In the percentage density analysis, the sample size for terminals intoning round vesicles was extremely small. Therefore multiply labeled terminals were used to further characterize nigrotectal terminals in all six animals, In a total of 49 multiply labeled terminals sampled, none eontained a majority of round vesicles, U~tr~tructaral characteristics of ~igrotectaz terrn~~a~s Labeled nigrotectal terminals in the intermediate gray layer displayed the following characteristics: (1) Vesicles. The majority of vesicles were pleomorphic (Fig. 4). Vesicles were distributed either throughout the profiles or clustered. The clusters were often adjacent to the synaptic specialization. (2) Contacts. Labeled terminals appeared to form symmetricat synaptic junctions (Fig. 5). (3) Size. Labeled terminals ranged from 0.6 to 3.0pm in mean diameter (X = 1.42 pm _+0.38 SD, n = 251). Heavily labeled terminals with more than one silver grain were also
531
Nigrotectal projection in the cat
Fig. 1. Brightfield photomicrograph of a [3H]proline injection site in the substantia nigra of cat 82-106. This section corresponds approximately with section (C) in Fig. 2. Scale bar = 3 mm.
measured and their size distribution and mean diameter did not differ significantly from the terminals labeled with only one silver grain. (Fig. 6). (4) Terminal boutons. Most terminals appeared to be end-boutons (Fig. 4B), although approximately 4% of labeled terminals were identified as en-passant boutons (Fig. 4A). (5) Postsynaptic site. Approximately 95% of the labeled nigrotectal terminals contacted only one postsynaptic profile. A few contacted two or even three profiles. Most terminals synapsed with dendrites, identified by the regular array of microtubules and/or the presence of ribosomes (Fig. 4D). Only a few terminals contacted cell bodies (6.4%; Fig. 7), initial axonal segments (2%) and vesicle-filled profiles (0.8%; Fig. 4C). The mean diameter of dendrites postsynaptic to nigrotectal terminals was 2.00 pm f 0.83 SD (N = 45) (Fig. 8). Profiles postsynaptic to terminals heavily labeled with more than one silver grain were also measured, and their size distribution and mean diameter did not
differ significantly from the profiles postsynaptic terminals labeled with only one silver grain.
DISCUSSION
The present results support two main conclusions which contribute to our understanding of the nigrotectal pathway. The first is that nigrotectal terminals contain pleomorphic vesicles. This result is consistent with pharmacological and physiological evidence indicating that the nigrotectal tract has an inhibitory, The second conclusion conGABAergic influence. 16*4’ cerns the identity of the postsynaptic processes contacted by the nigrotectal axons. The vast majority (over 90%) of nigrotectal terminals contact medium or small dendrites. This result suggests that the nigrotectal tract does not contribute heavily to the population of GABAergic terminals which previous studies have shown cover the cell bodies and prox-
Table 1. Percentage density analysis of silver grains in the stratum griseum intermediale of 82-138 (left side)
% distribution Profiles containing round vesicles Profiles containing pleomorphic vesicles Myelinated axons Dendrites Cilia Cell bodies Blood vessels Unidentified
to
2.9 13.7 43.5 12.3 4.3 4.4 0.5 18.4
%
%
area
density
3.7 5.9 41.9 12.6 6.1 6.5 3.1 20.2
0.8 2.3 1.0 1.0 0.7 0.7 0.2 0.9
M. BEWANel cd.
Fig. 2. Schematic representation of five sections through the injection sites in animals 82406 and 82-109 plotted using bright field illumination. ICY,inferior colliculus; MG, medial geniculate body; ON, oculomotor nerve; P, cerebral podnnde; PG, periaqueductaI gray; PO, pons; R, red nucleus; RF, reticular formation; SC, superior colSicuhq SN, substantia n&a. Arrows indicate the needk track.
