Immunocytochemical localization of the ?2 subunit of the gamma-aminobutyric acidA receptor in the...

THE JOURNAL OF COMPARATIVE NEUROLOGY 350260-271 (1994)

Immunocytochemical Localization of the pz Subunit of the Gamma-Aminobutyric

Acid* Receptor in the Rat Brain

JUAN I. MORENO, MARTA A. PIVA, CELIA P. MIRALLES, AND ANGEL L. DE BLAS Division of Molecular Biology and Biochemistry, School of Biological Sciences,

University of Missouri-Kansas City, Kansas City, Missouri 641 10-2499

ABSTRACT An antiserum to the P 2 subunit of the rat gamma-aminobutyric acid (GABAA) receptor was

prepared by immunizing a rabbit with a fusion protein expressed in bacteria. The fusion protein had the large, intracellular loop expanding between the putative M3 and M4 transmembrane domains of the pz subunit fused to staphylococcal protein A (SP,4). The antiserum immunoprecipitated both the solubilized and the affinity-purified GABAA receptors. The anti-pz antibodies were affinity purified on immobilized P 2 intracellular loop peptide. The antibodies recognized a 55-57 kDa peptide in immunoblots of either crude membranes from rat cerebral cortex or affinity-purified GABAA receptors from bovine cerebral cortex. Immunocytochemistry with the affinity-purified antibody has revealed for the first time the localization of the Pa subunit in the rat brain. A comparative study of the regional and cellular immunoreactivities of the affinity-purified anti-P2 antibody and the monoclonal antibody 62-3G1 (which recognizes both Pz and P 3 subunits) is presented. The procedure described for generating and preparing specific anti-Pz subunit antibodies that are valuable for immunocytochemistry could be extended to other GABAA receptor subunits. o 1994 ~ i ~ e y - ~ i s s , Inc.

Key words: antibody, benzodiazepine receptor, fusion protein, GABAA receptor, immunopreeipitation

The brain gamma-aminobutyric acid type A (GABAA) receptor (GABAAR)/benzodiazepine receptor (BZDR) is a heteropentameric membrane protein (Nayeem et al., 1994) that forms a C1- channel. To date, six isoforms of CL subunit (al-ad, three of P (P1-P3), three of y (yl-y3), one of 6 (611, and two of p (pl and p2) have been cloned from mammalian brain (Schofield et al., 1987; Ymer et al., 1989; Olsen and Tobin, 1990; Burt and Kamatchi, 1991; Luddens and Wisden, 1991; Wilson-Shaw et al., 1991; Cutting et al., 1992; De Lorey and Olsen, 1992). All subunits have a long extracellular amino-terminal domain, four putative hydro- phobic transmembrane a-helix domains (Ml-M4), a large intracellular loop (IL) located between M3 and M4, and a short extracellular carboxy-terminal domain. We had previously made monoclonal (mAbs) and polyclonal antibodies to several subunits of the GABAAR/BZDR for studying the subunit composition and distribution of this receptor through the brain (Vitorica et al., 1987,1988; De Blas et al., 1988; Khan et al., 1993, 1994a,b; Miralles et al., 1994; Gutikrrez et al., 1995a,b). Our mAb 62-3G1 to the affinity- purified GABA*R/BZDR recognized an extracellular epitope that is present in the amino end of the pz and P3 subunits (p2/3) but not in p1 (Ewert et al., 1992). The mAb 62-3G1 has been used for the combined immunocytochemical localization of the Pz and p3 subunits of the GABAAR in

the rat brain (De Blas et al., 1988). In this communication, we report the making of a novel antiserum to the large intracellular loop of the Pz subunit (p21L) that recognizes the native GAEAAR. The affinity-purified antibodies have been used for the biochemical characterization and the immunocytochemical localization of the Pz subunit of the GABAAR. We have selected the PzIL as the immunogen, because the amino acid sequence of this region is highly subunit-specific (Ymer et. al, 1989; Olsen and Tobin, 1990). In addition, the conformational structure of the bacterially expressed pzIL is expected to be closer to that of the native receptor than to the synthetic peptides used by most groups for generating conventional antisera to pz. To the best of our knowledge, this is the first time that this approach has been used for raising antibodies aiming to localize GABAAR subunits by immunocytochemistry. The procedure presented in this communication could be of general use for making specific antibodies to other subunits that both immunoprecipitate the native brain GABAAR and are good immunocytoche mistry reagents.

