Brain Research, 529 (1990) 57-78

57

Elsevier BRES 15888

The G A B A and substance P input to dopaminergic neurones in the substantia nigra of the rat J.P. Bolam and Y. Smith MRC Anatomical Neuropharmacology Unit, University Department of Pharmacology, Oxford, U.K. (Accepted 27 March 1990)

Key words: Immunocytochemistry; Synapse; Glutamate decarboxylase; Tyrosine hydroxylase

In order to examine the synaptic input to dopaminergic neurones in the substantia nigra from GABAergic terminals and terminals that contain substance P, double and triple immunocytochemical studies were carded out at the light and electron microscopic levels in the rat. In a first series of experiments sections of the substantia nigra were incubated to reveal axon terminals containing either substance P or glutamate decarboxylase and then incubated to reveal dopaminergic neurones using tyrosine hydroxylase immunocytochemistry. Examination of this material in the light microscope revealed that many substance P- and glutamate decarboxylase-immunoreactive boutons were associated with the dopaminergic cells. In the electron microscope it was found that the perikarya and dendrites of the dopaminergic neurones received symmetrical synaptic input from terminals that displayed immunoreactivity for substance P or glutamate decarboxylase. A small proportion of the substance P-positive boutons formed asymmetrical synapses. In a second series of experiments sections of the substantia nigra were processed by the pre-embedding immunocytochemical technique for tyrosine hydroxylase and then the post-embedding immunogold technique for gamma-aminobutyric acid (GABA). Examination in the electron microscope revealed that the tyrosine hydroxylase-positive neurones received symmetrical synaptic input from many GABA-positive terminals. Quantitative analyses demonstrated that a minimum of 50-70% of all boutons afferent to the dopaminergic neurones display glutamate decarboxylase or GABA immunoreactivity. Triple immunocytochemical studies i.e. pre-embedding immunocytochemistry for tyrosine hydroxylase and substance P, combined with post-embedding immunogold staining for GABA, revealed that some of the substance P-immunoreactive boutons that were in contact with the dopaminergic neurones also displayed GABA immunoreactivity. In a third series of experiments the combination of anterograde transport of lectin-conjugated horseradish peroxidase or biocytin with post-embedding GABA immunocytochemistry demonstrated that at least one of the sources of GABA-containing terminals in the substantia nigra is the striatum. The results of the present study: (1) demonstrate that dopaminergic neurones in the substantia nigra receive symmetrical synaptic input from GABAergic and substance P-containing terminals, (2) show that a proportion of these terminals contain both substance P and GABA and (3) suggest that the major synaptic input to dopaminergic neurones is from GABAergic terminals and that a part of this innervation is derived from the striatum.

INTRODUCTION T h e dopaminergic neurones of the substantia nigra that project to the striatum are critical for the normal functioning o f the basal ganglia. The release of dopamine f r o m their terminals in the striatum represents a major m o d u l a t o r y feedback mechanism of information flow t h r o u g h the basal ganglia. Thus the output of the cortico-striato-pallidal and the cortico-striato-nigral pathways are m o d u l a t e d by the dopaminergic neurones that m a k e direct synaptic contact with the projection neurones of the striatum 23. T h e functional importance of this system is exemplified by the m a r k e d m o t o r disturbances that occur in Parkinson's disease and the models of Parkinson's disease involving the selective destruction of nigral dopaminergic neurones 92. Within the striatum the release of dopamine from

terminals in synaptic contact with striatal output neurones 23, cholinergic neurones s'43 or other neurone types 4°-42'44'86, is dependent on the activity of nigral axons and ultimately on the activity of the afferent synaptic input to the dendrites and perikarya of the parent neurones in the substantia nigra. It is thus important to know the origin, chemistry and pattern of afferent synaptic input to dopaminergic neurones in order to more fully understand the role of the dopaminergic system in the functional organization of the basal ganglia. The substantia nigra receives many afferent inputs of diverse origin and chemical nature that all have the potential to make synapses with the dopaminergic neurones. These include excitatory inputs from the cortex 24' 47,53,80 and the subthalamic nucleus 1°,37'67, y-aminobutyric acid ( G A B A ) and peptide-containing inputs from the striatum 26, G A B A - c o n t a i n i n g input from the globus

Correspondence: J.P. Bolam, MRC Anatomical Neuropharmacology Unit, University Department of Pharmacology, South Parks Road, Oxford OX1 3QT, U.K. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

58 pallidus 19'63'65, a s e r o t o n e r g i c input from the dorsal r a p h e 75'9° a n d a cholinergic input from the brainstem 2'25. In a d d i t i o n to these, t h e r e is the possibility that dopam i n e r g i c n e u r o n e s r e c e i v e input from local G A B A e r g i c i n t e r n e u r o n e s o r p r o j e c t i o n n e u r o n e s 54. Several of these nigral afferents have b e e n shown to m a k e direct synaptic c o n t a c t with n e u r o n e s of the pars c o m p a c t a identified as s t r i a t o n i g r a l n e u r o n e s by r e t r o g r a d e labelling from the s t r i a t u m o r as d o p a m i n e r g i c n e u r o n e s by i m m u n o c y t o c h e m i s t r y for d o p a m i n e o r tyrosine hydroxylase. Thus, t h e y h a v e b e e n shown to receive input from the s t r i a t u m 69's7 a n d the globus pallidus 65 in the rat and from the nucleus t e g m e n t i p e d u n c u l o p o n t i n u s in the cat 78. On the basis o f c h e m i c a l criteria they have been shown to r e c e i v e s y n a p t i c input from t e r m i n a l s that display immun o r e a c t i v i t y for substance p4,9,35,46,5o, serotonin52, n e u r o t e n s i n 91, e n k e p h a l i n ( u n p u b l i s h e d observations) a n d t h e s y n t h e t i c e n z y m e for G A B A , g l u t a m a t e decarb o x y l a s e ( G A D ) 2t's2. O f all the afferents to the substantia nigra the input t h a t is p r o b a b l y q u a n t i t a t i v e l y a n d functionally the most i m p o r t a n t is that f r o m the striatum. The whole of the s t r i a t u m p r o j e c t s to the substantia nigra in a t o p o g r a p h ical m a n n e r w h e r e it t e r m i n a t e s on the d o p a m i n e r g i c n e u r o n e s (see a b o v e ) a n d the o u t p u t n e u r o n e s of the pars r e t i c u l a t a 71'79'88. In a d d i t i o n to m a n y o t h e r a p p r o a c h e s , i m m u n o h i s t o c h e m i c a l studies c o m b i n e d with selective lesions o f striatal n e u r o n e s 56'6° o r r e t r o g r a d e t r a n s p o r t of a x o n a l t r a c e r s t8'z° suggest that the main transmitter in this p a t h w a y is G A B A . T h e r e is also evidence that s u b s t a n c e P o r o t h e r tachykinins and d y n o r p h i n are p r e s e n t in this p a t h w a y l'a3,26,Sl's3. It is these afferents that, at least in p a r t , directly o r indirectly i m p a r t i n f o r m a t i o n to the d o p a m i n e r g i c n e u r o n e s that enables t h e m to p e r f o r m their m o d u l a t o r y f e e d b a c k in the striatum. D e s p i t e the functional i m p o r t a n c e of the G A B A e r g i c a n d s u b s t a n c e P-containing striatal afferents to the

substantia nigra detailed knowledge of the synaptic relationships of these inputs to the dopaminergic neurones is still lacking. The main objective of the present experiments was therefore to examine in detail, the synaptic organization of the G A B A - and substance P-containing afferent input to dopaminergic neurones of the substantia nigra of the rat. A second objective was to a t t e m p t to d e m o n s t r a t e directly that at least some of the striatai terminals in the substantia nigra contain G A B A and in view of the partial co-localization of substance P and G A B A in the projection neurones of the striatum 58, to d e t e r m i n e whether G A B A and substance P are co-localized in nigral terminals. Some of the results of these studies have been published in abstract form elsewhere 4.