Nigrotectal projection in the cat
82437 Fig. 3. Schematic representation of four sections through the injection sites in animals 82-137 and 82-138 plotted using bright field illumination. Legend same as in Fig. 2, with the addition of C, cuneiform nucleus.
imal dendrites of neurons in the intermediate gray layer. 27 In the sections which follow, we will discuss the evidence for each of these conclusions and their implications for our understanding of tectal circuitry.
Morphology
of nigrotectal
terminals
The present results indicate that the majority of nigrotectal terminals contain pleomorphic vesicles.
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M. BEHANet al
Fig. 4. (A) Heavily labeled en-passant nigrotectal bouton from 82-154. (B) Labeled nigrotectal end bouton from 82-109:curved arrow shows myelin sheath ending at the preterminal; straight arrow shows synaptic specialization. (C) Labeled nigrotectal terminal from 82-154 contacting a vesicle-containing profile (P). (D) Two different labeled nigrotectal terminals from 82-138 contacting the same postsynaptic dendrite. Scale bar for (A)-(D) = 1 pm.
535
Nigrotectal projection in the cat
Fig. 5. On the left is a labeled nigrotectal terminal from 82- 138 contacting a dendrite with the silver grain overlying a symmetrical specialization. On the right (closed arrow) is an unlabeled terminal contacting the same dendrite with an asymmetrical specialization. Scale bar = 1 pm.
40
SINGLE MULTIPLE
GRAIN
X = 1.42 “0.38
GRAINS
X = 1.47 +_0.39
30
20
10
MEAN DIAMETER
(rm)
Fig. 6. The size of labeled nigrotectal terminals in six animals is plotted here. Open bars show terminals with one overlying silver grain. Closed bars show terminals with two or more overlying silver grains.
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M. BEHAN et al.
Fig. 7. (A) and (B) show serial sections through a heavily labeled nigrotectal terminal from 82-l 54 contacting a cell body. Scale bar = 1pm.
Most of the nigrotectal terminals also appear to form symmetrical synaptic contacts. However, without serial sections, it is impossible to state with certainty that all nigrotectal contacts are symmetrical. For example, Rapisardi et nl.,” in a study of F profiles in the lateral geniculate nucleus, have shown that terminals which contain for the most part flattened vesicles may have postsynaptic densities ranging from extensive to thin. Postsynaptic densities in the superior colliculus are, in general, modest. However, the postsynaptic densities associated with nigrotectal terminals are much less extensive than, for example, those of retinal terminals in the superficial tectal layers.? The latter contain round vesicles and form distinct asymmetrical contacts. Our results concerning the morphology of the nigrotectal terminals are consistent with the results of
previous anatomical studies of this pathway. For example, the morphology of terminals in the deep layers of the superior colliculus that degenerate following kainic acid lesions in the substantia nigra has been studied in the cat.42 The degenerating terminals appear to form symmetrical synapses and to contain pleomorphic vesicles. Vincent et uI.,~’ using the electron microscope autoradiographic technique to label nigrotectal fibers in the rat, observed a similar morphology. Pleomorphic vesicles and symmetrical contacts are properties of GABAergic terminals in a wide variety of structures, including the lateral geniculate nucleus,‘O the perigeniculate nucleus,30 visual cortex,” hippocampus and superior colliculus2 of the cat, somatosensory cortex of the monkey,15 and the dorsal cochlear nucleus,32 and neostriatum’ of the
531
Nigrotectal projection in the cat
5i=2.00-'0.83 N = 45
6
MEAN DIAMETER
(pn)
Fig. 8. The size distribution of dendrites postsynaptic to labeled nigrotectal terminals in six animals is plotted here. Only dendrites which were clearly cut in cross section were included.
