SYNAPSE 23:107-114 (1996)

Striatal Efferents Preferentially Innervate Neurons in the Ventral Pallidum Containing GABAA Receptor a 1 Subunit-Like Immunoreactivity

JOSEF MARKSTEINER, WALmR KAUFMA”, KRISTIAN PFALLER, WERNER SIEGHART, AND ALOIS SARIA

Neurochemical Unit, Clinic of Psychiatry, A-6020 Innsbruck (J.M., W.K., AS.), Department of Biochemical Psychiatry, Clinic of Psychiatry, A-1010, Vienna, Austria (W.S.), Department of Histology and Embryology,

University of Innsbruck (K.P)

KEY WORDS GABAA receptor, Immunocytochemistry, Substance P, Ventral pallidum

ABSTRACT The y-aminobutyric acid-A receptor consists of several subunits. In this immunohistochemical study we investigated the regional distribution of the a1 and a2 subunits with subunit-specific antibodies in the ventral pallidum, and compared the staining patterns to those of substance P (SP). a1 subunit antigenic sites were found to be localized to pallidal neurons, varicosities, and varicose fibers. a1 immunopositive fibers mainly appeared “tubulus-like” due to the intense staining of the membranes of the long pallidal dendrites. Double labelling of a1 subunit and substance P revealed that a1 subunit-like immunoreactive (IR) dendrites and somata of the pallidal neurons were often invested by SP-IR striatal efferents. Subcellularly, the dendritic and somatic membranes of pallidal neurons were strongly immunopositive for the a1 subunit, whereas only a few axon terminals exhibited a1-IR. or2-IR was restricted to a low number of ventral pallidal neurons. The distributional patterns obtained for the a1 and a2 subunits suggest that striatal efferent neurons directly influence pallidal neurons displaying a distinct GABAA subunit composition, which may be of pharmacological importance since the alpxy2-subunits containing receptors have mainly a benzodiazepine type I pharmacology. o 1996 Wiley-Liss, Inc.

INTRODUCTION Gamma-aminobutyric acid A (GABAA) receptors in

the mammalian central nervous system are members of a family of ligand-gated ion channels consisting of heterooligomeric glycoprotein complexes in synaptic and extrasynaptic membranes. Molecular cloning stud- ies have identified five subunits and isoforms thereof, namely, 6a, 4p, 4y, 6, and 2p (Harvey et al., 1993; See- burg et al., 1990). The composition and functional prop- erties of the GABAA receptors vary within the central nervous system (Olsen and Tobin, 1990). There is an extensive literature concerning the distribution of GABA, subunit antigens in the central nervous system (Persohn et al., 1992; Benke et al., 1991; Nicholson et al., 1992; Thompson et al., 1992; Baude et al., 1992; Gao et al., 1993; Zhang et al., 1991a). In situ hybridization studies have revealed the cellular localization of GABAA a1 subunit mRNAs in the rat forebrain (Zhang et al., 1991b) and in the lower brain stem of the rat (Hironaka et al., 1990). 0 1996 WILEY-LISS, INC.

The aim of the present study was to localize the a1 and a2 subunits with immunocytochemical methods in the ventral pallidum, and to study whether a preferen- tial relationship of striatal efferents exists for either the a1 or the a2 subunit of the GABAA receptor complex. For the ventral pallidum, substance P (SP) was used as a neuronal marker (Heimer et al., 1982; Zahm and Heimer, 1988; Otsuka and Yoshioka, 1993) displaying a specific arrangement of fibers, termed “woolly fibers” for their distinctive morphology (Heimer et al., 1982; Haber and Nauta, 1983).

A distinct GABAA subunit composition of pallidal neu- rons in relation to striatal efferents may be of pharmaco- logical importance, since the alpxy2-containing recep- tors are believed to have a benzodiazepine type I pharmacology, whereas a2-, 013-, and 1x5-containing re- ceptors have a benzodiazepine type I1 pharmacology (Hadingham et al., 1993; Pritchett et al., 1989).

Received June 12, 1995; accepted in revised form November 21, 1995.

108 J. MARKSTEINER ET AL.

MATERIALS AND METHODS Twelve male Sprague-Dawley rats (Forschungsinsti-

tut fur Versuchstierzucht, Himberg, Austria) were used for this study. They were deeply anesthetized with thio- pental (150 mgkg, i.p.). The brains were perfused via the ascending aorta with Tyrode’s solution, pH 7.4, fol- lowed by 250 ml4% paraformaldehyde in 100 mM phos- phate-buffered saline (PBS), pH 7.4, postfixed for 2 h in the same fixative, and then transferred to 20% su- crose in PBS for a t least 24 h. Brains were quickly frozen by immersion in cold (-50°C) isopentane and stored a t -70°C.