MATERIALS AND METHODS

Double immunocytochemistry Preparation of tissue sections. All immunocytochemical analyses were carried out on female albino Wistar rats (160-250 g). The rats were deeply anaesthetized with chloral hydrate (350 mg/kg; 3.5% m 0.9% sodium chloride solution). They were then perfused through the aorta with the aid of a peristaltic pump, initially with Ca2+-free Tyrode's solution followed by two fixative solutions that consisted of: (1) 300 ml of 0.01-0.15% glutaraldehyde and 3% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4; PB) that was administered over a period of 6-9 min followed by (2) 100 ml of 4% paraformaldehyde in the same buffer, administered over a period of 10-15 min. After perfusion the brain was removed, on some occasions post-fixed for up to 2 h in the second fixative, the mesencephaion was dissected out, washed in PB and then in phosphate-buffered saline (0.01 M phosphate, pH 7.4; PBS). The dissected mesencephalon was sectioned at 50-70 ~m on a vibrating microtome in PBS, collected in glass scintillation vials and washed several times in PBS. The sections of the mesencephalon were subjected to double immunocytochemical staining using either the double pre-embedding technique of Levey et alY or a combination of pre-embedding and post-embedding immunocytochemistry (see Bolam and InghamS). Double pre-embedding immunocytochemistry. For the double pre-embedding staining the peroxidase-antiperoxidase (PAP) method of SternbergerTM was utilized. The sections of the mesencephalon were processed as follows: incubation in the primary

Fig. 1. Light micrographs of sections of the ventral mesencephalon incubated to reveal substance P immunoreactivity (DAB as chromogen) and tyrosine hydroxylase immunoreactivity (BDHC as chromogen) as well as control sections. TH-immunoreactive structures are identified by the granular reaction product formed by the BDHC (arrowheads) whereas substance P-immunoreactive structures are identified by the amorphous DAB reaction product (arrows). A: high power light micrograph of the border between the most ventral part of the substantia nigra pars compacta (SNC) and the substantia nigra pars reticulata (SNR). TH-positive perikarya are present in the SNC and dendrites in both the SNC and SNR (arrowheads). Substance P-immunoreactive structures are more abundant in the SNR and are identified by the amorphous and more dense reaction product (arrows). The substance P-positive boutons (SP-bouton or SP) are often seen in association with TH-immunoreactive dendrites (TH-den) and perikarya or non-immunoreactive dendrites (den). B: high power light micrograph of the border between the most ventral part of the SNC and the SNR in a section that was incubated through the whole double immunostaining protocol but with the omission of the antiserum against substance P. Only the granular stained TH-immunoreactive perikarya and dendrites can be detected in this section. C-F: light micrographs of the border between the ventral tegmental area (VTA) and the interpeduncular nucleus (IP) from a section incubated to reveal both substance P and tyrosine hydroxylase immunoreactivities (C,E,F) and a control section (D). C: the VTA contains granular stained TH-positive cells (arrowheads, shown at high power in E) whereas the IP contains the amorphously stained substance P-positive cells (arrows); some examples of substance P-positive cells in the IP are shown at high power in E D: section that was incubated through the whole double immunostaining protocol but with the omission of the antiserum against TH. The IP contains substance P-positive cells whereas the VTA contains no TH-immunoreactive structures. A,B,E and F are at the same magnification, bar = 10 pm; C and D are at the same magnification, bar = 20 pm.

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antibody (see below) diluted in PBS for 18-36 h at 4 °C with constant, gentle shaking; 3 x 10 rain washes in PBS; incubation in the secondary antibody diluted 1:50 in PBS for 1-2 h; 3 x 10 rain washes in PBS; incubation in PAP diluted 1:100 in PBS for 1-2 h;

..... !i,~:i~

3 x 10 rain washes in PBS followed by 10 min wash in Tris-HC1 buffer (0.05 M, pH 7.4-7.6; TB). After pre-incubation in a 0.05% solution of 3,3"-diaminobenzidine tetrahydrochloride (DAB; Sigma Chemical Co.) in TB for 10 min the antigen was localized by

¸

F

60 addition of hydrogen peroxide, to a final concentration of 0.001%. The reaction allowed to continue for 5-10 min and was terminated by 2 × 5 min washes in TB. After extensive washes in PBS the second antigen, i.e. tyrosine hydroxylase, was localized by following the same sequence of incubations except that the chromogen for the peroxidase reaction was benzidine dihydrochloride (BDHC) rather than DAB. Thus after incubation in PAP the sections were washed once in PBS and 2 × 10 min in 0.01 M phosphate buffer at pH 6.8. They were then incubated in a solution of B D H C (0.01% in 0.01 M PB, pH 6.8) containing 0.025% sodium nitroprusside for 10-15 min. Hydrogen peroxide, to a final concentration of 0.005%, was added and the sections were incubated for a further 2-6 min. The reaction was stopped by several washes in the low pH phosphate buffer (for details see refs. 5, 45). Antibody preparations. The primary antibodies that were used to label populations of axonal terminals in the substantia nigra were directed against: glutamate decarboxylase ss, prepared in sheep and used at a dilution of 1:1000 or 1:2000 and substance P (SP) TM, monoclonal and used at a dilution of 1:1000 or 1:1500. The primary antibody against tyrosine hydroxylase (TH) used to label the dopaminergic neurones was raised in rabbit and used at a dilution of 1:1000 (ref. 81). For the G A D antiserum the secondary antibody was rabbit anti-sheep IgG and the PAP prepared in goat (Dakopatts). For the monoclonai antibodies against SP the secondary antibody was rabbit anti-rat IgG (Dakopatts) and the PAP prepared in rat (Sternberger-Meyer, Inc.). For the antiserum against TH goat anti-rabbit IgG and rabbit PAP (ICN Immunobiologicals) were used. Control incubations were carried out first, to ensure that cross-reactivity was not occurring between the different antibody solutions and secondly, to ensure that the BDHC reaction product did not take on the appearance of, or become deposited on, the D A B reaction product. Thus in each experiment the whole of the double immunocytochemical protocol was carried out but with omission of each of the primary antibodies in turn. Pre- and post-embedding immunocytochemistry. In order to examine the synaptic organization of GABA-containing boutons in contact with dopaminergic neurones, some rats were perfused with fixative containing a higher concentration of glutaraldehyde (up to 1%). Vibrating microtome sections of the mesencephalon were then incubated to reveal TH immunoreactivity using the PAP method and D A B as the chromogen for the peroxidase reaction as described above. They were then prepared for electron microscopy and mounted on slides as described below. Different regions of the substantia nigra containing TH-immunoreactive structures were re-embedded in blocks of resin and ultrathin sections were collected on coated single-slot gold grids. These sections were then immunostained to reveal G A B A using a rabbit antibody (code: G A B A 9 ) 3°,7°,72 ' The incubations for G A B A immunocytochemistry were carried out as described in detail elsewhere 32. In brief, the grids with sections attached were floated on drops of filtered (Millipore, 0.22 /~m pore size) solutions according to the following protocol: 1% aqueous periodic acid, 10 min; brief immersion of grids in 3 separate vials of double distilled boiled water; 1% aqueous sodium periodate, 10 min; 5% and then 1% normal goat serum in Tris (0.01 M)/phosphate (0.01 M)/buffered isotonic saline, pH 7.4 (TPBS) for 30 and 10 min respectively; after washes, 1.5 h in a 1:1000 or 1:2000 dilution of G A B A antiserum diluted in 0.05% polyethylene glycol in 50 mM Tris HC1 buffer (pH 7.5 or 0.02 M Tris HCI (pH 8.2) containing 0.1% bovine serum albumin and 20% glycerol), after several washes the primary antibody was localized by incubation for 2 h in goat anti-rabbit IgG conjugated to colloidal gold particles of 10 or 15 nm in diameter (1:10 dilution; BioCell, Cardiff or Janssen Pharmaeeutica). The sections were stained first with aqueous 1% uranyl acetate (1 h) and then with lead citrate and examined in the electron microscope. In order to test the possibility of the co-existence of G A B A and substance P in boutons forming synaptic contacts with the dopa-

minergic neurones, some sections of the substantia nlgra were processed first to reveal substance P and TH by the double pre-embedding method and then uttrathin sections of regions containing both substance P- and TH-labelled elements were immunostained to reveal GABA. To test the specificity of the protocol, some ot the ullrathin sections were incubated through the whole protocol but with the omission of the primary antiserum. In order to test for crossreactivity in the substantia nigra some sections were incubated with G A B A antiserum pre-adsorbed to either G A B A or glutamate conjugated to polyacrylamide beads with glutaraldehyde 77.