rat. In all of these areas, immunocytchemical localization of glutamate decaboxylase was confined to terminals displaying pleomorphic vesicles and symmetrical contacts. A similar morphology has been described in the cat for terminals in the superior colliculusz9 and visual cortexI that show selective uptake of tritiated GABA. Further evidence that the nigrotectal pathway is GABAergic is shown by kainic acid lesions in the substantia nigra pars reticulata which result in a significant decrease in glutamate decarboxylase activity in the deep layers of the superior colliculus.41 Moreover, microinjections of bicuculline, a GABA antagonist, into the superior colliculus block the inhibition resulting from nigral stimulation and produce abnormal spontaneous saccades. In contrast, injections of mucimol, a GABA agonist, selectively suppress saccade-related cells in the superior colliculus, mimicking the inhibition that results from nigral stimulation. I6 Thus, both morphological and pharmacological evidence are consistent with the view that the nigrotectal tract is GABAergic. Identity of the postsynaptic process
The majority of terminals labeled in the present experiments contact relatively small processes (2.00 pm + 0.83 mean diameter). Only a few (5%) were found contacting cell somata. This result is
consistent with previous anatomical studies that showed degenerating terminals synapsing with predominantly small and medium-sized dendrites in the superior colliculus, following lesions in the substantia nigra pars reticula. 42 As to the identity of these processes, they might be proximal processes of small neurons or the distal processes of large neurons, since a variety of cell sizes are present in the deep tectal layers.12~28~31 At the present time, the identity of these small dendritic processes remains to be determined. There is evidence, however, that at least some of them are the distal dendrites of tectal efferent neurons. Karabelas and Moschovakis24 have recorded intracellularly from deeper tectal neurons following nigral stimulation, and their results indicate a monosynaptic response. Possible role of nigrotectal input on predorsal bundle neurons
An important question that remains is the nature of the influence of the nigrotectal tract on the cells of origin of the predorsal bundle. There is physiological evidence that the substantia nigra exerts a monosynaptic inhibitory influence on tectospinal neurons,24 and as discussed above, several lines of evidence suggest that this influence is GABAergic. Recently, Lu et al. *’ have shown that predorsal
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M. BEHAK et uf.
bundle cells have somata and proximal dendrites that are contacted by many glutamate decarboxylase immunoreactive terminals. Our present results indicate that the majority of glutamate decarboxylase immunoreactive terminals are not contributed by the nigrotectal tract, since nigrotectal terminals preferentially make synaptic contacts with smaller dendritic processes. Thus, predorsal bundle cells appear to be contacted by at least two sources of GABAergic terminals: one-probably arising from cells in substantia nigra-that contacts distal dendrites, and a second-of unknown origin(s), such as the intrinsic GABAergic neurons within the superior colliculusthat heavily innervates the somata and proximal
dendrites. The distinction between axodendritic and axosomati~ GABAergic terminals on predorsal bundle cells may reflect differences in the mode of action of these inputs, as recent studies have demonstrated for hippocampal and visual cortical ceIls.‘.“5 However, in the superior colliculus further studies are needed to clarify the origin and significance of these GABAergic synaptic connections. Ackno~~edge~nts-This
study was supported by grant EYO4478 to M. Behan, NSO9623 to W. C. Hall and NS 17619 and EY05490 to C.-S. Lin. We are grateful to Lisa Klatt and Peter Appell for their excellent technical assistance. Our thanks to N. B. Cant, R. E. Kalil and P. H. Smith for helpful comments on the manuscript
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Neuroscience Vol. 21, No. 2, pp. 529-539, 1987 Printed in Great Britain
0 1987 IBRO
THE NIGROTECTAL PROJECTION IN THE CAT: AN ELECTRON MICROSCOPE AUTORADIOGRAPHIC STUDY M. BEHAN,* C.-S. LJNt and W. C. HALLt *Neuroscience Training Program and School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706, U.S.A. and tDuke University Medical Center, Durham, NC 27710, U.S.A. Abstract-Recent evidence indicates that the nigrotectal tract plays an important role in regulating the premotor responses of cells in the in the intermediate gray layer of the superior colliculus. The purpose of the present study was to characterize the ultrastructure of nigrotectal terminals and of their postsynaptic targets in the intermediate gray layer. Nigrotectal terminals were identified in the electron microscope by labeling them autoradiographically, following injections of tritiated proline into the substantia nigra pars reticulata. The majority of nigrotectal terminals contain a high proportion of pleomorphic vesicles and form symmetrical synaptic contacts. Most of these terminals synapse with small dendritic profiles (2.00 pm + 0.83 SD), which may be the distal dendrites of neurons in the intermediate gray layer. Less than 10% of the labeled contacts are made with cell bodies or initial axonal segments.