Accepted June 16, 1994 Address reprint requests to Angel L. de Blas, PhD, Division of Molecular

Biology and Biochemistry, 109 Biological Sciences Building, University of Missouri-Kansas City, Kansas City, MO 64110-2499.

O 1994 WILEY-LISS, INC.

ANTIBODIES TO GABAA RECEPTORS 261

MATERIALS AND METHODS Materials

LN-methyl-3Hlflunitrazepam (FNZ; 20 Ciimmol) and [me- thylamine-3H1muscimol (71.4 Ci/mmol) were from Du Pont-New England Nuclear. The Ro7-198611 was a gener- ous gift form Dr. Norman Gilman of Hoffmann-La Roche, Inc. The rat PI, pz, and p3 cDNAs were from Dr. Peter Seeburg, University of Heidelberg, Germany. The pRIT33 plasmid (Nilsson and Abrahmsen, 1990) was from Dr. Lars Abrahmsen, Royal Institute of Technology, Stockholm, Sweden. Factor Xa was from Pierce.

Expression of SPA-PJL, SPA-P21L, and SPA-P3IL fusion proteins

The 374-bp DNA encoding the rat p21L was synthesized by the polymerase chain reaction using a rat pz cDNA as the template. Residues 13-39 of the forward oligonucleotide TCGTGGAATTCCAACTACATCTTCTTTGGGAGAGGA- CCC corresponded to nucleotides 1,055-1,081 of the rat pz mRNA (Ymer et al., 1989). The oligonucleotide primer also included an EcoRI site in the reading frame of pRIT33. Residues 17-40 of the reverse complementary oligonucleotide CGGGATCCCGTCATTACCGATCAATGGCGTTC- ACATCAGT corresponded to nucleotides 1,429-1,406 of the mRNA. It also contained a BamHI site and two stop codons. Therefore, the amplified DNA contained an EcoRI site at the 5' end and a BamHI site a t 3' end. These unequal termini ensured the right orientation of this fragment when it was subcloned into pRIT33. The staphylococcal protein A (SPA)-p21L genic construction was verified by DNA sequencing (Sanger et al., 1977). The transformation of Escherichia coli N4830-1 with pRIT33 was done according to Hanaham (1985). The thermoinducible expression of the fusion protein was done by growing the cells in LB medium at 28°C to 1.5-2.0 ODGo0. An equal volume of Terrific Broth (Tartof and Hobbs, 1987) at 56°C was added and incubated at 42°C for 1.5-2.0 hours.

A similar procedure was followed for SPA-p31L and SPA-PIIL expression. Residues 13-32 of the forward oligonucleotide CGTGGGAGCTCGAACTATATTTTCTTTG- GACG corresponded to nucleotides 1,052-1,071 of the rat p3 mRNA (Ymer et al., 1989). It also contained a Sac1 site in the reading frame of pRIT33. Residues 12-35 of the reverse complementary primer CGGGATCCTCATCTGTCTATG- GCATTCACATCGGT corresponded to nucleotides 1,420- 1,397 of the rat P3 mRNA. The primer also included a stop codon and a BamHI site. For SPA-PIIL expression, the forward oligonucleotide was TCGTGGAATTCCAATTA- CATTTTCTTCGGAAAAGGC, and the reverse complementary was TCGTGGAATTCCTTATCACTTGTCTATG- GAGTTCACGTCAGT. Residues 13-36 of the forward primer and 16-42 of the reverse complementary corresponded to nucleotides 1,059-1,082 and 1,432-1,406, respectively, of the rat mRNA (Ymer et al., 1989). The forward oligonucleotide also contained an EcoRI site in the reading frame of pRIT33, and the reverse complementary had a BamHI site and two stop codons. The expressed SPA fusion proteins consisted of an SPA moiety in the N terminal and an IL moiety (PIIL, p21L, or p31L) in the C terminal.