Anterograde labelling of striatonigral terminals combined with GABA immunostaining Striatonigral terminals were anterogradely labelled by injections of either wheatgerm agglutinin conjugated to horseradish peroxidase (WGA/HRP) (7% solution in 0.9% sodium chloride solution) or by injections of biotinylated lysine36 (biocytin, Sigma Chemical Co.; 5% solution in TB). Anaesthetized rats received unilateral or bilateral stereotaxie injections in the striatum. The injections were made using a micropipette syringe and volumes of 50-t00 nl for the WGA/HRP and 400-450 nl for the biocytin were administered over three sites. After a survival period of 24 h the animals were re-anaesthetized and perfused through the aorta initially with Ca2+-free Tyrode's solution and then with a mixture of 2.5% glutaraldehyde and 0.5-1% paraformaldehyde in PB. The brains were removed, dissected into blocks containing the injection sites and blocks containing the substantia nigra. They were then sectioned at 70/~m on a vibrating microtome. The injected and transported WGA/HRP was revealed by incubation in a solution of DAB and hydrogen peroxide as described above for the immunostaining or using o-tolidine (dimethylbenzidine) as the chromogen stabilized with DAB-cobalt 62 as described in detail elsewhere 5. The sections were then prepared for electron microscopy (see below). For the biocytin-injected animals, the injected and transported material was revealed by incubation in a 1:100 dilution of an avidin-biotin-peroxidase complex (Vector) for 2-18 h and then in the DAB/H20 2 solution as described above. The sections were then prepared for electron microscopy (see below). Areas of the substantia nigra that contained anterogradely labelled terminals from both sets of animals were re-embedded in blocks of resin and ultrathin sections collected on Pioloform-coated single-slot gold grids. The sections were then immunostained to reveal G A B A as described above.

Preparation for electron microscopy On completion of the immunostaining or visualization of the anterograde marker, the sections were treated with 1% osmium tetroxide for 20-40 min and then washed several times in PB. They were then dehydrated in an ascending series of alcohols (including 1% uranyl acetate in the 70% ethanol) and two changes of propylene oxide before being transferred to an electron microscopy resin (Durcupan ACM, Fluka), mounted on microscope slides and cured at 60 °C for 48 h. Following light microscopic analysis, regions of interest were re-embedded in blocks of resin, sectioned on an ultramicrotome (Reichart OMU2), collected on Formvar- or Pioloform-coated single slot grids, stained with lead citrate 59 and examined in a Philips 410 electron microscope.

Analysis of material All the sections were examined in the light microscope for the distribution and relationships of immunostained structures. Areas for subsequent electron microscopic analysis were sometimes photographed, their positions in the sections noted and then cut out from the microscope slide and either re-embedded in resin in the form of a block or glued to the surface of pre-cured blocks. The tissue was then sectioned and the ultrathin sections examined in the electron microscope. In some cases the same structures that were identified in the light microscope were examined in the electron microscope by correlated light and electron microscopys. In other

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Fig. 2. Light micrographs of sections of the substantia nigra incubated to reveal GAD immunoreactivity (DAB as cl~omogen) and TH immunoreactivity (BDHC as chromogen) as well as a control section. Throughout this and the subsequent figures TH-immtmoreactive structures are indicated by arrowheads and GAD-immunoreactive structures by arrows. A: fight micrograph of the substantia nigra pars compacta showing three TH-labelled perikarya and isolated segments of TH-labelled dendrites (arrowheads). The neuropil contains many GAD-positive boutons that appear to be randomly dispersed. The TH-positive perikarya have many GAD-positive terminals associated with them (arrows). Many of the GAD-positive boutons were found to form synapses when examined in the electron microscope. The GAD-positive bouton labelled 4A is shown at the electron microscopic level in Fig. 4A. GAD-positive boutons were also associated with TH-positive dendrites (GAD, TH at lower right of the micrograph). A non-immunoreactive perikaryon (star) also has GAD boutons associated with it. B: light micrograph of a similar region as in A from a section that was incubated through the whole double immunostaining protocol but with the omission of the antiserum against GAD. Note that TH-immunoreactive perikarya and dendrites are present but not the GAD-immunoreactive boutons. C,D: light micrographs of the substantia nigra pars reticulata showing TH-immunoreactive perikarya (TH) and dendrite (arrowhead), a non-immunoreactiveperikaryon (star) and perikarya that contain the DAB reaction product and so are probably GAD-immunoreactive (G). The neuropil is densely filled with GAD-immunoreactiveboutons that outline non-stained dendrites in longitudinal section or form 'rosettes' around dendrites in cross-section (arrows). The non-immunoreactive perikarya and GAD-positive perikarya are also outlined by the GAD-positive boutons. Contrast the density and pattern of staining of the GAD boutons with that observed in the pars compacta (A). The TH-positive perikarya are also apposed by GAD-positive boutons (arrows) but to a similar degree to that found in the pars compacta. Bar = 25/~m for A-D.

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Fig. 3. Electron micrographs of sections of the substantia nigra incubated to reveal substance P immunoreactivity (DAB as chromogen, SP) and tyrosine hydroxylase immunoreactivity (BDHC as chromogen; reaction product indicated by arrowheads). A: electron micrograph of part of a TH-immunoreactive perikaryon identified by the presence of granules of the TH immunoreaction product formed by the BDHC (TH-ir). The perikaryon is apposed by two boutons that contain the DAB reaction product thus identifying them as substance P-immunoreactive (SP-ir). B: high power electron micrograph of the two substance P-positive boutons (SP) shown in A. The upper of the two boutons forms symmetrical synaptic contact (arrow) with the TH-immunoreactive perikaryon. Note the crystalline appearance of the TH-immunoreaction product formed by the BDHC (TH-ir). C,D: two TH-immunoreactive dendritic shafts (TH-ir) that receive symmetrical synaptic input (arrows) from substance P-immunoreactive boutons (SP). Non-immunoreactive boutons are indicated by asterisks. Bar in A = 1 urn; B-D are at the same magnification, bar ~ 0.5 ~m.

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Fig. 4. Electron micrographs of sections of the substantia nigra incubated to reveal G A D immunoreactivity (DAB as chromogen; GAD) and tyrosine hydroxylase immunoreactivity (BDHC as chromogen; granules of reaction product indicated by TH). A: electron micrograph of part of one of the TH-immunoreactive neurones shown in Fig. 2A (neurone apposed by bouton labelled 4A). The proximal dendrite of this TH-positive neurone is apposed by three boutons, two are non-immunoreactive (stars) and one is immunoreactive for GAD that is shown at higher magnification in B. "Another bouton in the neuropil is also immunoreactive for GAD. B: high power electron micrograph of the GAD-immunoreactive bouton shown in A. The bouton forms symmetrical synaptic contact (arrow) with the TH-immunoreactive proximal dendritic shaft. C - E : electron micrographs of TH-positive dendrites receiving synapses (arrows) from GAD-immunoreactive (GAD) and non-immunoreactive boutons (asterisks). Bar in A = 2/~m; B = 0.5/~m; C - E are at the same magnification, bar = 0.5 jum.

64 cases, regions close to the surface of the section (i.e. within the depth of penetration of the immunoreagents) were systematically scanned for structures that displayed immunoreactivity. In an attempt to quantify the GABAergic input to dopaminergic neurones, TH-immunoreactive structures were systematically examined in electron microscopic sections. All the axonal boutons, whether immunoreactive or not, that were either apposed to, or in synaptic contact with the TH-immunoreactive elements were noted. From this, a rough estimate of the proportion of GAD- or GABA-immunoreactive boutons in contact with the dopaminergic neurones was obtained. In order to quantify the immunoreactivity in GABA-immunostained sections the surface area of individual boutons was measured with the aid of a digitizing pad connected to a Macintosh computer and the density of gold particles overlying them calculated. These values were compared to the density of immunogold particles overlying TH-positive dendrites in the same micrograph or, if TH-positive dendrites were not present, they were compared to the density overlying unstained dendrites.

