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Glutamic acid decar☐ylase and enkephalin immunoreactive axon terminals in the rat neostriatum synapse with striatonigral neurons
Brain Research, 365 (1986) 151-158
151
Elsevier BRE 21315
Glutamic acid decarboxylase and enkephalin immunoreactive axon terminals in the rat neostriatum synapse with striatonigral neurons N. ARONIN. K. CHASE and M. DIFIGLIA Departments of Medicine and Physiology, Universi O' of Massachusetts Medical Center, Worcester, MA 01605 and *Department of Neurology, Massachusetts General Hospital, Fruit Street, Boston, MA 02114 (U.S.A. )
(Accepted September 18th, 1985) Key words: Leu-enkephalin - - glutamate decarboxylase - - caudate nucleus - - immunocytochemistry
Synaptic interactions between striatal projection neurons and axon terminals containing immunoreactive glutamic acid decarboxylase (GAD) or Leu-enkephalin were examined in the rat neostriatum using a combined method of horseradish peroxidase retrograde transport from the substantia nigra and immunohistochemistry at the electron microscopic level. Results showed that numerous immunoreactive GAD and enkephalin boutons formed synapses with the cell bodies and dendrites of medium-sized striatonigral neurons. These findings demonstrate that within the neostriatum GABA and enkephalin directly influence caudate output pathways.
G a m m a - a m i n o b u t y r i c acid ( G A B A ) and enkephalins are measurable in high concentrations within the neostriatumg,35,45 where they are candidate transmitters. Both substances are releasable from neostriatal tissue in vitro 22-32 and in vivo ~1,~0, exhibit numerous binding sites within the caudate nucleus ~2,24,28, and produce inhibitory electrophysiological effects 4,19,25. i m m u n o h i s t o c h e m i c a l studies have shown that glutamic acid decarboxylase ( G A D ) , the synthesizing enzyme for G A B A , and immunoreactive enkephalins are located in medium-sized neostriatal spiny neurons13,3t-34-37, which have long efferent axons with collaterals that terminate locally and form synaptic contacts within caudate neuropil 5A4AS.32,42. Recent evidence from a human postmortem study 30 and the high p r o p o r t i o n of caudate efferent neurons known to exist 7A0,21 suggested that one important functional connection in the neostriaturn might involve immunoreactive G A D and/or enkephalin boutons synapsing with striatofugal cells. The synapses made by immunoreactive G A D or Leu-enkephalin axon terminals with striatonigral neurons were investigated at the ultrastructural level in the present study using a c o m b i n e d m e t h o d of
horseradish peroxidase retrograde transport and immunohistochemistry. S p r a g u e - D a w l e y rats (n = 3) were anesthetized with pentobarbital (35 mg/kg) and 0.5 ~1 of 30% complexed wheat germ a g g l u t i n i n - h o r s e r a d i s h peroxidase ( W G A - H R P ; Sigma Chemical Co., St. Louis, M O ) was pressure injected over 1 h into the substantia nigra. Injections were m a d e stereotactically29 and the needle was introduced at a 45 ° angle from the horizontal plane in o r d e r to maximize correct placement of the injected lectin into the substantia nigra, Twenty h later the animals were perfused intracardially for 20 rain with cold fixative (500 ml of 4% p a r a f o r m a l d e h y d e and 0.1% glutaraldehyde buffered to pH 7.4), followed with a perfusion of phosphate buffer (pH 7.2) for 10 rain. The brain was placed in phosphate buffer for 1 h. Sections (35 ~ m ) from the anterior caudate ipsilateral to the injected substantia nigra were cut on a Vibratome. The protocol of B o w k e r et al.S was used to intensify the reaction product in cells which contained transported W G A - H R P . In brief, incubation of the tissue in 0.5% CoCI 2 (5 rain) was followed by t r e a t m e n t with 3 ' , 3 ' - d i a m i n o b e n z i d i n e ( D A B ; Sigma) in phosphate
Correspondence: M. DiFiglia, Department of Neurology, Warren Bldg. 370, Massachusetts General Hospital, Fruit St., Boston. MA
02114, U.S.A. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
152
Fig. 1. Labeled striatonigral neurons in the caudate nucleus exhibit punctate granules of HRP reaction product (arrowheads) in their cytoplasm. Some reaction granules appear in the emerging dendrite (arrow) of one cell. An unlabeled cell appears at ringed arrow. Bar = 20 um.