Purification of SPA-PlIL, SPA-PJL, and SPA-Q3IL fusion proteins

SPA-pIIL, SPA-pzIL, and SPA-p31L fusion-protein purification was done according to Martson et al. (1984) with

several modifications aiming to reduce proteolytic degradation. Briefly, a 4.5 g bacterial pellet (from 1.5 liters of culture) was vigorously suspended in 100 ml of 0.5% Triton X-100,100 mM EDTA in buffer A (50 mM Tris-HC1, pH 8.0, 50 mM NaCl), and immediately centrifuged at 14,OOOg for 5 minutes at 2°C. The pellet was stored at -70°C. The frozen bacteria were simultaneously thawed and suspended in 50 ml of 8 M urea, 100 mM EDTA, 20 mM EGTA, 1 mM phenylmethylsulfonyl fluoride (PMSF) in buffer A. The suspension was strongly stirred in an ice-water bath during 30 minutes, and 150 ml of ice-cold 100 mM potassium phosphate, pH 10.7, 100 mM EDTA, 1 mM PMSF was added, followed by vigorous agitation for 30 minutes. The suspension was centrifuged at 9,OOOg for 30 minutes at O'C, and the proteins present in the supernatant were precipi- tated by adding ammonium sulfate to a final concentration of 50% w/v. The protein was recovered by centrifugation at 9,OOOg for 30 minutes at O"C, and the pellet was dissolved in 50 ml of TST (50 mM Tris-HC1, pH 7.6, 150 mM NaC1, 0.05% Tween 20,20 mM EDTA) followed by centrifugation at 14,00Og, for 5 minutes at 0°C. The SPA-plIL, SPA-pzIL, or SPA-P31L fusion protein present in the supernatant was purified by affinity chromatography on I@-Sepharose 6 FF (Pharmacia-LKB). The sample was loaded onto a 3 ml column followed by washing with 150 bed volumes of TST and two bed volumes of 5 mM ammonium acetate, pH 5, at 4°C. The fusion protein was eluted with 0.5 M acetic acidiammonium acetate buffer, pH 3.4. The amount of fusion protein in the extract was normally larger than the capacity of the column, therefore, the same procedure was repeated using the same column with the flow-through until no more fusion protein could be purified. For immunizations, the eluate containing the SPA-P21L fusion protein was lyophilized and dissolved in phosphate-buffered saline (PBS).

Antibodies to SPA-PzIL fusion protein A New Zealand rabbit was immunized subcutaneously

with 1 mg of SPA-P21L fusion protein in complete Freund's adjuvant distributed in multiple injections sites. The following immunizations were done in incomplete Freund's adjuvant at 4-week intervals. The rabbit was bled the tenth day after each immunization, and the antibody titer was monitored by both enzyme-linked immunosorbent assay (ELISA) with immobilized pzIL and immunoprecipitation of the solubilized GABAAR from rat cerebral cortex membranes (Khan et al., 1993). The serum used in this study was collected after ten immunizations. It has been reported that the SPA moiety stimulates the immune response to SPA fusion proteins (Lowenadler et al., 1986).

Specific cleavage of the fusion proteins and purification of the PlIL, PJL,

and P3IL peptides To ensure that the fusion-protein preparation was free

from bacterial proteases, the affinity-purified SPA-PzIL fusion protein was purified one more time on the same IgG-Sepharose 6 FF column. The eluate was lyophilized and dissolved in 50 mM Tris-HC1, pH 8.3, 0.3 M NaCl, 6 mM CaClZ. Factor Xa was added at 1500 (enzyme: fusion protein) molar ratio, followed by incubation for 16 hours at 25°C. Factor Xa was inactivated at 65°C during 10 minutes, and the mixture was passed through an IgG-Sepharose 6 FF column. The latter retained the protein-A moiety of the cleaved fusion protein as well as the noncleaved fusion

262 J.I. MORENO ET AL.

Fig. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the staphylococcal protein A (SPA)-P2 intracellular loop (IL) fusion protein. Lane 1: Molecular weight marker proteins. Lane 2: Five micrograms of affinity-purified SPA-PJL fusion protein. Lane 3: Five micrograms of protein A. The 12% polyacrylamide gel was stained with Coomassie brilliant blue. The marker proteins are phos- phorylase b (94 m a ) , bovine serum albumin (67 m a ) , ovalbumin (43 kDa), carbonic anhydrase (30 m a ) , soybean trypsin inhibitor (20 kDa), a-lactalbumin (14.4 kDa).

protein. The nonretained p21L peptide was lyophilized and dissolved in water. The p31L and plIL peptides were purified following the same procedure.