Several lines of evidence support the view that the nigra. Some of these results have been presented in substantia nigra pars reticulata influences the eye abstract form4 movement-related responses of neurons in the interEXPERIMENTAL PROCEDURES mediate gray layer of the superior colliculus.22~2*~40~41 The substantia nigra pars reticulata projects to the Six normal adult cats were used. The cats were anesthedeeper layers of the superior colliculus,‘3~23~36 and tized with pentobarbital administered intravenously, and then received two 0.2 ~1 injections of a mixture of anatomical studies have shown a close correspon[‘Hlproline and [3H]leucine (25pCi/pl each) into each dence between the terminal distribution of the nigrosubstantia nigra pars reticulata. The syringe needle was tectal tract, and the location of cells or origin of the guided stereotaxically into the substantia nigra by an predorsal bundle, the pathway which projects to approach that was 20” from vertical. The needle track penetrated the lateral geniculate nucleus and did not encontralateral brainstem gaze centers.28 Moreover, croach on the superior colliculus. Following a 24 h survival physiological studies suggest that the nigrotectal tract period, the animal was reanesthetized, given 1 ml of sodium may serve to gate the eye movement related bursts of heparin intravenously and perfused intracardially. The perimpulses in cells of the intermediate gray layer. In fusate consisted of 1.5 1 of 1% glutaraldehyde and 1% particular, Hikosaka and Wurtz,“-” have reported paraformaldehyde in 0.1 M Sorensen’s phosphate buffer, pH 7.4, with 0.002% CaCl, at 37°C. This solution was that many nigrotectal cells have a high level of followed by 21 of buffered 2% glutaraldehyde and 2% spontaneous activity which decreases before saccades paraformaldehyde at 37°C. After the perfusion, the brain to a visual or auditory stimulus. Since the nigrotectal was refrigerated at 4”C, undisturbed in the skull. Two hours projection has an inhibitory influence on cells in later the brain was exposed and stored in the more dilute the deeper layers of the superior colliculus,6s7*24this fixative overnight at 4°C. The following day the brain was removed from the skull, and the superior colliculus and decrease in nigral activity may serve to disinhibit the caudal diencephalon were dissected out and cut into sections cells of the intermediate gray layer. There is also 1OOpm thick with an Oxford Vibratome. The tissue was evidence that the inhibitory influence of the nigrosubsequently postfixed in 2% 0~0, in phosphate buffer tectal tract is mediated by gamma aminobutyric acid with 5% sucrose, washed, dehydrated and flat-embedded in Epon-Araldite. (GABA). GABAergic neurons are present in the Selected sections containing the injection sites in animals substantia nigra pars reticulata,33s3?and at least some 82-106, 82-109, 82-137 and 82-138 were mounted on gelaof the nigrotectal terminals are GABAergic.*,4’ Furtinized glass slides and dipped in Kodak NTB-2 photothermore, the activity of deep tectal neurons and graphic emulsion. The tissue was exposed for 6 weeks after saccadic eye movements are strongly influenced by which the sections were developed in D19 at 15°C counter stained with Cresyl Violet and cover slipped. For electron GABA agonists and antagonists.16 In the present study, we extend these findings by microscopic autoradiography, squares from the embedded 100 pm sections were cut from the intermediate gray layer describing the ultrastructure of nigrotectal terminals of the superior colliculus in the middle of its rostrocaudal and their postsynaptic targets, after the terminals extent, and remounted on resin blocks. Thin sections from these squares were cut and placed on celloidin-coated glass have been labeled autoradiographically following slides, and then coated with a monolayer of Ilford L4 injections of tritiated proline into the substantia *Author to whom correspondence should be addressed. Abbreoiurion:
GABA, gamma aminobutyric acid.