153 buffer (pH 7.2) containing 0.01%
H202 (15
min). In
all cases, the region of the suhstantia nigra contained a heavy deposit of D A B reaction product. The adjacent ventral tegmental region contained a light precipitate of peroxidase reaction product. A modification of the Sternberger peroxidase antiperoxidase m e t h o d 39 was used as previously described from this l a b o r a t o r y 2. Tissue was incubated in 3% normal goat serum in phosphate buffer for 30 rain and then in antiG A D antiserum (from Dr. J.-Y. Wu), or in anti-leuenkephalin antiserum (from Dr. E c k a r d W e b e r ) at a dilution of 1:300, for 18 h at 4 °C. Sections were then treated with goat anti-rabbit antiserum (Miles), 1:30 dilution for 45 min, followed by P A P complex (Miles) at a dilution of 1:30 for 45 min. The G A D antiserum was generated against a highly purified enzyme43, 44 and has been used successfully in immunohistochemical studies in the caudate 3. The Leu-enkephalin antiserum has the following cross-reactivities with related peptides in r a d i o i m m u n o a s s a y at 50% displacement with 1 ~tM concentration of peptide: Leu-enkephalin, 100%; Met-enkephalin, 0.35%; dynorphinl 7, < 0.04%; dynorphinl 6, < 0.02%; dynorphinl_13, < 0.01%; dynorphinl_ s, < 0.01%; dynorphin~_~7, < 0.001%; and ct-Neo-endorphin, < 0.001% 4l. Deletion of the primary antiserum resulted in absence of immunohistochemical staining. Preincubation of the L e u - e n k e p h a l i n antiserum with Leu-enkephalin (50 ~g/ml) for 6 h eliminated specific immunohistochemical labeling. In both of these control conditions only the r e t r o g r a d e labeling of caudate neurons was observed. Following the immunohistochemistry tissue sections were treated with 1% osmium for 1 - 2 h, stained en bloc with 1% uranyl acetate and then e m b e d d e d flat in E p o n between two sheets of A k l a r . Results were the same in all 3 animals at the light and electron microscopic levels. Light microscopic results showed that H R P reaction granules were visi-
ble in the cell bodies and emerging dendrites of numerous medium-sized neurons throughout the caudate nucleus (Fig. 1). Regions of the caudate containing a high density of retrogradely labeled cells and immunoreactive axon terminals were cut out and r e m o u n t e d on blank Epon blocks. Thin sections with a gold interference color were cut and m o u n t e d on formvar-coated slot grids. Lead staining was not used. At the ultrastructural level electron-dense bodies were observed within the cytoplasm of medium-sized neurons and were used to identify retrogradely labeled cells. As many as 8 dense H R P - l a b e l e d granules could be found in a single section through a neuron (Fig. 2). Most of the reactive granules ranged from 0.2 to 0.4 u m in size, were spherical in shape, appeared uniformally electron dense in thicker sections and in thinner sections had the a p p e a r a n c e of multivesicular bodies (Figs. 4, 5, 9, 10). All the HRP-labeled neurons identified at the ultrastructural level contained unindented nuclei and a cytoplasm relatively sparse in organelles. Dense bodies also appeared in primary (Figs. 3 - 5 ) and secondary dendrites (Figs. 10A, B) some of which gave rise to spines. The presence of dense bodies p e r m i t t e d the identification of distal processes of retrogradely labeled neurons in cross section. Rarely axon terminals containing a large m e m b r a n e bound dense granule were also observed. These axons were considered to be anterogradely labeled or to belong to collaterals of retrogradely m a r k e d caudate neurons. Immunohistochemical staining for G A D and Leuenkephalin was localized to axon terminals 0 . 5 - 1 . 0 u m in size. The immunohistochemical reaction product was different from the t r a n s p o r t e d W G A - H R P product and consisted of a fine precipitate dispersed diffusely throughout individual boutons. Reaction product was present mostly on the m e m b r a n e s of synaptic vesicles in lightly labeled boutons and also
Fig. 2. A medium-sized caudate neuron has an unindented nucleus and cytoplasm sparse in organelles. The cytoplasm contains 8 dense bodies (arrowheads) which identify the cell as a striatal projection neuron. An axosomatic synapse is present at the arrow. Bar = 2 u m Fig. 3. Part of another medium-sized striatonigral neuron contains large HRP reaction granules which are present in the perikaryal cytoplasm and in an emerging dendrite (arrowheads). Bar = 2 I~m. Fig. 4. Portion of a primary dendrite is postsynaptic at arrow to immunoreactive GAD axon terminal. Axon terminal A does not exhibit GAD immunoreactivity. Large dense body at arrowhead marks the dendrite as belonging to a striatonigral neuron. Bar = 0.5 urn.