RESULTS

Light microscopic observations Appearance of immunostaining. The B D H C reaction p r o d u c t t h a t was u s e d to localize T H - i m m u n o r e a c t i v e s t r u c t u r e s h a d a g r a n u l a r a p p e a r a n c e (Figs. 1 A , B , C , E a n d 2) t h a t was b l u e b e f o r e o s m i u m t r e a t m e n t . A f t e r t r e a t m e n t with o s m i u m the r e a c t i o n p r o d u c t b e c a m e g r e y / b l a c k . T h e r e a c t i o n p r o d u c t f o r m e d when D A B was u s e d as the c h r o m o g e n for the p e r o x i d a s e reaction d i s p l a y e d the typical b r o w n colour a n d a m o r p h o u s t e x t u r e (Fig. 1 D , F ) . T h e two reaction products were easily d i s t i n g u i s h a b l e f r o m each on the basis of both the c o l o u r a n d t e x t u r e (see Figs. 1 and 2). In e a c h case the i m m u n o s t a i n e d structures were p r e s e n t in the m o s t superficial layers o f the sections b e c a u s e t e c h n i q u e s to e n h a n c e the p e n e t r a t i o n of the i m m u n o r e a g e n t s w e r e not used. In general, the d e p t h of p e n e t r a t i o n o f structures l a b e l l e d with the B D H C reaction p r o d u c t was less t h a n that o f those labelled with DAB reaction product. Substance P and tyrosine hydroxylase. T h e sections of

the substantia nigra that were incubated to reveal SP-immunoreactive structures and then T H - i m m u n o r e active structures showed the distribution of immunostaining that has been described previously. Thus the substantia nigra pars compacta (SNC) contained a dense accumulation of T H - i m m u n o r e a c t i v e neurones and dendrites (Fig. 1A,B) that e x t e n d e d into the more medial ventral t e g m e n t a l area (Fig. 1C,E). The substantia nigra pars reticulata (SNR) also contained many TH-immunoreactive structures (Fig. 1A,B; see also G A D & T H i m m u n o s t a i n e d sections Fig. 2C,D). These consisted of the ventrally e x t e n d e d dendrites of the SNC cells as well as the p e r i k a r y a and dendrites of a small population of i m m u n o r e a c t i v e cells located in the caudoventral SNR. The SP immunoreactivity revealed using D A B as the c h r o m o g e n was in the form of axons and terminals and gave an overall brown a p p e a r a n c e to the surface of the S N R (Fig. 1A). Many of the SP-immunoreactive structures were m o r e discretely stained and had the appearance of distinct terminals or axon swellings (see Fig. 1A). The SNC also contained many SP-immunoreactive terminals, albeit at a much lower density than in the SNR, and these were generally discretely labelled terminals (Fig. 1A). The medially located interpeduncular nucleus was also rich in SP-immunoreactive structures including cell bodies, dendrites and axons (Fig. 1C,D and F). The two sets of immunoreactive structures were interspersed with each o t h e r both in the SNC and S N R and were easily distinguishable. The SP-immunoreactive terminals often showed associations with non-immunoreactive structures, particularly with structures that app e a r e d to be dendrites (Fig. 1A), but also with perikarya. In addition m a n y of the immunoreactive terminals were closely a p p o s e d to dendrites o r p e r i k a r y a that were i m m u n o r e a c t i v e for T H , i.e. contained the grey/black granular reaction product (Fig. 1A). In some cases several SP-immunoreactive terminals were associated with a single T H - i m m u n o r e a c t i v e dendrite or perikaryon.

Fig. 5. Electron micrographs of sections of the substantia nigra incubated to reveal substance P immunoreactivity (SP arrows; DAB as chromogen) and tyrosine hydroxylase immunoreactivity (TH arrowheads; BDHC as chromogen) by pre-embedding methods and then GABA by the post-embedding immunogold method (electron dense gold particles, small arrows). A: a small TH-positive dendrite is postsynaptic to two boutons. One of the boutons displays substance P immunoreactivity (SP) and appears to make an asymmetrical synapse (arrows). The second bouton forms symmetrical synaptic contact (arrow) and has many immunogold particles overlying it (some of which are indicated by small arrows) thus identifying it as GABA-immunoreactive (GABA). The density of immunogold particles overlying this bouton is 4.8 times greater than the density overlying the TH-positive dendrite. B: two boutons that are immunoreactive for substance P and have a high density of immunogold particles overlying them (some of which are indicated by small arrows) thus identifying them as also being GABAimmunoreactive (GABA & SP). The upper bouton is in symmetrical synaptic contact with a dendritic shaft. The density of immunogold particles overlying the boutons is 5.3 and 4.9 (upper and lower) times greater than the density overlying the unstained dendrite to which they are apposed. C: three boutons that contain the DAB reaction product and have a relatively high density of immunogold particles overlying them thus identifying them as immunoreactive for both substance P and GABA (GABA & SP). Two of the boutons are apposed to a non-immunoreactive dendrite and the other to a TH-immunoreactive dendrite (TH arrowhead). The density of immunogold particles overlying the boutons is 8-15 times greater than the density overlying the TH-positive dendrites. Note that the TH immunoreaction product formed by the BDI-IC in this dendrite and in the lower left of the micrograph, has only low electron density due to the post-embedding procedure. Compare the electron density of the BDHC reaction product with that in Figs. 3 and 4. Bar in A ~ 0.5 am; B and C are at the same magnification, bar = 0.5 am.

65

Glutamate decarboxylase and tyrosine hydroxylase. The distribution of BDHC-stained structures i.e. THimmunoreactive perikarya and dendrites, was similar to

that described above (Fig. 2A,B). The GAD-immunoreactive structures revealed using DAB as the chromogen, were extensively distributed throughout the mesen-

66

Fig. 6. A: electron micrograph of a TH-immunoreactive dendrite (TH arrowheads) that is apposed by two GABA-immunoreactive boutons (GABA, small arrows), a non-immunoreactive bouton (asterisk) and a bouton that is immunoreactive for both substance P and GABA (GABA & SP). The density of immunogold particles overlying the boutons is 16--31.5 times greater than the density overlying the TH-positive dendrite. Each of the boutons except one forms symmetrical synaptic contact with the TH-immunoreactive dendritic shaft. B,C: electron micrographs of sections incubated to reveal TH-immunoreactive structures using DAB as the chromogen for the peroxidase reaction and then incubated by the immunogold method to reveal G A B A . Two TH-positive dendrites (TH) are embedded in areas rich in GABA-immunoreactive boutons (b). Non-immunoreactive boutons are also present (asterisks) and the one in B forms an asymmetrical synapse with the TH-positive dendrite. The density of immunogold particles overlying the GABA-positive boutons is 12-16 times greater than the density overlying the TH-positive dendrite. Bar in A :- I!~5,urn: B and C are at the same magnification, bar = 0.5/~m.

67 cephalon. Within the substantia nigra, the GAD-immunoreactive structures consisted predominantly of axons or punctate boutons (Fig. 2A,C,D) but lightly stained neuronal perikarya were also detected (Fig. 2C,D). The bouton staining was very intense and present at a high density. Within the SNR they were distributed in an ordered fashion, outlining unstained dendrites and giving a rosette-like appearance in cross-section (Fig. 2C,D). In addition, unstained perikarya (Fig. 2C) and perikarya lightly immunoreactive for GAD (Fig. 2C,D) were surrounded by the immunoreactive boutons. In contrast to this, in the pars compacta of the substantia nigra the GAD-immunoreactive terminals were distributed randomly and did not surround or outline other structures (Fig. 2A). They did show associations with other structures (dendrites and perikarya) but the density was much lower than in the reticulata (compare Fig. 2A with 2C and D). As with the sections double-stained for substance P and TH, the DAB-stained (i.e. GAD-immunoreactive) structures were easily distinguishable from the THimmunoreactive structures on the basis of their colour, texture and location (Fig. 2A,C,D). There was a marked and consistent association between the TH-immunoreactive structures and the GAD-immunoreactive boutons. All TH-immunoreactive perikarya and proximal dendrites that were at the same depth in the tissue as the GAD-immunostained structures had many GAD-immunoreactive boutons associated with them (Fig. 2A,C,D). The number and density of terminals in contact with TH-immunostained structures although high, were much lower than for non-immunostained structures in the SNR. This was substantiated in the electron microscopic study and indeed it was noted that the density of all afferent boutons was much lower for TH-positive neurones than for non-immunoreactive neurones. Control sections. Controls for the double immunocytochemical reactions were carried out to ensure that false-positives did not occur i.e. that the reaction product from one reaction-did not become associated with, or take on the appearance, of the other reaction product. The most important control was to incubate throughout the double protocol but with the omission of each of the primary antibodies in turn. This resulted only in the staining of structures with BDHC-type reaction product if the first primary antibodies were omitted (Figs. 1B and 2B) and only DAB-type reaction product, if the second primary antibody was omitted (Fig. 1D,F). These sections also acted as controls for non-specific binding of the secondary antibodies and of the PAP complexes. They also demonstrate that the goat anti-rabbit antiserum, used to localize the TH-immunoreactive sites in the second reaction, does not recognize the rabbit anti-rat

antibodies (used for the first antigen) after the peroxidase reaction. Omission of both primary antibodies resulted in sections that contained no reaction products associated with neuronal structures. Reaction product was associated, however, with red blood cells and occasional vascular endothelial cells.