Immunocytochemistry Free-floating sections (60 pm) were processed as pre-

viously described (Marksteiner e t al., 1992,1995). After incubation with 10% normal goat serum (Dakopatts, Copenhagen, Denmark), sections were incubated with primary antiserum (anti-al, 1:750; anti-aZ, 1:750; anti- SP, 1:1,000). The sections were then processed with appropriate secondary antibodies (1: 100, Dakopatts) and afterwards with PAP complex (1:200, Dakopatts) for 90 min each a t room temperature. The peroxidase reaction was developed by using 3,3’-diaminobenzidine tetrahydrochloride (DAB, Sigma, Munich, Germany) as a chromogen (40 mg DAB in 100 ml50 mM Tris-HC1, pH 7.6), and then 0.006% hydrogen peroxide was added for 6-7 min. After each incubation step (except preincu- bation), three washes (10 min each) with Tris-HC1 were included. All buffers and antibody dilutions contained 0.4% Triton X-100.

Double-labelling experiments Incubation procedures were performed as described

by Wouterlood (1988). Specifically, sections were incu- bated for 48 h (at 4°C) in a mixture of rat anti-SP antise- rum and rabbit anti-a1 antiserum, followed by an incu- bation with horse anti-rat secondary antibodies (1: 100, Dakopatts). Subsequently, the sections were incubated with the appropriate PAP complex (1:200, Dakopatts), and were then reacted with Ni-DAB (400 mg nickel- ammonium sulfate in 50 mM TBS, pH 8.0, 20 pl HzOz), yielding a blue-black precipitate reaction product. Thereafter, the sections were incubated for 1 h at room temperature with swine anti-rabbit secondary antibod-

(~1-1R c~2-1R ac ax CPU d GABA PBS

SP Tu w

so

Abbreviations

a 1 subunit-like immunoreactivity a2 subunit-like immunoreactivity anterior commissure axon caudate putamen dendrite y-aminobutyric acid phosphate-buffered saline soma substance P olfactory tubercle ventral pallidum

ies (1:200, Dakopatts), followed by an incubation with the appropriate PAP complex (1:200, Dakopatts). The sections were then treated with DAB (5 mg DAB in 10 ml 50 mM Tris-HC1, pH 7.61, resulting in a brown reaction product. Controls using no primary antiserum and antisera preadsorbed with the synthetic peptide (10 pM, 24 h, 4°C) were included in each experiment.

Immunofluorescence Fourteen- pm-thick cryostat sections were processed

for the indirect immunofluorescence method (Coons, 1958). Briefly, after blocking nonspecific binding with 10% normal swine serum and 0.1% bovine serum albu- min in 50 mM Tris-HC1-buffered saline (TBS), sections were incubated with a mixture of rat SP- (1:200) and rab- bit al-antisera (1:400) for 48 h at 4°C. Afterwards, sec- tions were parallel-incubated with Texas red conjugated swine anti-rat antibodies (150, Amersham, Bucking- hamshire, UK) and fluorescein-conjugated donkey anti- rabbit antibodies (1:50, Amersham) for 3 h at room tem- perature. All antisera were diluted in TBS containing 0.1% TritonX-100 and 0.1% bovine serum albumin. Incu- bations were performed in humid chambers. The sec- tions were embedded in Glycergel (Dakopatts) and ex- amined in an Olympus BX6OF microscope equipped for fluorescence microscopy, using a WB (BP450-480) and a WG (BP510-550) filter (Olympus, Hamburg, Germany).

Electron microscopy Brains processed for electron microscopy were per-

fused with 250 ml of 4% paraformaldehyde and 0.05% glutaraldehyde over 30 min (Somogyi and Takagi, 1982). After perfusion, several tissue blocks of the ven- tral pallidum were cut with a 60-pm sections Vibratome Micro-cut H 12001 (Tulin, Austria). Immunocytochem- istry was performed as described for light microscopy, with the difference that no Triton X-100 was used. After 3,3’-diaminobenzidine reaction, the sections were treated with 1% OsOl in 0.1 M phosphate buffer for 45 min, washed in distilled water, and placed for 30 min in uranyl acetate (0.5% in 70% alcohol). After dehydra- tion in graded ethanol and propylene oxide, the sections were infiltrated with epoxy resin (Durcupan ACM, Fluka, Buchs, Switzerland) overnight at room tempera- ture and flat-embedded on glass slides for light micro- scopic assessment. The resin was polymerized by heat- ing in an incubator at 60°C for 48 h. Areas of interest were reembedded for electron microscopic sectioning. Serial ultrathin sections were cut on a Reichert ultrami- crotome (Jung CM 3000; Leica, Nussloch, Germany) and collected on copper single-slot grids. Afterwards they were examined in a Philips CM 120 electron micro- scope (Eindhoven, Netherlands).