154
Fig. 5. Medium-sized striatonigral neuron marked by dense bodies (arrowheads) in the soma and primary dendrite is postsynaptic to 4 immunoreactive GAD boutons which synapse with the cell body (ringed-arrow), and dendrite (arrow and crossed-arrows). Synapses at arrow and ringed-arrow are shown at higher magnification in Figs. 6 and 8 respectively. Bar = 2/~m.
155
Fig. 10. Serial sections of a dendrite which is postsynaptic at arrow (especially in B) to an immunoreactive Leu-enkephalin bouton. The dendrite contains a dense body (arrowheads) which appears as large in A as the adjacent mitochondrion and identifies the dendrite as belonging to a striatonigral neuron. The dendrite is probably a secondary or more distal branch since its diameter is relatively small. Ringed arrow in A shows synapse formed by unlabeled axon with another dendrite. Bar = 0.5 urn. within the a x o p l a s m of darkly l a b e l e d terminals.
Synaptic interactions b e t w e e n r e t r o g r a d e l y label-
E l e c t r o n m i c r o s c o p i c e x a m i n a t i o n s h o w e d that G A D
ed n e u r o n s and i m m u n o r e a c t i v e G A D or e n k e p h a l i n
or l e u - e n k e p h a l i n
t e r m i n a l s con-
axon t e r m i n a l s w e r e f o u n d relatively easily in tissue
tained clear, p l e o m o r p h i c vesicles and m a d e synaptic
sections t r e a t e d for b o t h h i s t o c h e m i c a l reactions.
contacts on s o m a t a , p r i m a r y d e n d r i t e s , the shafts of
Numerous
distal d e n d r i t e s and spines. Synaptic j u n c t i o n s w e r e
apsed with the s o m a t a of r e t r o g r a d e l y l a b e l e d neu-
immunoreactive
s y m m e t r i c for all i m m u n o r e a c t i v e
GAD
boutons
immunoreactive
GAD
terminals
syn-
rons (Fig. 8). F r e q u e n t l y in single sections as m a n y as
(Fig. 6) and m o s t of the L e u - e n k e p h a l i n c o n t a i n i n g
three
immunoreactive
GAD
axon t e r m i n a l s con-
axons (Fig. 9). O c c a s i o n a l l y the synapses f o r m e d by
tacted the cell b o d y of a striatonigral cell. P r i m a r y
e n k e p h a l i n - p o s i t i v e t e r m i n a l s with spines a p p e a r e d
d e n d r i t e s w e r e also f r e q u e n t l y postsynaptic to G A D
asymmetric. A l t h o u g h colchicine was not used, im-
i m m u n o r e a c t i v e b o u t o n s (Fig. 4). Figs. 5, 6 and 8
m u n o h i s t o c h e m i c a l localization of G A D and leu-en-
s h o w a n e u r o n which r e c e i v e d G A B A e r g i c input to
k e p h a l i n to cell b o d i e s was o b s e r v e d .in s o m e tissue
b o t h its cell b o d y and p r i m a r y d e n d r i t e . A relatively
sections and f r e q u e n t l y c o e x i s t e d in n e u r o n s which
large p r o p o r t i o n of the e n k e p h a l i n i m m u n o r e a c t i v e
also e x h i b i t e d H R P
r e t r o g r a d e r e a c t i o n granules.
t e r m i n a l s also c o n t a c t e d the s o m a t a (Fig. 7) and den-
I m m u n o r e a c t i v e n e u r o n s c o n t a i n e d u n i n d e n t e d nu-
drites (Figs. 9 and 10) of striatonigral n e u r o n s . It ap-
clei.
p e a r e d that, at least qualitatively, the r e l a t i v e distri-
Fig. 6. Serial section through portion of primary dendrite shown in Fig. 5. Immunoreactive GAD axon terminal forms a symmetric synapse at arrow with emerging dendrite of striatal efferent neuron shown in Fig. 5. Note the dense bodies at arrowheads which identify the cell as projecting to the substantia nigra. Bar = 0.5 Bm. Fig. 7. Portion of a retrogradely labeled cell body which contains two large dense bodies in its cytoplasm is postsynaptic at arrow to an immunoreactive Leu-enkephalin axon terminal and at ringed-arrow to an unlabeled bouton. Both synaptic junctions are symmetric. Bar = 0.5/~m. Fig. 8. Axosomatic symmetric synapse (arrow) formed by immunoreactive GAD bouton with striatonigral neuron shown in Fig. 5. Bar = 0.5/~m. Fig. 9. Immunoreactive Leu-enkephalin axon terminal forms symmetric synapse with cell body of the striatal projection neuron shown in Fig. 7. HRP reaction granule appears at arrowhead. Bar = 0.5 um.