Affinity purification of the antibodies One milligram of the affinity-purified P21L peptide was

immobilized in 3 ml of Affi-gel 10 (BioRad) according to the supplier's recommended procedure. A 2 ml sample of rabbit anti-SPA-PJL serum was passed twice through 1 ml of gel by gravity followed by washing with 150 bed volumes of 1 x TST and 10 vol of 0.2 x TST. The antibodies were eluted by a discontinuous pH gradient: pH 5, 4, and 3 (0.2 M acetic acid/ammonium acetate), pH 2 and 1 (0.2 M acetic acid/ HCl), and pH 0 (1 M HCl). The fractions were collected on 1 M Tris-HC1, pH 8.0, in ice-water bath, and the pH was quickly adjusted to 7.0 with NaOH. Bovine serum albumin (BSA) was added to a final concentration of 2 mgiml. The presence of antibodies in the different fractions was monitored by both ELISA with immobilized pzIL and immunoprecipitation of solubilized GABAAR.

Immunocytochemistry The procedure has been described elsewhere (De Blas,

1984; De Blas et al., 1984, 1988). Briefly, 60-day-old Sprague-Dawley rats were perfused under anesthesia (80 mgikg ketamine hydrochloride, 8 mgikg xylazine) with either periodate/lysine/4% paraformaldehyde/O. 1% glutar- aldehyde (PLP) fixative (McLean and Nakane, 1974) or Zamboni's fixative (15% aqueous solution of picric acid, 4%

n x

80 W

73 c 3 0 60 rn N

40 r- I

u 2 0 m

U I I I I

0 10 20 3 0

Antiserum (PI)

Fig. 2. Immunoprecipitation of the solubilized gamma-aminobutyric acid (GABAA) receptor from rat cerebral cortex with anti-SPA- PzIL antiserum. Inoreasingvolumes of serum wereused for immunopre- cipitating the solubilized receptor (1.7 mgiml of solubilized membrane protein in a final volume of 500 ~ 1 ) . The receptor present in either the pellets (solid circles) or the supernatants (open circles) was determined by [3Hlflunitrazepam (FNZ) binding. No immunoprecipitation was observed with the preimmune serum.

TABLE 1. Immunoprecipitation of the GABA,R/BZDR

Affinity -purified receptor from

bovine cerebral cortex'

Immuno- Ligand precipitation ('36)

PHIFNZ 88.2 k 4.2 13Hlmuscimol 89.7 ? 3.2

Solubilized receptor from rat cerebral cortex2

Cumulative immunoprecipitation ('36)

First Second Third

73.3 2 4.5 89.8 i 1.5 93.2 i 1.1 75.9 ? 3.9 86.5 f 3.4 93.4 i 0.2

'Immunoprecipitation was done with 20 ~1 of anti-P21L antiserum and 0.7 pmoles of affinity-purified receptor. Data are mean k SD of two values. "Each of the three sequential immunoprecipitations was done with 20 pl of anti-&IL antiserum. The second and third immunoprecipitations were done with the supernatant of the previous immunoprecipitation. Data are mean i SD of two values.

paraformaldehyde in 0.1 M phosphate buffer, pH 7.4). The brains were frozen and sliced (26 pm) with a freezing microtome. The free-floating tissue sections were incubated for 24 hours at 4°C with either mAb 62-3G1 culture medium diluted 100-fold in 0.3% Triton X-100, 0.1 M phosphate buffer, pH 7.4, or affinity-purified anti-P21L (0.11 pg/ml) diluted in the same buffer. The localization of the antibody bound to the tissue was revealed after incubation with a biotin-labeled secondary antibody followed by avidin-biotin-horseradish peroxidase complex (ABC procedure; Vector Laboratories) or by a peroxidase-antiperoxi- dase (PAP) technique. The immunoreactivity of the tissue sections with mAb 62-3G1 (which was raised by immunizing a BALB/c mouse with purified GABAA receptors; Vitorica et al., 1988) was displaced by incubating the mAb with affinity-purified GABAAR (De Blas et al., 1988). Simi- larly, immunoreactivity of the tissue sections with the anti-PzIL antibody was blocked with the purified PZIL peptide as shown below. However, a mixture of purified

263 ANTIBODIES TO GABAA RECEPTORS

A

Fig. 3. Cleavage of SPA-p,IL fusion protein with factor X,, purification of the p21L, and immunoblots with affinity-purified antibodies to pzIL. A: Thirteen percent SDS-PAGE stained with Coomassie brilliant blue. Lane 1: Molecular weight marker proteins. Lane 2: Control containing 5 kg of SPA-p21L fusion protein incubated with buffer in absence of factor Xa. Lane 3: One and a half micrograms of affinity- purified pzIL peptide after cleavage of SPA-pzIL with factor Xa. Lane 4: Eight micrograms of SPA-p21L fusion protein treated with factor Xa

PIIL and p31L peptides did not affect the immunoreactivity of the anti-P21L antibody with the tissue sections.