Electron microscopic observations Appearance of the reaction products. The reaction product formed by the DAB was typically amorphous and electron dense (Figs. 3-6). At low magnification the immunostained structures had the appearance of being homogeneously filled with the reaction product (Figs. 3A, 4A) but at higher magnification it was apparent that it was particularly associated with the internal plasma membrane and the membranes of subcellular organelles (Figs. 3B-D, 4B-E, 5, 6). The reaction product formed by the BDHC in the TH-immunoreactive structures was in the form of electron dense granules or crystals that were apparently randomly dispersed throughout the cytoplasm (Figs. 3, 4). The granules of BDHC reaction product were generally more electron dense than the DAB reaction product (Figs. 3, 4) except in the sections that were subjected to the post-embedding immunostaining (Figs. 5, 6). The two reaction products were easily distinguishable from each other on the basis of the difference in electron density, form (crystalline or amorphous) and location (attached to membranes or not) (see Figs. 3-6). The sections that were incubated by the post-embedding immunogold procedure had many gold particles overlying them. The gold particles were unevenly distributed; high concentrations were identified overlying sub-populations of neuronal structures. Individual structures were identified as immunopositive if the density of gold particles overlying it was at least 4 times greater than overlying TH-positive or unstained dendrites and that this occurred in at least two adjacent sections (Figs. 5-7). In practice, when background staining was present, the density of immunogold particles overlying GABA-positive boutons was 4-31.5 times greater than that overlying TH-positive or unstained dendrites in the vicinity. Omission of the primary antiserum or adsorption against GABA conjugated to polyacrylamide beads, abolished all the immunostaining. Adsorption of the antiserum against glutamate conjugated to polyacrylamide beads did not affect the immunostaining. Substance P-immunoreactive terminals. The terminals that displayed immunoreactivity for substance P were found throughout the pars reticulata and pars compacta of the substantia nigra. They were of medium size, contained many vesicles and occasional mitochondria. The shape of the vesicles was difficult to assess because

68 of the peroxidase reaction product but could best be described as of a slightly pleomorphic shape. When membrane specializations were detected they were mainly of the symmetrical type (Figs. 3, 5, 6A) but many contacts were observed that exhibited asymmetrical specializations and post-junctional dense bodies (Fig.

5A). The majority of synaptic contacts involving substance P-positive boutons were with dendritic shafts. A common post-synaptic target of the substance P-positive boutons were structures that displayed immunoreactivity for TH. A total of 58 boutons were identified in contact with TH-positive structures and the majority of

69 which formed symmetrical synaptic specializations (Figs. 3, 5, 6). As with the overall distribution, the majority of contacts identified in searches of the nigra were with TH-positive dendritic shafts (Figs. 3C,D, 5, 6A) and only a small proportion in contact with perikarya (Fig. 3A,B) (this of course may reflect the relative densities of perikarya and dendrites in the SN). The same THpositive structures that received input from the substance P-positive boutons were also post-synaptic to non-immunoreactive boutons (Figs. 3C, 5A, 6A).

Glutamate decarboxylase-immunoreactive

terminals.

Both divisions of substantia nigra possessed many boutons that displayed immunoreactivity for glutamate decarboxylase (Fig. 4). The penetration of the immunoreagents used to reveal the G A D was the least of all the antigens studied and thus the GAD-positive terminals were found in the most superficial layers of the ultrathin sections. The boutons showed quite a variability in size and they contained many packed vesicles, the shape of which was difficult to judge. They also often contained mitochondria and there was an association between large boutons and the number of mitochondria. The GAD-positive synaptic boutons formed symmetrical membrane specializations (Fig. 4) although on rare occasions asymmetrical synapses were observed. The majority of terminals that were identified formed synaptic contacts with dendritic shafts but, as mentioned above this may simply reflect the overall relative density of dendrites and perikarya. Within the region of the penetration of the immunoreagents the majority of boutons in contact with individual structures displayed G A D immunoreactivity. Many of the GAD-positive boutons made synaptic contact with TH-immunoreactive dendrites and perikarya. In fact, within the region of the penetration of the immunoreagents for G A D , virtually all TH-immunoreactive structures received input from GAD-positive boutons. It was often the case that when identified in a single ultrathin section, a high proportion of the termi-

nals afferent to TH-positive structures were immunoreactive for G A D (Fig. 4). GABA-immunoreactive terminals. The sections that were immunostained by the post-embedding method for G A B A contained many immunoreactive terminals that displayed the morphological characteristics described above for the GAD-positive boutons. Thus they showed a wide variation in size and often contained mitochondria. Since the immunogold particles did not obscure the membranes of vesicles, they could be identified as being pleomorphic. Virtually all the synaptic specializations identified were of the symmetrical type and they represented a high proportion of all nigral boutons. In sections that were both pre-embedding stained for T H using either DAB (Fig. 6B,C) or BDHC (Fig. 6A) as the chromogen and post-embedding stained for GABA, numerous GABA-positive terminal boutons were seen forming symmetrical synapses predominantly with dendrites and less frequently with perikarya that displayed T H immunoreactivity.

Substance P, tyrosine hydroxylase and GABA immunostaining. In each experimental run of double preembedding immunostaining, ultrathin sections of the substantia nigra were tested for G A B A immunoreactivity. Since relatively low concentrations of glutaraldehyde were used to retain both substance P and T H immunoreactivities, G A B A immunoreactivity was often absent or very low. However, in some cases GABA immunoreactivity was present, albeit at a low level, in sections that also contained both substance P- and TH-immunostained structures (Figs. 5 and 6A). In sections of this type GABA-immunoreactive terminals were often identified in contact with TH-immunoreactive dendrites or perikarya (Figs. 5A,C, 6A). On several occasions THimmunoreactive dendrites were seen to receive input from both GABA-immunoreactive and substance Pimmunoreactive terminals (Figs. 5A, 6A). Substance P-immunoreactive terminal or pre-terminal boutons were sometimes seen to be also immunoreactive for GABA

Fig. 7. Electron micrographs of anterogradely labelled structures combined with post-embedding immunocytochemistryfor GABA. A: an anterogradely labelled bouton forming synaptic contact (arrow) with a dendrite in the substantia nigra. The bouton is identified as of striatal origin by a granule of peroxidase reaction product (HRP; DAB as chromogen) (that was partially lost during processing). The bouton also has many immunogold particles overlying it (some indicated by small arrows) thus identifying it as GABA-immunoreactive. The density of immunogold particles overlying the anterogradely labelled bouton is 9.9 times greater than the density overlying the postsynaptic dendrite. B: a similar bouton anterogradely labelled by HRP/WGA transported from the striatum (HRP) and immunoreactivefor GABA (some of the immunogold particles are indicated by small arrows). In this case the chromogen for the peroxidase reaction was o-toluidine (dimethyl benzidine). The density of immunogold particles overlying the anterogradely labelled bouton is 29 times greater than the density overlyingthe postsynaptic dendrite. C: an anterogradely labelled bouton (bio & GABA) in the globus pallidus followinginjections of biocytinin the striatum. The bouton also has many immunogold particles overlyingit thus identifyingit as GABA-immunoreactive.The density of immunogoidparticles overlying the biocytin-labelled bouton is 4 times greater than the density overlying the postsynaptic dendrite. A second bouton (b) is also immunoreactive for GABA but does not contain the peroxidase reaction product. D: three myelinated fibres in the substantia nigra, the lower one is immunoreactive for GABA (GABA), the one in the middle is anterogradely labelled with biocytin from the striatum and is immunoreactive for GABA (bio & GABA) and the upper one is neither immunoreactive nor anterogradely labelled (asterisk). The apparently high background staining in this figure is staining associated with large numbers of GABA-containing axons. A,C and D are at the same magnification, bar = 0.5/~m; bar in B = 0.5/~m.