156 bution of synaptic contacts to the cell bodies and dendrites of striatal projection neurons was similar for immunoreactive G A D and enkephalin boutons. Our findings provide direct morphological evidence that GABAergic boutons and Leu-enkephalin containing axons synapse with striatofugal cells. G A B A and enkephalins are thought to have inhibitory roles in the basal ganglia 19 and to influence neurons in the nigra directly via axon terminals of striatofugal projection cells. The present findings show that G A B A and enkephalins may also affect nigral function indirectly in the neostriatum via monosynaptic contacts with striatonigral neurons. It is likely that most of the retrogradely labeled caudate neurons identified in the present study project to the substantia nigra, where the densest accumulation of peroxidase activity was found following the H R P - W G A injections. The effective site of peroxidase uptake is likely to be smaller than the overall spread of the injection38 which extended beyond the boundaries of the substantia nigra in our study. However, it is possible that some of the retrogradely labeled cells observed terminate in the ventral tegmental area. The retrogradely labeled neurons in this study shared features of medium-sized striatonigral neurons which have been previously describedS,42 and demonstrated in combination with Golgi impregnations to be of the densely spiny type 3s. Additionally, combined Golgi-electron microscopic studies have shown that the medium spiny cell has an unindented nucleus 15,16 and receives mainly symmetric axosomatic inputs from terminals with pleomorphic vesicles15,36. The cell of origin of the GABAergic terminals observed here cannot be identified with certainty, but most of the presynaptic boutons probably are derived from local axon collaterals of medium-sized spiny cells. This speculation is supported by immunohistochemical electron microscopic observations of Ribak et al. 34. There is, however, neuroanatomical evidence that some GABA-containing neurons may belong to another less frequent cell type. Using a combined Golgi-autoradiography method and uptake of [3H]GABA as the marker for GABAergic neuronal elements, Bolam et al. 6 found that only neurons with indented nuclei, which are known to belong to the aspiny type15,16, contained the silver grains of the in-
jected label. The majority of immunohistochemical evidence which shows a high density of immunoreactive G A D neurons in the neostriatum27, 34, together with earlier studies demonstrating a G A B A striatal output pathway9,18, 26 supports the likelihood that most of the GABAergic terminals identified in this study belong to the medium spiny cell type. Although G A D and enkephalin immunoreactivity was examined in separate tissue sections in this study, it is likely that many of the immunoreactive axon terminals we observed actually contained both G A D and enkephalin. Recent studies from our laboratory3 and those of others 27 have demonstrated that immunoreactive G A D and enkephalins coexist in numerous medium-sized neostriatal neurons. Also the morphological appearance of both populations of immunoreactive boutons and their relative distribution of synaptic contacts to striatonigral neurons appeared very similar. Observations in Huntington's disease brain point to the presence of synaptic interactions between G A B A or enkephalin-containing terminals with striatal output cells. Penny and Young 30 have described a 50-70% decrease in G A B A , benzodiazepine and opiate receptor binding in striatal tissue in a postmortem brain of a Huntington's disease patient. The extent of change in receptor binding together with neuropathologica120 and neurochemicaP, 17 studies, which indicate that the medium-sized spiny striatofugal cell is a principal target of the degenerative process in Huntington's disease, are consistent with the likelihood that synaptic contacts onto projecting neurons are numerous and functionally important in human brain. The GABAergic neostriatal projection neuron has been proposed to play a central role in the function of the cortical-striatofugal pathway22. An important feature of this speculation is that local axon collaterals of striatal GABAergic output neurons inhibit the activity of other projecting cells, thereby allowing the maintenance of a topographical delimitation of the cortical-striatonigral pathway. The findings of this study provide a neuroanatomical basis for this type of functional organization. This work was supported by NIH Grants AM 01126-04 to N.A. and NS 16367 to M.D. The authors greatly appreciate the generosity of Dr. Eckard
157 W e b e r of t h e U n i v e r s i t y of O r e g o n , H e a l t h S c i e n c e s
of t h e P e n n s y l v a n i a S t a t e U n i v e r s i t y , H e r s h e y M e d i -
Center, Portland OR, for providing the Leu-enkeph-
cal C e n t e r , H e r s h e y , P A , w h o c o n t r i b u t e d t h e G A D
alin a n t i s e r u m a n d a r e v e r y t h a n k f u l to D r . J . - Y . W u
antiserum.