Specificity of antisera Specificity of the a1 and a2 antisera was demon-

strated by several lines of evidence. Anti-peptide a1 (1-9) and C-terminal nonapeptide anti-az (416-424)

GABAA RECEPTOR a1 IMMUNOREACTIVIW 109

Fig. 1. Photomicrographs of an aspect of the ventral pallidum illustrate the distribution of al-IR (brown) and SP-IR (dark blue) in double-labelled coronal sections. The brown reaction product is often associated with SP-IR “woolly fibers” (indicated by arrowheads). Top: al-IR perikarya do not display SP-IR but were outlined by SP-IR. Bar, 10 km (A and B). Bottom: The contribution of e l - IR to this assessment of fibers is mainly due to the immunoreactive pallidal dendrites.

antibodies were purified from the sera of immunized rabbits by affinity chromatography on thiopropyl-seph- arose 6B coupled to the immunizing peptide. Purified antibodies selectively recognized the peptides a1 and a2, respectively, and the GABAA receptor purified from rat brain in an ELISA (enzyme linked immunosorbent

assay). In addition, these antibodies were able to precip- itate GAB% receptors from brain membrane extracts, and in Western blots identified a single protein with an apparent MW 51 kDa which could be blocked in the presence of the peptide (Zezula and Sieghart, 1991; Zimprich et al., 1991; Fenelon et al., 1995).

110 J. MARKSTEINER ET AL.

Fig. 2. Double immunofluorescence micrographs of an aspect of the ventral pallidum after incubation with the a1 antiserum (A, C) and SP antiserum (B, D). al-IR perikaryon (A, indicated by arrowhead) is invested by SP-IR varicosities (B). al-IR fibers and varicosities (C) are innervated by SP-IR terminals (D). Bar, 10 wm.

The monoclonal substance P antiserum was pur- chased from Seralab, Sussex, England. This antibody recognizes the COOH-terminal end of substance P (Cuello et al., 1979).

No specific al-, a2-, or SP-IR was observed in control sections of brains incubated with the antisera pread- sorbed with the respective synthetic peptides (10 FM).

RESULTS In general, in the ventral pallidum, al-IR profiles

appeared in the form of particles of varying size, vari- cose fibers, and immunopositive neurons. The neurons had varied shapes, some being spherical, and others triangular or polygonal, with a diameter ranging from 10-30 pm, giving rise to thick dendrites (Fig. 1 top, bottom). The majority of a1-IR neurons had a diameter >20 pm (Fig. 2A). At the light microscopic level, a high number of al-IR fibers appeared “tubulus-like,” mainly

due to the intense staining of the membranes of the long pallidal dendrites. The staining pattern for al-IR closely followed the same delineations of the ventral pallidum as SP-IR, but displayed a lower density com- pared to SP-IR (compare Fig. 3A to Fig. 3B). Double immunofluorescence revealed that these a1-IR fibers were smaller in diameter than SP-IR “woolly fibers” (Fig. 2C,D). Perisomatic, as well as peridendritic, SP- immunostaining of cw 1-IR neurons was frequently ob- served in double-labelling experiments (Figs. 1 bottom, 2). The staining pattern of a2-IR was low in density and was complementary to that of c w l - and SP-IR (Fig. 3C). It consisted mainly of immunopositive neurons with single varicose fibers. Double labelling with SP- IR revealed only a few cells invested with substance P- containing efferents.

Subcellularly, al-IR was localized to dendritic and somatic membranes, both at synaptic and extrasynaptic

GABA, RECEPTOR a1 IMMUNOREACTMTY 111

Fig. 3. Distributions of al-IR (A), SP (B), and d - I R (C) are shown in coronal sections. A high density of al-IR is observed in the area of the ventral pallidurn (W), whereas the caudate putamen (CPu) dis- plays only a low density of immunostaining. The density of u2-IR is low-to-very low, except for a2-IR perikarya. In the area of the olfactory tubercle (Tu), a2-IR shows a high density of fibers compared to the rare staining for al-IR. Bar, 200 Frn (A-C).

sites (Fig. 4A-C). The axodendritic synapses were the most numerous, and symmetric synapses were mainly detected. Also, the case of axosomatic synapses, sym- metric contacts predominated. The 3,3'-diaminobenzi- dine immunoreaction product was most dense at the synaptic junctions and extended diffusely into the cyto- plasma. The membranes of only a few axons were al-IR. In the soma, immunopositive staining was additionally seen in subcellular compartments as in the endo- plasmatic reticulum (Fig. 4 0 . Substance P-IR was pres- ent in axon terminals, and was found to be in vesicles (Fig. 4D).