70 TABLE I Proportion of boutons in contact with TH-immunoreactive structures that display GAD or GABA immunoreactivity

A total of 389 and 168 TH-positive profiles with boutons in synaptic contact or apposed to the membrane were examined in GABA- and GAD-stained sections, respectively. In the GABA-stained sections the figure represented about 70% of the total number of TH-positive profiles identified. Boutons were categorized as GABA-positive in single sections only. GA BA

Synaptic boutons Immunopositive Immunonegative Apposed boutons Immunopositive Immunonegative All boutons Immunopositive Immunonegative

GAD

Number

Percent

Number

Percent

298 124

70.6 29.4

93 101

47.9 52.1

222 82

73.0 27.0

131 97

57.5 42.5

520 206

71.6 28.6

224 198

53.1 46.9

(Figs. 5B,C, 6A). These double-stained boutons were identified in contact with non-immunoreactive dendrites (Fig. 5B,C) or dendrites that displayed immunoreactivity for T H (Fig. 6A). In some cases, boutons that displayed immunoreactivity for both substance P and GABA, boutons that displayed immunoreactivity for only G A B A and boutons that were immuno-negative for substance P and G A B A were identified in contact with the same TH-immunoreactive dendrites (Fig. 6A). Proportion o f GABAergic terminals in contact with TH-immunoreactive structures. In an attempt to assess the proportion of GABAergic synaptic terminals afferent to TH-positive structures in the substantia nigra two sets of sections were examined in the electron microscope, sections stained for both G A D and T H and a series of sections stained for T H (using DAB as chromogen) and then by the post-embedding method for GABA. In the former case, only those ultrathin sections or those parts of ultrathin sections that were within the depth of penetration of both sets of immunoreagents were examined in the electron microscope. A total of 169 THimmunoreactive structures were identified with boutons in synaptic contact or simply apposed in single sections. A high proportion of these boutons (in the region of 50%) displayed immunoreactivity for G A D (see Table I). Similarly, in the case of sections that were stained for TH and then for G A B A (see Fig. 6B,C), in which the problem of penetration of the immunoreagents does not occur, 389 TH-positive structures were found with boutons in synaptic contact with or apposed to the membrane (which accounted for about 70% of TH-positive structures identified). Of these, approximately 70% were immunoreactive for G A B A (see Table I).

Anterograde labelling of striatonigral terminals and post-embedding G A B A immunocytochemistry. Following the injection of WGA/HRP into the striatum, anterogradely labelled terminal fields and axons of passage were identified in the globus pallidus and the substantia nigra pars reticulata. At the electron microscopic level, the peroxidase reaction product was in a different form to that seen in immunostained terminals whether the chromogen for the reaction was DAB or stabilized o-tolidine. It was less homogeneous, was often present in membrane limited bodies and often had a detrimental effect on the structure of the terminals (compare Figs. 3-6 with 7A,B). The morphology of the anterogradely labelled terminals was similar to that of some of the terminals that displayed GAD or GABA immunoreactivities i.e. medium-size, packed with vesicles of a slightly pleomorphic shape and symmetrical synaptic specializations. In the sections in which there was a good retention of GABA immunoreactivity, the anterogradely labelled terminals were identified as GABA-positive (Fig. 7A,B). In the animals injected with biocytin, anterogradely labelled axons and terminal fields were identified in the globus pallidus and substantia nigra pars reticulata with a resolution and discrete staining that was indistinguishable from the type of staining obtained with Phaseolus vulgaris leucoagglutinin (Fig. 7C,D). In the electron microscope the distribution of the peroxidase reaction product (Fig. 7C,D) was similar to that of immunostained terminals (Figs. 3-6) rather than that of structures labelled by the anterogradely transported WGA/HRP (Fig. 7A,B). The morphology of the terminals and the type of synaptic specialization both in the globus pallidus and the substantia nigra were similar to those of the GAD- or GABA-immunoreactive boutons as well as the terminals that were anterogradely labelled after intrastriatal injection of WGA/HRP. In the GABA-immunostained sections, the intensity of staining of the biocytin made it difficult to identify G A B A immunoreactivity within the biocytin-labelled structures. However, on some occasions axon terminals and myelinated axons in the globus pallidus and substantia nigra were identified as GABA-positive (Fig. 7C,D).

DISCUSSION The results of the present experiments help to elucidate the chemical nature and the pattern of synaptic input to dopaminergic neurones in the substantia nigra of the rat. They demonstrate first, that nigral dopaminergic neurones receive symmetrical synaptic input from terminals that display immunoreactivity for substance P. Secondly, they show that dopaminergic neurones in the

71 SNC and SNR receive a dense symmetrical synaptic input from GABAergic terminals, identified by immunoreactivity for either GAD or GABA and that this input represents at least 70% of their afferent synapses. Thirdly, they demonstrate that some of the substance P-immunoreactive boutons also display GABA immunoreactivity and at least some of these make synapses with the dopaminergic neurones. Finally the results demonstrate directly that one of the origins of GABA-positive terminals that make synaptic contacts in the substantia nigra is the striatum. Technical considerations

In order to have direct information on the synaptic contacts between chemically characterized structures it is necessary to apply techniques that enable the chemical nature of both the pre- and post-synaptic structures to be identified in the same ultrathin sections that are examined in the electron microscope. The reason for this is that in double immunocytochemical techniques applied at the light microscopic level only, it is impossible to state that two structures are in direct synaptic contact. Furthermore, the comparison of sections incubated separately for different antigens only allows indirect correlations to be made. For these reasons we applied a double immunocytochemical technique that is applicable to both light and electron microscopy6'45 in which both antigens are localized using immunoperoxidase methods but revealed with different chromogens, the reaction products of which are distinguishable at both the light and electron microscopic levels. A problem with this approach however, is the possibility of cross-reactivity of the antibodies or the deposition of the reaction product formed by the second chromogen at the site of the reaction product formed by the first chromogen. To control for these possibilities the whole of the double immunocytochemical procedure was carried out with the omission of each primary antibody in turn. In these experiments only the DAB reaction product was formed when the TH antiserum (i.e. the second primary antiserum) was omitted and only the BDHC reaction product formed when the first primary antiserum (substance P or GAD) was omitted. This demonstrates that there is no cross-reactivity between the two sets of antisera and that the second chromogen (BDHC) does not have access to the peroxidase molecules deposited by the first set of antibodies. This lack of cross-reactivity and the masking of the peroxidase molecules and indeed the masking of immunoglobulins, has been described before even when the two primary antibodies were raised in the same species of origin 6,45. The second approach that was used to give direct information about the chemical nature of both pre- and

post-synaptic structures was to combine pre-embedding immunocytochemistry for TH with post-embedding immunocytochemistry for GABA. In this procedure the question of cross-reactivity of antisera does not arise but as a general method, is of limited application. The reason for this is that post-embedding immunogold procedures are best suited to antisera against glutaraldehyde conjugated amino acids; they therefore require fixation with high concentrations of glutaraldehyde, i.e. a fixation that generally does not favour the retention of antigenicity for peptides and proteins. Thus compromises in fixation have to be made in order to retain the antigenicity of each of the antigens that are being examined in the tissue. This was the case in the present experiments, but it was possible to consistently obtain good immunoreactivity for both TH and GABA in the same tissue. However, in the cases where pre-embedding immunocytochemistry for TH and substance P was combined with post-embedding immunocytochemistryfor GABA, all three antigens were only rarely labelled because of the compromises that had to be made in the fixation. Substance P-immunoreactive input to dopaminergic neurones

The detection of substance P-immunoreactive terminals in the substantia nigra that form both symmetrical and asymmetrical synaptic specializations confirms earlier ultrastructural observations in single immunocytochemical studies in the rat 73 and monkey17. The present results also confirm and extend previous double immunocytochemical studies that showed an association 9 and synaptic contacts35'46 between substance P-immunoreactive terminals and TH-positive dendrites and perikarya in the rat substantia nigra. In the present study we demonstrate that the substance P-positive input to the rat substantia nigra is more widespread than previously suggested and that the substance P-immunoreactive terminals form synaptic contacts with all parts of the dopaminergic neurones so far examined i.e. perikarya, large diameter proximal dendrites and small diameter distal dendrites, in both the pars compacta and pars reticulata. The morphology of the substance P-immunoreactive boutons forming symmetrical synapses with the THpositive structures was very similar to the terminals identified in the substantia nigra following either intrastriatal injection of anterograde tracer69 (see also Fig. 7 of the present study) or following lesions of striatal neurones28'29'69'87. The similarities included the size of the bouton, the type and density of synaptic vesicles and the membrane specializations. This observation together with the extensive neurochemical evidence that substance P or other tachykinins are present in the striatonigral