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151
Elsevier BRE 21315
Glutamic acid decarboxylase and enkephalin immunoreactive axon terminals in the rat neostriatum synapse with striatonigral neurons N. ARONIN. K. CHASE and M. DIFIGLIA Departments of Medicine and Physiology, Universi O' of Massachusetts Medical Center, Worcester, MA 01605 and *Department of Neurology, Massachusetts General Hospital, Fruit Street, Boston, MA 02114 (U.S.A. )
(Accepted September 18th, 1985) Key words: Leu-enkephalin - - glutamate decarboxylase - - caudate nucleus - - immunocytochemistry
Synaptic interactions between striatal projection neurons and axon terminals containing immunoreactive glutamic acid decarboxylase (GAD) or Leu-enkephalin were examined in the rat neostriatum using a combined method of horseradish peroxidase retrograde transport from the substantia nigra and immunohistochemistry at the electron microscopic level. Results showed that numerous immunoreactive GAD and enkephalin boutons formed synapses with the cell bodies and dendrites of medium-sized striatonigral neurons. These findings demonstrate that within the neostriatum GABA and enkephalin directly influence caudate output pathways.
G a m m a - a m i n o b u t y r i c acid ( G A B A ) and enkephalins are measurable in high concentrations within the neostriatumg,35,45 where they are candidate transmitters. Both substances are releasable from neostriatal tissue in vitro 22-32 and in vivo ~1,~0, exhibit numerous binding sites within the caudate nucleus ~2,24,28, and produce inhibitory electrophysiological effects 4,19,25. i m m u n o h i s t o c h e m i c a l studies have shown that glutamic acid decarboxylase ( G A D ) , the synthesizing enzyme for G A B A , and immunoreactive enkephalins are located in medium-sized neostriatal spiny neurons13,3t-34-37, which have long efferent axons with collaterals that terminate locally and form synaptic contacts within caudate neuropil 5A4AS.32,42. Recent evidence from a human postmortem study 30 and the high p r o p o r t i o n of caudate efferent neurons known to exist 7A0,21 suggested that one important functional connection in the neostriaturn might involve immunoreactive G A D and/or enkephalin boutons synapsing with striatofugal cells. The synapses made by immunoreactive G A D or Leu-enkephalin axon terminals with striatonigral neurons were investigated at the ultrastructural level in the present study using a c o m b i n e d m e t h o d of
horseradish peroxidase retrograde transport and immunohistochemistry. S p r a g u e - D a w l e y rats (n = 3) were anesthetized with pentobarbital (35 mg/kg) and 0.5 ~1 of 30% complexed wheat germ a g g l u t i n i n - h o r s e r a d i s h peroxidase ( W G A - H R P ; Sigma Chemical Co., St. Louis, M O ) was pressure injected over 1 h into the substantia nigra. Injections were m a d e stereotactically29 and the needle was introduced at a 45 ° angle from the horizontal plane in o r d e r to maximize correct placement of the injected lectin into the substantia nigra, Twenty h later the animals were perfused intracardially for 20 rain with cold fixative (500 ml of 4% p a r a f o r m a l d e h y d e and 0.1% glutaraldehyde buffered to pH 7.4), followed with a perfusion of phosphate buffer (pH 7.2) for 10 rain. The brain was placed in phosphate buffer for 1 h. Sections (35 ~ m ) from the anterior caudate ipsilateral to the injected substantia nigra were cut on a Vibratome. The protocol of B o w k e r et al.S was used to intensify the reaction product in cells which contained transported W G A - H R P . In brief, incubation of the tissue in 0.5% CoCI 2 (5 rain) was followed by t r e a t m e n t with 3 ' , 3 ' - d i a m i n o b e n z i d i n e ( D A B ; Sigma) in phosphate
Correspondence: M. DiFiglia, Department of Neurology, Warren Bldg. 370, Massachusetts General Hospital, Fruit St., Boston. MA
02114, U.S.A. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
152
Fig. 1. Labeled striatonigral neurons in the caudate nucleus exhibit punctate granules of HRP reaction product (arrowheads) in their cytoplasm. Some reaction granules appear in the emerging dendrite (arrow) of one cell. An unlabeled cell appears at ringed arrow. Bar = 20 um.