Other procedures Brain membranes from rat cerebral cortex were prepared

as described elsewhere (Mernoff et al., 1983). The Ro7- 19871 1 affinity-purified receptor from bovine brain was prepared as described by Siege1 et al. (1983). The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) was carried out according to Laemmli (1970). Immunoblotting of the protein expressed in bacteria was done according to Towbin et al. (1979). For blocking the Fc-mediated binding of the antibody to the SPA, the nitrocellulose filter was incubated with 2 mgiml of human IgG in TST for 1 hour at room temperature before adding the first antibody. Incubations with the antibodies were also done in the presence of 1 mg/ml of human IgG. Immunoblots of the Ro7-1987/ 1 affinity-purified receptor and membranes from cerebral cortex were done according to De Blas and Cherwinski (1983). The ELISA procedure was performed as described by De Blas et al. (1984b).

RESULTS Expression and purification of SPA-PzIL

fusion protein A denaturation-renaturation procedure that included

treatment of the Triton-washed bacteria with urea and alkaline buffer was the most successful for the extraction of the fusion proteins. Other standard extraction methods

(see Materials and Methods). Lane 5: Five micrograms of protein A incubated with factor Xa. B: Immunoblot with affinity-purified anti- bzIL antibodies. The gel shown in A was destained by repeated washes in 25 mM Tris-glycine, 20% methanol, and the protein bands were transferred to a nitrocellulose membrane. The blocking of the Fc- mediated antibody binding to SPA was done as indicated in Materials and Methods.

(such as sonication and freeze-thawing) were less successful in terms of both low yield and higher protein degradation. Figure 1 shows that the apparent molecular weight (46 kDa) of the purified fusion protein, was the expected one (46 kDa = 32.5 kDa of SPA + 14 kDa of PJL). The quality of our fusion proteins preparation was comparable to the best SPA fusion proteins expressed in E. coli (Monaco et al., 1987; Lundeberg et al., 1990; Nilsson and Abrahmsem, 1990) or in Staphylococcus aureus (Lowenadler et al., 1986; Moreno et al., 1991). The yield was 6-7 mg purified SPA-P21L fusion protein per liter of bacterial culture.

Immunoprecipitation of the GABA*/BZDR Figure 2 shows that approximately 80% of L3H1FNZ

binding activity of the solubilized GABAAR/BZDR from crude membranes could be immunoprecipitated with 20 p1 of anti-Pz1L antiserum. Receptor immunoprecipitation was totally blocked by adding purified PzIL peptide at a final concentration of 0.15 p M (not shown). This result indicated that the expressed peptide contained all the GABAARI BZDR epitopes recognized by the polyclonal antibodies. Table 1 shows that 93% of the solubilized receptors from crude membranes of rat cerebral cortex (determined by either [3H]FNZ or [3H]muscimol binding) could be precipi- tated with the anti-PzIL antiserum. Similar results were obtained with affinity-purified receptor from bovine cerebral cortex (Table 1). We have previously shown that maximal immunoprecipitation of the affinity-purified receptor could be accomplished with one single immunoprecipitation (Kahn et al., 1993, 1994). However, the quantification

264

of the solubilized GABAAR from crude membranes requires three sequential immunoprecipitations (Khan et al., 1993, 1994).

Purification of the p2IL The fusion protein contained a factor-Xa-recognition

amino acid sequence located between the SPA and the PzIL moieties. Therefore, it was cleaved by factor X, (Fig. 3A, lane 4). No endogenous proteolytic activity was detected after incubating the purified fusion protein in the absence of factor X, (Fig. 3A, lane 2). AEinity chromatography on IgG-Sepharose resulted in the purification of the pzIL peptide free of protein A (Fig. 3A, lane 3). In SDS-PAGE, the PzIL peptide migrated faster than the 14.4 kDa cu-lactalbumin marker. Nevertheless, the molecular weight of the p21L was determined to be 14 kDa by gel permeation HPLC (datanot shown). The yield was 1.0-1.5 mg of purified PzIL peptide per liter of bacterial culture.