72 pathway 1"13'26"39'51suggests that the major origin of those substance P-immunoreactive boutons that form symmetrical synapses with the TH-positive elements is the striatum. This is made more likely by the demonstration that striatal neurones that express substance P immunoreactivity are of the medium-size densely spiny class 33 i.e. the class that projects to the substantia nigra TM. The origin of the substance P-positive terminals that form asymmetrical synaptic specializations with dopaminergic and non-dopaminergic structures remains a matter of conjecture. It is unlikely that they are derived from the striatum as anterograde labelling methods have invariably identified striatonigral terminals as forming symmetrical synaptic specializations 2s'29'69"87 (see also present study). Similarly, the local axon collaterals of striatonigral terminals form symmetrical synaptic contacts 68'89. The synaptic terminals of known origin in the substantia nigra that form asymmetrical synaptic specializations are those from the cortex 47"53'8° (cat) and those from the nucleus tegmenti pedunculopontinus pars compacta (TPC) TM (cat). It is unlikely that the substance Pcontaining terminals are derived from the cortex. However, since some of the cholinergic neurones in the TPC also contain substance p84,85, it is possible that neurones derived from this region give rise to those substance P-positive terminals. Consistent with this is the demonstration that choline acetyltransferase-immunoreactive terminals in the substantia nigra that presumably arise from the TPC make asymmetrical synaptic specializations 3'48'49. A further possible origin that may also be considered is the raphe, as serotonin-immunoreactive boutons also form asymmetrical specializations 52. However direct evidence of the origin of these substance P-containing terminals awaits the application of double immunocytochemical analyses of retrogradely labelled neurones. It is interesting to note that some enkephalinimmunoreactive terminals form asymmetrical synaptic specializations in the substantia nigra of the primate 31 and, in the rat, some of them have been identified in contact with TH-immunoreactive dendrites (unpublished). In addition to the similarities in morphology of the substance P-positive terminals and striatonigral terminals, the substance P-positive boutons observed in this study were similar to that of a population of GADimmunoreactive boutons in contact with the TH-positive structures. Indeed, when conditions were optimal, it was possible to detect G A B A immunoreactivity (by the post-embedding method) in boutons that displayed substance P immunoreactivity (see below).

GABAergic input to doparninergic neurones The detection of a large number of GAD- and G.ABA-immunoreactive terminals in the substantia nigra

is consistent with previous light microscopic and ultrastructural studies of GAD-immunoreactive structures 54' 56~57.60.61. Our observations are in keeping with all of these studies in that there is a dense distribution of GAD-positive terminals that form symmetrical synaptic contacts with all parts of nigral neurones. Furthermore our results also demonstrate the presence of endogenous G.ABA in terminals that form symmetrical synaptic contacts with dopaminergic and non-dopaminergic neurones in the substantia nigra. The demonstration of many GAD-immunoreactive terminals in symmetrical synaptic contact with TH-immunoreactive structures confirms and extends some preliminary ultrastructura121'~2 and light microscopic findings 57. From the light microscopic examination of the sections that were stained for both GAD and TH it was evident that a large proportion of the afferent terminals associated with the TH-positive perikarya and dendrites, whether located within the pars compacta or the pars reticulata, were G.AD-immunoreactive..Analysis of the same sections by electron microscopy confirmed this. In an attempt to assess what proportion of the afferent terminals to the dopaminergic neurones was G.AB.Aergic, systematic analyses of TH-immunoreactive structures were made. In this way it was observed that in the region of 50% of the synaptic boutons apposed to the dopaminesynthesizing neurones were G.ABAergic. The major drawback of this approach relates to the penetration of the immunoreagents. Thus, as no penetration-enhancing techniques were used in our study, the immunostained structures were confined to the outer few microns of the sections. Furthermore, from analysis of the ultrathin sections in the electron microscope it was evident that the depth of penetration of TH-immunostained structures was not always the same as the GAD-immunostained structures. The TH-immunostained perikarya and dendrites that were examined for GAD-positive input were therefore selected very carefully. The sample is therefore likely to be biased, in that it will be an underestimate of the true number of GAD boutons in contact with TH-immunoreactive structures, the figures presented in the table must therefore be considered as a minimum value for the proportion of input to dopamine-synthesizing neurones that contain GAD. Because of these limitations the analysis was repeated using a combination of pre-embedding immunocytochemistry for TH (DAB as the chromogen) and post-embedding immunogold staining for G.AB.A. With this combination there is no question of penetration of the immunoreagents for the GAB.A, the results are less likely to be an underestimate of the true density. Indeed, the values obtained with this approach are higher than those obtained with the double pre-embedding method; in the region of 70% of the

73 terminals afferent to the TH-positive structures were immunoreactive for GABA. This value is still likely to be an underestimate because although 1% glutaraldehyde was included in the fixative it is possible that the GABA was not retained in all GABAergic terminals. Nevertheless, the essential finding is that a high proportion of the terminals that make contact with dopaminergic neurones, whether located in the pars compacta or in the pars reticulata of the substantia nigra display GABA immunoreactivity. Dopaminergic neurones are thus likely to be under a powerful inhibitory control from GABAergic neurones. It is of interest to note that in the present study we observed only very few GAD-positive or GABA-positive terminals making asymmetrical synapses. This apparent rarity of asymmetric GABAergic synapses in the substantia nigra is consistent with the observations of Van den Pol et al. s2 and Flumerfelt et al. 21 who did not report any asymmetrical synapses. In contrast, our observations are not consistent with the findings of Ribak et al. 61 who reported that 15% of GAD-positive synapses were asymmetrical and the findings of Nitsch and Riesenberg 54 who observed occasional GAD-positive boutons forming asymmetrical synapses. An explanation for these discrepancies is that in the present study and in the two other studies in which asymmetrical synapses were not reported, the search for GAD- or GABA-positive synapses was directed towards dopaminergic neurones i.e. THpositive structures were identified first of all and then examined for GABAergic input. In contrast the two studies that identified significant numbers of GADpositive asymmetrical synapses were not specifically directed towards dopaminergic neurones but to the substantia nigra as a whole. It is thus likely that GABAergic boutons that form asymmetrical synapses do so predominantly with non-dopaminergic neurones, presumably located mainly in the pars reticulata.

Origin of GABAergic terminals There are at least three possible sources of GABAergic terminals in contact with the dopaminergic neurones in the substantia nigra; the striatum, the globus pallidus and local nigral neurones. There is a substantial body of evidence implicating GABA as a transmitter in the striatonigral pathway (for references see Graybiel and Ragsdale26). Morphological studies have demonstrated that striatonigral neurones identified by retrograde labelling from the substantia nigra display immunoreactivity for G A B A TM or GAD 2°. Furthermore the number of GAD-immunoreactive axons and terminals in the substantia nigra is markedly reduced by destruction of the striatum 54'56'6°. In the present study we have demonstrated directly that a population of

terminals in the substantia nigra that are derived from neurones in the striatum, display GABA immunoreactivity. This was done by combining the anterograde transport of HRP/WGA or biocytin with post-embedding immunocytochemistry for GABA. Following such experiments the anterogradely labelled terminals in the substantia nigra were found to be immunoreactive for GABA. Although we observed the GABA-positive striatonigral terminals forming symmetrical synapses with elements in the substantia nigra, the conditions of fixation were not suitable to enable the procedure to be combined with TH immunocytochemistry. Because of this we were unable to directly demonstrate GABApositive striatonigral terminals forming synapses with the dopaminergic neurones. However it is clear from other anatomical experiments that dopaminergic neurones represent one of the targets of striatonigral terminals69'a7 so it is likely that the targets of at least some GABApositive striatonigral terminals identified in our study, are the dendrites and perikarya of dopaminergic neurones. Experiments are in progress in which the anterograde transport of biocytin is combined with pre-embedding immunostaining for TH and post-embedding immunostaining for GABA to determine directly whether GABA-containing striatonigral terminals synapse with dopaminergic neurones. It should be remembered however, that the striatal input to the substantia nigra is mostly directed towards its ventral aspect i.e. the pars reticulata and although part of the dendritic tree of some of the dopaminergic neurones in the pars compacta descend ventrally into the reticulata where they may receive input from striatal terminals, it is likely that most striatal terminals innervate non-dopaminergic neurones of the pars reticulata. Thus although a high proportion of the terminals afferent to the dopaminergic neurones may be derived from the striatum, most of the striatal input to the substantia nigra is likely to innervate the perikarya and dendrites of non-dopaminergic neurones in the pars reticulata. A second possible source of the GAD- or GABAimmunoreactive terminals in synaptic contact with the nigral dopaminergic structures is the globus pallidus. The results of anatomical studies combining retrograde labelling from the substantia nigra and GAD immunocytochemistry have shown that pallidonigral neurones display GAD immunoreactivity in both the rat and the cat 19'23. These observations confirm earlier neurochemical studies that indicated that the pallidum is one of the sources of GAD and GABA in the substantia nigra 26. We have recently extended these findings in the rat, demonstrating that pallidonigral terminals, identified by the anterograde transport of Phaseolus vulgaris leucoagglutinin, have a characteristic morphology and display GABA immuno-