153 buffer (pH 7.2) containing 0.01%
H202 (15
min). In
all cases, the region of the suhstantia nigra contained a heavy deposit of D A B reaction product. The adjacent ventral tegmental region contained a light precipitate of peroxidase reaction product. A modification of the Sternberger peroxidase antiperoxidase m e t h o d 39 was used as previously described from this l a b o r a t o r y 2. Tissue was incubated in 3% normal goat serum in phosphate buffer for 30 rain and then in antiG A D antiserum (from Dr. J.-Y. Wu), or in anti-leuenkephalin antiserum (from Dr. E c k a r d W e b e r ) at a dilution of 1:300, for 18 h at 4 °C. Sections were then treated with goat anti-rabbit antiserum (Miles), 1:30 dilution for 45 min, followed by P A P complex (Miles) at a dilution of 1:30 for 45 min. The G A D antiserum was generated against a highly purified enzyme43, 44 and has been used successfully in immunohistochemical studies in the caudate 3. The Leu-enkephalin antiserum has the following cross-reactivities with related peptides in r a d i o i m m u n o a s s a y at 50% displacement with 1 ~tM concentration of peptide: Leu-enkephalin, 100%; Met-enkephalin, 0.35%; dynorphinl 7, < 0.04%; dynorphinl 6, < 0.02%; dynorphinl_13, < 0.01%; dynorphinl_ s, < 0.01%; dynorphin~_~7, < 0.001%; and ct-Neo-endorphin, < 0.001% 4l. Deletion of the primary antiserum resulted in absence of immunohistochemical staining. Preincubation of the L e u - e n k e p h a l i n antiserum with Leu-enkephalin (50 ~g/ml) for 6 h eliminated specific immunohistochemical labeling. In both of these control conditions only the r e t r o g r a d e labeling of caudate neurons was observed. Following the immunohistochemistry tissue sections were treated with 1% osmium for 1 - 2 h, stained en bloc with 1% uranyl acetate and then e m b e d d e d flat in E p o n between two sheets of A k l a r . Results were the same in all 3 animals at the light and electron microscopic levels. Light microscopic results showed that H R P reaction granules were visi-
ble in the cell bodies and emerging dendrites of numerous medium-sized neurons throughout the caudate nucleus (Fig. 1). Regions of the caudate containing a high density of retrogradely labeled cells and immunoreactive axon terminals were cut out and r e m o u n t e d on blank Epon blocks. Thin sections with a gold interference color were cut and m o u n t e d on formvar-coated slot grids. Lead staining was not used. At the ultrastructural level electron-dense bodies were observed within the cytoplasm of medium-sized neurons and were used to identify retrogradely labeled cells. As many as 8 dense H R P - l a b e l e d granules could be found in a single section through a neuron (Fig. 2). Most of the reactive granules ranged from 0.2 to 0.4 u m in size, were spherical in shape, appeared uniformally electron dense in thicker sections and in thinner sections had the a p p e a r a n c e of multivesicular bodies (Figs. 4, 5, 9, 10). All the HRP-labeled neurons identified at the ultrastructural level contained unindented nuclei and a cytoplasm relatively sparse in organelles. Dense bodies also appeared in primary (Figs. 3 - 5 ) and secondary dendrites (Figs. 10A, B) some of which gave rise to spines. The presence of dense bodies p e r m i t t e d the identification of distal processes of retrogradely labeled neurons in cross section. Rarely axon terminals containing a large m e m b r a n e bound dense granule were also observed. These axons were considered to be anterogradely labeled or to belong to collaterals of retrogradely m a r k e d caudate neurons. Immunohistochemical staining for G A D and Leuenkephalin was localized to axon terminals 0 . 5 - 1 . 0 u m in size. The immunohistochemical reaction product was different from the t r a n s p o r t e d W G A - H R P product and consisted of a fine precipitate dispersed diffusely throughout individual boutons. Reaction product was present mostly on the m e m b r a n e s of synaptic vesicles in lightly labeled boutons and also
Fig. 2. A medium-sized caudate neuron has an unindented nucleus and cytoplasm sparse in organelles. The cytoplasm contains 8 dense bodies (arrowheads) which identify the cell as a striatal projection neuron. An axosomatic synapse is present at the arrow. Bar = 2 u m Fig. 3. Part of another medium-sized striatonigral neuron contains large HRP reaction granules which are present in the perikaryal cytoplasm and in an emerging dendrite (arrowheads). Bar = 2 I~m. Fig. 4. Portion of a primary dendrite is postsynaptic at arrow to immunoreactive GAD axon terminal. Axon terminal A does not exhibit GAD immunoreactivity. Large dense body at arrowhead marks the dendrite as belonging to a striatonigral neuron. Bar = 0.5 urn.