DISCUSSION In the present study we found clear evidence that a

high density of al-IR is present in the pallidal areas, compared to the relatively low density in the nucleus accumbens and in the caudate putamen. In contrast, for a2-IR a dense immunostaining was observed in the caudate putamen, and a low density in the ventral pal- lidum. The localization of the different alpha subunits of GAl3AA receptor is in line with previous immunocyto- chemical and in situ hybridization studies (Zhang et al., 1991b; Zimprich et al., 1991; Wisden et al., 1992; Fritschy and Mohler, 1995).

The intensity of immunoreactivity for the a1 and a2 subunits was not strongly dependent on the fixation procedure, since increasing concentrations of glutaral- dehyde over 0.05% or the duration of fixation did not reduce the density of staining.

In the ventral pallidurn, al-IR perikarya had the ap- pearance of typical pallidal neurons (Millhouse, 1986). The shape and size of al-IR perikarya were similar to neurons containing GABA and glutamic acid decarbox- ylase (Ribak et al., 1980; Oertel and Mugnaini, 1984). This is in line with previous studies showing a selective association of the a1 subunit with GABAergic neurons (Fritschy et al., 1992; Gao et al., 1993, 1995) rather than with cholinergic neurons of the rat basal forebrain (Henderson, 1995). In adjacent sections we did not find clear evidence for a colocalization of both alpha subunits in the same cell. In this context, it is of interest that a developmental switch of al- and a2-IR was particularly evident in pallidal areas, and that the initially dense ol2-IR was gradually replaced by al-IR (Fritschy et al., 1994). The ventral striatopallidal system is a part of the basal ganglia (Heimer et al., 1982). The basal ganglia- thalamocortical projection system forms multiple reentrant circuits (Alexander, 1986; Alexander and Crutcher, 1990). Output from the ventral striatopallidal system is channeled via the mediodorsal thalamus to prefrontal cortical areas and the anterior part of the cingulate gyrus, and is an example of a reentrant loop. Our study, like others, indicates that d - I R is particu- larly dense in pallidal areas and other brain regions involved in the motor circuits, as, for instance, the sub- stantia nigra. Further studies are necessary to clarify

112 J. MARKSTEINER ET AL.

Fig. 4. Electron micrographs showing immunoreactivity for the a1 subunit (A-C) and SP-IR (D) in the ventral pallidum. al-IR is present in dendrites (d) and somata (so), with the highest density at the synaptic junctions. Membranes of axon terminals (ax) are labelled to a lesser extent. The plasma mem- brane also displays a1-IR (C, indicated by arrowheads). In contrast to the localization of al-IR, SP-IR is found only in the axon terminals (D). Bar, 0.5 p,m (A, B, D), 1 p,m (C).

GABAA RECEPTOR a1 IMMUNOREACTMTY 113

whether cil-IR is attributed to certain reentrant loops. As a consequence, a distinct pharmacological profile in respect of GABA may exist for the different circuits in the basal ganglia.

At the light microscopic level, al-IR fibers frequently appeared as “tubulus-like.” Subcellular localization of the al-antigenic sites revealed that the dendritic mem- branes displayed al-IR. The double-labelling experi- ments indicated axodendritic and axosomatic contacts of substance P-immunopositive fibers with al-IR pal- lidal neurons, with mainly symmetric synapses. How- ever, electron microscopical studies have indicated that both pallidal neurons and cholinergic neurons of the forebrain receive GABA- and substance P-containing synaptic contacts (Bolam et al., 1986; Zaborszky et al., 1986; Ingham et al., 1988). Generally, subcellular local- ization of a1-IR in the ventral pallidum was similar to that obtained in the cerebellar cortex (Baude et al., 1992; Somogyi et al., 1989).

The distribution pattern of 1x1-IR obtained in this study may very well correlate with the localization of the GABAA receptor complex, since in the brain the a1 subunit is most widely codistributed with p2 subunit mRNA. Furthermore, this subunit composition colocal- izes very often with y2 mRNA (Pritchett et al., 1989; Wisden et al., 1992; Gutierrez et al., 1994). In most brain regions where high levels of expression of these specific subunit mRNAs for the GAB% receptor were observed, high densities of (3H)zolpidem binding sites were also found (Duncan et al., 19951, embodying the functional profile of benzodiazepine type I receptors (Duncan et al., 1995; Sigel et al., 1991).

In conclusion, SP-containing striatal efferents pre- ferentially innervate pallidal neurons containing the GABAA-receptor cil subunit, which conveys character- istics of a type I benzodiazepine receptor (Pritchett et al., 1989).

ACKNOWLEDGMENTS This work was supported by the Austrian Science

Foundation (grant SFB F 00206). We thank Professor Pavelka, Department of Histology, University of Inns- bruck, Austria, for providing the electron microscopic facilities.

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