emulsion.26 After 30-70 weeks exposure, the slides were developed in D19 at 15°C. The emulsion-sectioncelloidin sandwich was floated off on water and the sections were collected on mesh grids. Sections were stained in an LKB
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automatic stainer for 20 min each in uranyl acetate and lead citrate at 30°C. Sections were viewed and photographed on a Phillips 410 electron microscope. Injection sites in animals 82-106, 82-109, 82-137 and 82-138 were plotted with a microprojector in bright field ilIumination. Electron microscopic autoradiographs from animals 82-138 (left side) were analysed by the percentage density method described by Bachman and Salpeter.2,3sA circle with 50% probability of enclosing the site of the radioactive disintegration was drawn around the center of each silver grain, and the identity of profiles contained within the circle ascertained. Eight different categories of profiles were identified: profiles containing round vesicles, profiles containing pleomorphic vesicles, myelinated axons, dendrites, glia. cell bodies, blood vessels and unidentified. Often the circle drawn around each grain enclosed more than one profile type. When this occurred, the grain was divided among the profiles according to the relative area occupied by each protile category in the tissue, using the method of Wiiliams.43 Every silver grain in a 1mm2thin section was analysed in this fashion for the percentage distribution of grains. To calculate the percentage area occupied by each profile category, a montage of six micrographs was taken from the upper left hand corner of each of four randomly chosen grid squares, in the same thin section that was used for the silver grain analysis. The celluiar elements underlying 25 random points on a transparent overlay placed on each of the 24 micrographs were recorded. Percentage density was obtained by dividing percentage distribution by percentage area. If the distribution of grains over the entire tissue is random, then percentage density should have a value of 1. Thus, values greater than 1 indicate that the particular profile is selectively labeled. The size distribution of labeled terminals in all six animals was obtained by entering the outlines of terminals with synaptic specializations on a digitizing tablet interfaced with a Harris 6024/5 computer. The size distribution of postsynaptic dendrites was measured by entering the outlines of only those dendrites which were clearly cut in cross-section, and in which transverse sections of microtubules could be identified. RESULTS
Injection sites Injection sites from animals 82-106,82-109,82-137 and 82-138 were plotted after a six week exposure, with the maximum spread of labet or “halo” inch&d in each drawing. Sections were not available from cases 82- 116 and 82- 154 for plotting the injection sites as all the tissue was processed for electron microscopy. In 82-106, only one injection was made. This injection was we11 localized in the substantia nigra, deep to the rostra1 third of the superior colliculus (Figs I and 2). The injection sites in 82-109 were centered in the rostra1 pole of the tegmentum. Tissue was analysed from the right side of 82-109 only. There was some spread of iabel along the needle track on the right side which may have involved the su~uneifo~ nucleus (Fig. 2). The injection sites in 82-137 were concentrated at the diencephalic-mesencephalic boundary. Tissue was taken from the left side only, as the injection on the right side was too superficial to involve the substantia nigra (Fig. 3). However, the injection site on the right
side may have included a small part of the subcuneiform nucleus and the pardbigeminal nucleus.” Only the tissue from the left side of 82- I38 was used