154
Fig. 5. Medium-sized striatonigral neuron marked by dense bodies (arrowheads) in the soma and primary dendrite is postsynaptic to 4 immunoreactive GAD boutons which synapse with the cell body (ringed-arrow), and dendrite (arrow and crossed-arrows). Synapses at arrow and ringed-arrow are shown at higher magnification in Figs. 6 and 8 respectively. Bar = 2/~m.
155
Fig. 10. Serial sections of a dendrite which is postsynaptic at arrow (especially in B) to an immunoreactive Leu-enkephalin bouton. The dendrite contains a dense body (arrowheads) which appears as large in A as the adjacent mitochondrion and identifies the dendrite as belonging to a striatonigral neuron. The dendrite is probably a secondary or more distal branch since its diameter is relatively small. Ringed arrow in A shows synapse formed by unlabeled axon with another dendrite. Bar = 0.5 urn. within the a x o p l a s m of darkly l a b e l e d terminals.
Synaptic interactions b e t w e e n r e t r o g r a d e l y label-
E l e c t r o n m i c r o s c o p i c e x a m i n a t i o n s h o w e d that G A D
ed n e u r o n s and i m m u n o r e a c t i v e G A D or e n k e p h a l i n
or l e u - e n k e p h a l i n
t e r m i n a l s con-
axon t e r m i n a l s w e r e f o u n d relatively easily in tissue
tained clear, p l e o m o r p h i c vesicles and m a d e synaptic
sections t r e a t e d for b o t h h i s t o c h e m i c a l reactions.
contacts on s o m a t a , p r i m a r y d e n d r i t e s , the shafts of
Numerous
distal d e n d r i t e s and spines. Synaptic j u n c t i o n s w e r e
apsed with the s o m a t a of r e t r o g r a d e l y l a b e l e d neu-
immunoreactive
s y m m e t r i c for all i m m u n o r e a c t i v e
GAD
boutons
immunoreactive
GAD
terminals
syn-
rons (Fig. 8). F r e q u e n t l y in single sections as m a n y as
(Fig. 6) and m o s t of the L e u - e n k e p h a l i n c o n t a i n i n g
three
immunoreactive
GAD
axon t e r m i n a l s con-
axons (Fig. 9). O c c a s i o n a l l y the synapses f o r m e d by
tacted the cell b o d y of a striatonigral cell. P r i m a r y
e n k e p h a l i n - p o s i t i v e t e r m i n a l s with spines a p p e a r e d
d e n d r i t e s w e r e also f r e q u e n t l y postsynaptic to G A D
asymmetric. A l t h o u g h colchicine was not used, im-
i m m u n o r e a c t i v e b o u t o n s (Fig. 4). Figs. 5, 6 and 8
m u n o h i s t o c h e m i c a l localization of G A D and leu-en-
s h o w a n e u r o n which r e c e i v e d G A B A e r g i c input to
k e p h a l i n to cell b o d i e s was o b s e r v e d .in s o m e tissue
b o t h its cell b o d y and p r i m a r y d e n d r i t e . A relatively
sections and f r e q u e n t l y c o e x i s t e d in n e u r o n s which
large p r o p o r t i o n of the e n k e p h a l i n i m m u n o r e a c t i v e
also e x h i b i t e d H R P
r e t r o g r a d e r e a c t i o n granules.
t e r m i n a l s also c o n t a c t e d the s o m a t a (Fig. 7) and den-
I m m u n o r e a c t i v e n e u r o n s c o n t a i n e d u n i n d e n t e d nu-
drites (Figs. 9 and 10) of striatonigral n e u r o n s . It ap-
clei.
p e a r e d that, at least qualitatively, the r e l a t i v e distri-
Fig. 6. Serial section through portion of primary dendrite shown in Fig. 5. Immunoreactive GAD axon terminal forms a symmetric synapse at arrow with emerging dendrite of striatal efferent neuron shown in Fig. 5. Note the dense bodies at arrowheads which identify the cell as projecting to the substantia nigra. Bar = 0.5 Bm. Fig. 7. Portion of a retrogradely labeled cell body which contains two large dense bodies in its cytoplasm is postsynaptic at arrow to an immunoreactive Leu-enkephalin axon terminal and at ringed-arrow to an unlabeled bouton. Both synaptic junctions are symmetric. Bar = 0.5/~m. Fig. 8. Axosomatic symmetric synapse (arrow) formed by immunoreactive GAD bouton with striatonigral neuron shown in Fig. 5. Bar = 0.5/~m. Fig. 9. Immunoreactive Leu-enkephalin axon terminal forms symmetric synapse with cell body of the striatal projection neuron shown in Fig. 7. HRP reaction granule appears at arrowhead. Bar = 0.5 um.