74 reactivity 64. This observation is consistent with the observations of Nitsch and Riesenberg 54 who suggested that a population of large GAD-positive boutons in the substantia nigra that persist following lesions of the striatum but not after hemitransection caudal to the globus pallidus, are derived from the pallidal complex. The major synaptic targets of the pallidonigral terminals have been identified by retrograde labelling from the thalamus, brainstem or superior colliculus, as the nigrofugal cells located in the pars reticulata 65. In addition to this target however, dopaminergic neurones in the ventral part of the pars compacta, identified by TH immunocytochemistry, were also found to receive input from pallidonigral terminals 65. Thus some of the GABAergic terminals in contact with the dopaminergic structures are derived from the pallidum. Indeed, in the present material some of the GAD- or GABA-positive terminals in contact with dopaminergic neurones had the morphological characteristics ascribed to pallidonigral terminals i.e. large boutons containing many pleomorphic vesicles, a large number of mitochondria and forming symmetrical synaptic specializations. The third possible source of GABAergic terminals in contact with dopaminergic neurones are GABAergic neurones located within the substantia nigra itself. The inhibitory output neurones of the pars reticulata give rise to local axon collaterals that arborize within the nigra 27 and have an inhibitory action 16. There is also a possibility that the substantia nigra contains small interneurones 22'34 that are immunoreactive for G A D 54. The possibility that these classes of nigral neurones give rise to GABAergic boutons within the nigra is made more likely by the fact that a large number of GAD-positive terminals are preserved following hemitransection just rostral to the substantia nigra i.e. at a level that would interrupt the striatonigral and pallidonigral pathways 54. Thus at least some of the GABAergic terminals identified in contact with dopaminergic neurones may be derived from local GABAergic neurones. Further work is necessary to determine directly whether local neurones make synaptic contact with dopaminergic neurones and if so, what the numerical significance is, and what is the pattern of input. Co-localization o f G A B A and substance P In some animals, when relatively high concentrations of glutaraldehyde were used that favoured immunostaining for G A B A by post-embedding immunogold, it was still possible to detect both T H and substance P by pre-embedding methods. As the conditions of fixation for each antigen were not optimal the staining for each was less complete than in ideal conditions of fixation. There was some difficulty in identifying TH-immunostained

structures because the electron density of the BDHC reaction product was reduced by the post-embedding immunogoid protocol. Despite these limitations, a population of substance P-immunoreactive axonal boutons and terminals were categorized as immunoreactive for G A B A and indeed some of these double-stained boutons were seen in synaptic contact with TH-positive dendrites. Since some of the GABA-positive terminals in the vicinity of double-stained boutons were not immunoreactive for substance P, despite the fact that they were within the depth of penetration of the substance P immunoreagents, indicates that GABA and substance P coexist in only a sub-population of GABAergic terminals. This is not surprising in view of the varied origins of GABA terminals in the substantia nigra (see above). The converse, i.e. only a sub-population of substance P terminals contain GABA, is not necessarily the case. The reason for this is that false-negative results often occur when attempting to detect GABA by post-embedding methods in neuronal structures that also contain a peroxidase reaction product, presumably because the peroxidase reaction products obscure the antigenic sites that are detected by the antibodies to GABA 65. The co-localization of substance P and GABA is not a surprising observation in view of the known co-localization in the striatonigral pathway (for references see Graybiel and Ragsdale 26) and indeed in medium-size neurones within the striatum 58. GABAergic projection neurones account for the majority of neurones in the striatum 7"38'66. It is therefore probable that most of the substance P-positive projection neurones also contain GABA. Thus, in the substantia nigra the dense innervation of the pars reticulata by substance P-positive terminals is likely to be a dense innervation by terminals in which substance P and GABA are co-localized. If this is the case, then it is likely that the role of substance P in the substantia nigra is not as a neurotransmitter as such, but rather as a neuromodulator, influencing the action or release of GABA. Comparison of the synaptic input to dopaminergic neurones to that of nigral reticulata neurones From the light microscopic study alone it is clear that there are marked differences between the synaptology of dopaminergic neurones (either in the pars compacta or the pars reticulata) and non-dopaminergic neurones of the substantia nigra. The simple fact that there are differences in the density of innervation of the pars compacta and the pars reticulata (e.g. more dense substance P innervation of the reticutata) would suggest a difference in synaptology. In fact, analysis at the single cell level by high resolution light microscopy, confirms this. In the double-stained sections the perikarya of

75 reticulata n e u r o n e s are apparently ensheathed by G A D i m m u n o r e a c t i v e b o u t o n s whereas TH-immunoreactive neurones, although associated with many GAD-positive b o u t o n s , are by no means ensheathed. This also appears to be the case for the dendrites of dopaminergic neurones versus those o f non-dopaminergic reticulata neurones. T h e G A D immunostaining in the reticulata is characterized by dense terminal staining in the form of rosettes, surrounding dendrites 56'57'6° whereas the dendrites of d o p a m i n e r g i c neurones have far less boutons associated with them. These differences are borne out by electron microscopic analyses. The n u m b e r and density of G A B A e r g i c terminals in synaptic contact with dopaminergic n e u r o n e s is lower than for reticulata neurones identified by retrograde labelling from the thalamus, superior colliculus or brainstem 64'65. O n e input to the substantia nigra that has been shown to have selectivity for one class of neurone is that from the globus pallidus. A l t h o u g h some pallidal terminals do m a k e contact with dopaminergic neurones, most of them are in contact with the perikarya and proximal dendrites of identified reticulata output neurone 64'65. This may also be the case for the striatal input to the nigra. Although pars c o m p a c t a n e u r o n e s identified as dopaminergic 37 or as nigrostriatal n e u r o n e s 69 receive input from the stria t u m , it is likely that the m a j o r target of the striatal terminals are the neurones of the reticulata since most striatonigral terminals innervate the reticulata, most of the G A D in the substantia nigra is in the reticulata and most of the input to reticulata cells is G A B A e r g i c . The input to the dopaminergic neurones is less dense and of a m o r e diverse origin. These differences in synaptology REFERENCES 1 Arai, H., Sirinathsinghji, D.J.S. and Ernson, P.C., Depletion in substance P- and neurokinin A-like immunoreactivity in substantia nigra after ibotenate-induced lesions of striatum, Neurosci. Res., 5 (1987) 167-171. 2 Beninato, M. and Spencer, R.E, A cholinergic projection to the rat substantia nigra from the pedunculopontine tegmental nucleus, Brain Research, 412 (1987) 169-174. 3 Beninato, M. and Spencer, R.E, The cholinergic innervation of the rat substantia nigra: a light and electron microscopic immunohistochemical study, Exp. Brain Res., 72 (1988) 178184. 4 Bolam, J.P., Substance P and 5HT input to dopamine neurons in the substantia nigra of the rat, Neurosci. Lett., Suppl. 9 (1987) $111. 5 Bolam, J.P. and Ingham, C.A., Combined morphological and histochemical techniques for the study of neural microcircuits. In A. BjOrklund, T. HOkfelt, EG. Wouterlood and A.N. van den Pol (Eds.), Analysis of Neuronal Microcircuits and Synaptic Interactions, Handbook of Chemical Neuroanatomy, Vol. 8, Elsevier, Amsterdam, 1990, pp. 125-197. 6 Bolam, J.P., Ingham, C.A., Izzo, EN., Levey, A.I., Rye, D.B., Smith, A.D. and Wainer, B.H., Substance P-containing terminals in synaptic contact with cholinerglc neurons in the neostriatum and basal forebrain: a double immunocytochemical study in

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