in the analysis. While there exists the possibility that a few of the labeled terminals are from the subcuneiform and/or parabigeminal nucleus, the following points argue against this. First, the spread of label into these areas was very restricted, and never included labeled ceil bodies. Secondly, labeled terminals in each animal were examined separately, and there was no indication of more than one type of termina1 in any of the six animals. Finally, neither of these areas, the subcuneiform or the parabigeminal nucleus, projects heavily to the intermediate gray layer.9,‘3 Identl~cat~on of ~igrotecta~ terminals Percentage density analysis. Percentage density analysis of.the distribution of silver grains in 82-138 (left side) following an injection of tritiated amino acid in the substantia nigra, was carried out for the following categories: (1) profiles containing round synaptic vesicles, (2) profiles containing pleomorphic synaptic vesicles, (3) myelinated axons, (4) dendrites or dendritic appendages, (5) glia, (6) cell bodies, (7) blood vessels, and (8) unidentified, a category which probably includes unmyelinated axons and preterminals. The analysis summarized in Table 1, was carried out on 535 silver grains overlying tissue from the inte~ediate gray layer of 82-138 (left side). A total of 573 points were used to generate the data for percentage area. Profiles in category 2, those containing mostly pleomorphic vesicles, were most heavily labeled, with a percentage density value of 2.3. Terminals containing mostly round vesicles with a percentage density value of 0.8, were unlabeled. Dendrites had a percentage density value of 1.0. Based on the percentage density analysis, these results suggest that most of the nigrotectal terminals contain pleomorphic vesicles. Grain density analysis. In the percentage density analysis, the sample size for terminals intoning round vesicles was extremely small. Therefore multiply labeled terminals were used to further characterize nigrotectal terminals in all six animals, In a total of 49 multiply labeled terminals sampled, none eontained a majority of round vesicles, U~tr~tructaral characteristics of ~igrotectaz terrn~~a~s Labeled nigrotectal terminals in the intermediate gray layer displayed the following characteristics: (1) Vesicles. The majority of vesicles were pleomorphic (Fig. 4). Vesicles were distributed either throughout the profiles or clustered. The clusters were often adjacent to the synaptic specialization. (2) Contacts. Labeled terminals appeared to form symmetricat synaptic junctions (Fig. 5). (3) Size. Labeled terminals ranged from 0.6 to 3.0pm in mean diameter (X = 1.42 pm _+0.38 SD, n = 251). Heavily labeled terminals with more than one silver grain were also
531
Nigrotectal projection in the cat
Fig. 1. Brightfield photomicrograph of a [3H]proline injection site in the substantia nigra of cat 82-106. This section corresponds approximately with section (C) in Fig. 2. Scale bar = 3 mm.
measured and their size distribution and mean diameter did not differ significantly from the terminals labeled with only one silver grain. (Fig. 6). (4) Terminal boutons. Most terminals appeared to be end-boutons (Fig. 4B), although approximately 4% of labeled terminals were identified as en-passant boutons (Fig. 4A). (5) Postsynaptic site. Approximately 95% of the labeled nigrotectal terminals contacted only one postsynaptic profile. A few contacted two or even three profiles. Most terminals synapsed with dendrites, identified by the regular array of microtubules and/or the presence of ribosomes (Fig. 4D). Only a few terminals contacted cell bodies (6.4%; Fig. 7), initial axonal segments (2%) and vesicle-filled profiles (0.8%; Fig. 4C). The mean diameter of dendrites postsynaptic to nigrotectal terminals was 2.00 pm f 0.83 SD (N = 45) (Fig. 8). Profiles postsynaptic to terminals heavily labeled with more than one silver grain were also measured, and their size distribution and mean diameter did not
differ significantly from the profiles postsynaptic terminals labeled with only one silver grain.
DISCUSSION
The present results support two main conclusions which contribute to our understanding of the nigrotectal pathway. The first is that nigrotectal terminals contain pleomorphic vesicles. This result is consistent with pharmacological and physiological evidence indicating that the nigrotectal tract has an inhibitory, The second conclusion conGABAergic influence. 16*4’ cerns the identity of the postsynaptic processes contacted by the nigrotectal axons. The vast majority (over 90%) of nigrotectal terminals contact medium or small dendrites. This result suggests that the nigrotectal tract does not contribute heavily to the population of GABAergic terminals which previous studies have shown cover the cell bodies and prox-
Table 1. Percentage density analysis of silver grains in the stratum griseum intermediale of 82-138 (left side)
% distribution Profiles containing round vesicles Profiles containing pleomorphic vesicles Myelinated axons Dendrites Cilia Cell bodies Blood vessels Unidentified