156 bution of synaptic contacts to the cell bodies and dendrites of striatal projection neurons was similar for immunoreactive G A D and enkephalin boutons. Our findings provide direct morphological evidence that GABAergic boutons and Leu-enkephalin containing axons synapse with striatofugal cells. G A B A and enkephalins are thought to have inhibitory roles in the basal ganglia 19 and to influence neurons in the nigra directly via axon terminals of striatofugal projection cells. The present findings show that G A B A and enkephalins may also affect nigral function indirectly in the neostriatum via monosynaptic contacts with striatonigral neurons. It is likely that most of the retrogradely labeled caudate neurons identified in the present study project to the substantia nigra, where the densest accumulation of peroxidase activity was found following the H R P - W G A injections. The effective site of peroxidase uptake is likely to be smaller than the overall spread of the injection38 which extended beyond the boundaries of the substantia nigra in our study. However, it is possible that some of the retrogradely labeled cells observed terminate in the ventral tegmental area. The retrogradely labeled neurons in this study shared features of medium-sized striatonigral neurons which have been previously describedS,42 and demonstrated in combination with Golgi impregnations to be of the densely spiny type 3s. Additionally, combined Golgi-electron microscopic studies have shown that the medium spiny cell has an unindented nucleus 15,16 and receives mainly symmetric axosomatic inputs from terminals with pleomorphic vesicles15,36. The cell of origin of the GABAergic terminals observed here cannot be identified with certainty, but most of the presynaptic boutons probably are derived from local axon collaterals of medium-sized spiny cells. This speculation is supported by immunohistochemical electron microscopic observations of Ribak et al. 34. There is, however, neuroanatomical evidence that some GABA-containing neurons may belong to another less frequent cell type. Using a combined Golgi-autoradiography method and uptake of [3H]GABA as the marker for GABAergic neuronal elements, Bolam et al. 6 found that only neurons with indented nuclei, which are known to belong to the aspiny type15,16, contained the silver grains of the in-
jected label. The majority of immunohistochemical evidence which shows a high density of immunoreactive G A D neurons in the neostriatum27, 34, together with earlier studies demonstrating a G A B A striatal output pathway9,18, 26 supports the likelihood that most of the GABAergic terminals identified in this study belong to the medium spiny cell type. Although G A D and enkephalin immunoreactivity was examined in separate tissue sections in this study, it is likely that many of the immunoreactive axon terminals we observed actually contained both G A D and enkephalin. Recent studies from our laboratory3 and those of others 27 have demonstrated that immunoreactive G A D and enkephalins coexist in numerous medium-sized neostriatal neurons. Also the morphological appearance of both populations of immunoreactive boutons and their relative distribution of synaptic contacts to striatonigral neurons appeared very similar. Observations in Huntington's disease brain point to the presence of synaptic interactions between G A B A or enkephalin-containing terminals with striatal output cells. Penny and Young 30 have described a 50-70% decrease in G A B A , benzodiazepine and opiate receptor binding in striatal tissue in a postmortem brain of a Huntington's disease patient. The extent of change in receptor binding together with neuropathologica120 and neurochemicaP, 17 studies, which indicate that the medium-sized spiny striatofugal cell is a principal target of the degenerative process in Huntington's disease, are consistent with the likelihood that synaptic contacts onto projecting neurons are numerous and functionally important in human brain. The GABAergic neostriatal projection neuron has been proposed to play a central role in the function of the cortical-striatofugal pathway22. An important feature of this speculation is that local axon collaterals of striatal GABAergic output neurons inhibit the activity of other projecting cells, thereby allowing the maintenance of a topographical delimitation of the cortical-striatonigral pathway. The findings of this study provide a neuroanatomical basis for this type of functional organization. This work was supported by NIH Grants AM 01126-04 to N.A. and NS 16367 to M.D. The authors greatly appreciate the generosity of Dr. Eckard
157 W e b e r of t h e U n i v e r s i t y of O r e g o n , H e a l t h S c i e n c e s
of t h e P e n n s y l v a n i a S t a t e U n i v e r s i t y , H e r s h e y M e d i -
Center, Portland OR, for providing the Leu-enkeph-
cal C e n t e r , H e r s h e y , P A , w h o c o n t r i b u t e d t h e G A D
alin a n t i s e r u m a n d a r e v e r y t h a n k f u l to D r . J . - Y . W u
antiserum.
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