to
2.9 13.7 43.5 12.3 4.3 4.4 0.5 18.4
%
%
area
density
3.7 5.9 41.9 12.6 6.1 6.5 3.1 20.2
0.8 2.3 1.0 1.0 0.7 0.7 0.2 0.9
M. BEWANel cd.
Fig. 2. Schematic representation of five sections through the injection sites in animals 82406 and 82-109 plotted using bright field illumination. ICY,inferior colliculus; MG, medial geniculate body; ON, oculomotor nerve; P, cerebral podnnde; PG, periaqueductaI gray; PO, pons; R, red nucleus; RF, reticular formation; SC, superior colSicuhq SN, substantia n&a. Arrows indicate the needk track.
Nigrotectal projection in the cat
82437 Fig. 3. Schematic representation of four sections through the injection sites in animals 82-137 and 82-138 plotted using bright field illumination. Legend same as in Fig. 2, with the addition of C, cuneiform nucleus.
imal dendrites of neurons in the intermediate gray layer. 27 In the sections which follow, we will discuss the evidence for each of these conclusions and their implications for our understanding of tectal circuitry.
Morphology
of nigrotectal
terminals
The present results indicate that the majority of nigrotectal terminals contain pleomorphic vesicles.
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M. BEHANet al
Fig. 4. (A) Heavily labeled en-passant nigrotectal bouton from 82-154. (B) Labeled nigrotectal end bouton from 82-109:curved arrow shows myelin sheath ending at the preterminal; straight arrow shows synaptic specialization. (C) Labeled nigrotectal terminal from 82-154 contacting a vesicle-containing profile (P). (D) Two different labeled nigrotectal terminals from 82-138 contacting the same postsynaptic dendrite. Scale bar for (A)-(D) = 1 pm.
535
Nigrotectal projection in the cat
Fig. 5. On the left is a labeled nigrotectal terminal from 82- 138 contacting a dendrite with the silver grain overlying a symmetrical specialization. On the right (closed arrow) is an unlabeled terminal contacting the same dendrite with an asymmetrical specialization. Scale bar = 1 pm.
40
SINGLE MULTIPLE
GRAIN
X = 1.42 “0.38
GRAINS
X = 1.47 +_0.39
30
20
10
MEAN DIAMETER
(rm)
Fig. 6. The size of labeled nigrotectal terminals in six animals is plotted here. Open bars show terminals with one overlying silver grain. Closed bars show terminals with two or more overlying silver grains.
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M. BEHAN et al.
Fig. 7. (A) and (B) show serial sections through a heavily labeled nigrotectal terminal from 82-l 54 contacting a cell body. Scale bar = 1pm.
Most of the nigrotectal terminals also appear to form symmetrical synaptic contacts. However, without serial sections, it is impossible to state with certainty that all nigrotectal contacts are symmetrical. For example, Rapisardi et nl.,” in a study of F profiles in the lateral geniculate nucleus, have shown that terminals which contain for the most part flattened vesicles may have postsynaptic densities ranging from extensive to thin. Postsynaptic densities in the superior colliculus are, in general, modest. However, the postsynaptic densities associated with nigrotectal terminals are much less extensive than, for example, those of retinal terminals in the superficial tectal layers.? The latter contain round vesicles and form distinct asymmetrical contacts. Our results concerning the morphology of the nigrotectal terminals are consistent with the results of
previous anatomical studies of this pathway. For example, the morphology of terminals in the deep layers of the superior colliculus that degenerate following kainic acid lesions in the substantia nigra has been studied in the cat.42 The degenerating terminals appear to form symmetrical synapses and to contain pleomorphic vesicles. Vincent et uI.,~’ using the electron microscope autoradiographic technique to label nigrotectal fibers in the rat, observed a similar morphology. Pleomorphic vesicles and symmetrical contacts are properties of GABAergic terminals in a wide variety of structures, including the lateral geniculate nucleus,‘O the perigeniculate nucleus,30 visual cortex,” hippocampus and superior colliculus2 of the cat, somatosensory cortex of the monkey,15 and the dorsal cochlear nucleus,32 and neostriatum’ of the
531
Nigrotectal projection in the cat
5i=2.00-'0.83 N = 45
6
MEAN DIAMETER
(pn)
Fig. 8. The size distribution of dendrites postsynaptic to labeled nigrotectal terminals in six animals is plotted here. Only dendrites which were clearly cut in cross section were included.
rat. In all of these areas, immunocytchemical localization of glutamate decaboxylase was confined to terminals displaying pleomorphic vesicles and symmetrical contacts. A similar morphology has been described in the cat for terminals in the superior colliculusz9 and visual cortexI that show selective uptake of tritiated GABA. Further evidence that the nigrotectal pathway is GABAergic is shown by kainic acid lesions in the substantia nigra pars reticulata which result in a significant decrease in glutamate decarboxylase activity in the deep layers of the superior colliculus.41 Moreover, microinjections of bicuculline, a GABA antagonist, into the superior colliculus block the inhibition resulting from nigral stimulation and produce abnormal spontaneous saccades. In contrast, injections of mucimol, a GABA agonist, selectively suppress saccade-related cells in the superior colliculus, mimicking the inhibition that results from nigral stimulation. I6 Thus, both morphological and pharmacological evidence are consistent with the view that the nigrotectal tract is GABAergic. Identity of the postsynaptic process
The majority of terminals labeled in the present experiments contact relatively small processes (2.00 pm + 0.83 mean diameter). Only a few (5%) were found contacting cell somata. This result is
consistent with previous anatomical studies that showed degenerating terminals synapsing with predominantly small and medium-sized dendrites in the superior colliculus, following lesions in the substantia nigra pars reticula. 42 As to the identity of these processes, they might be proximal processes of small neurons or the distal processes of large neurons, since a variety of cell sizes are present in the deep tectal layers.12~28~31 At the present time, the identity of these small dendritic processes remains to be determined. There is evidence, however, that at least some of them are the distal dendrites of tectal efferent neurons. Karabelas and Moschovakis24 have recorded intracellularly from deeper tectal neurons following nigral stimulation, and their results indicate a monosynaptic response. Possible role of nigrotectal input on predorsal bundle neurons
An important question that remains is the nature of the influence of the nigrotectal tract on the cells of origin of the predorsal bundle. There is physiological evidence that the substantia nigra exerts a monosynaptic inhibitory influence on tectospinal neurons,24 and as discussed above, several lines of evidence suggest that this influence is GABAergic. Recently, Lu et al. *’ have shown that predorsal
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bundle cells have somata and proximal dendrites that are contacted by many glutamate decarboxylase immunoreactive terminals. Our present results indicate that the majority of glutamate decarboxylase immunoreactive terminals are not contributed by the nigrotectal tract, since nigrotectal terminals preferentially make synaptic contacts with smaller dendritic processes. Thus, predorsal bundle cells appear to be contacted by at least two sources of GABAergic terminals: one-probably arising from cells in substantia nigra-that contacts distal dendrites, and a second-of unknown origin(s), such as the intrinsic GABAergic neurons within the superior colliculusthat heavily innervates the somata and proximal
dendrites. The distinction between axodendritic and axosomati~ GABAergic terminals on predorsal bundle cells may reflect differences in the mode of action of these inputs, as recent studies have demonstrated for hippocampal and visual cortical ceIls.‘.“5 However, in the superior colliculus further studies are needed to clarify the origin and significance of these GABAergic synaptic connections. Ackno~~edge~nts-This
study was supported by grant EYO4478 to M. Behan, NSO9623 to W. C. Hall and NS 17619 and EY05490 to C.-S. Lin. We are grateful to Lisa Klatt and Peter Appell for their excellent technical assistance. Our thanks to N. B. Cant, R. E. Kalil and P. H. Smith for helpful comments on the manuscript
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