Affinity purification of antibodies to p2IL and specific binding to p2IL peptide

Antibody purification from the antiserum was accomplished by affinity chromatography on immobilized PzIL peptide. The antibodies were eluted with a discontinuous pH gradient, and the activity of the fractions was monitored by both ELISA (using immobilized p21L peptide) and immunoprecipitation of the solubilized GABAAR. Two antibody peaks were collected at pH 3.4 and pH 2.3. The latter was used for immunocytochemistry and immunoblotting, because it showed the highest activity in ELISA and GABAAR immunoprecipitation. The specificity of the affinity-purified antibodies for the pzIL moiety of the fusion protein is also shown in Figure 3B. The affinity-purified antibodies reacted with pzIL (Fig. 3, lanes 2-4) but not with SPA (Fig. 3, lanes 43). The specificity of the affinity- purified anti-PzIL antibodies was also demonstrated by ELISA with immobilized PIIL, PJL, and p31L. The PIIL, pzIL, and PBIL were expressed as SPA fusion proteins, cleaved, and purified as described for p21L. The wells of ELISA plates were coated by incubation with 100 ng of the corresponding peptide. The bO5 for the wells coated with PzIL was 0.80 2 0.01, whereas those for the wells coated with PBIL and plIL were 0.15 +. 0.01 and 0.21 2 0.02, respectively.

Immunoblots with GABA*R/BZDR Figure 4A shows that the anti-P21L antiserum reacts

with a 55-57 kDa peptideb) of the affinity-purified GAB- AAR from bovine cerebral cortex. A peptide of similar mobility was also recognized by the mAb 62-3G1. Peptides of similar mobilities were also obtained in immunoblots of crude membranes from rat cerebral cortex (Fig. 4B) with the anti-pzIL antibody as well as with the mAb 62-3G1.

Immunocytochemistry In cerebellum, cerebral cortex, and inferior colliculus, the

affinity-purified anti-P21L antibody and the mAb 62-3G1 showed similar immunoreactivity (Fig, 5). In cerebellum, the immunoreactivity concentrated in the granule cell layer (Fig. 5A,C). We and others have previously shown that the GABAAR immunoreactivity is particularly concentrated in the glomeruli of the granule cell layer, which are rich in GABAergic synapses (De Blas et al., 1988; Somogyi et al., 1989). The binding of the anti-PzIL (0.11 kg/ml) to the tissue was blocked with 0.10 kg/ml of purified p21L peptide

A J.I. MORENO ET AL.

B

Fig. 4. Immun(ob1ot of purified GAB% receptors and brain membranes with anti-SPA-pZIL and monoclonal antibody (mAb) 62-3G1. A: Each strip contains the transfer of 0.7 pmoles of affinity-purified receptor from bovine cerebral cortex in 10 kg of protein. The antiserum to SPA-p21L (lane 1) and the mAb 62-3G1 culture medium (lane 2) wereused at 1:50 and 1:4 dilution, respectively. B: Each strip contained the transfer of 300 IJ-g of total membrane protein from rat cerebral cortex. The affinity-purified anti-p,IL antibody (lane 1) was used at a protein concentration of 1.4 pgiml, whereas the mAb 62-3G1 culture medium (lane 2) was used at 1:2 dilution. The SDS-PAGE was in 10% acrylamide.

(Fig. 5B). In cerebral cortex (Fig. 5D,E) and inferior colliculus (Fig. 5F,G), the immunoreactivity was strong in the neuropil through all layers (see also De Blas et al., 1988).

In the olfactory bulb, the anti-pzIL antibody (Fig. 6A) and the mAb 62-301 (Fig. 6C) showed similar immunoreactivity in all layers (glomerular, external plexiform, and mitral cell layer) except in the granule cell layer. Although mAb 62-3G1 labels most, if not all, granule cells, the anti-&IL did not bind to these cells (Fig. 6C,F vs. Fig. 6A,D,E). However, anti-P2IL specifically labeled short axon cells that are present in the granule layer. Some had prominent dendritic spines, and they resembled Blanes cells (Fig. 6D; Mori, 1987). The mAb 62-3G1 also labels short axon cells (Fig. 6F, arrow), although they are difficult to visualize due to the extensive labeling signal present on the more numer- ous granule cells. Figure 6B shows that the anti-PzIL immunoreactivity was blocked with purified PzIL peptide.

Contrary to the case with mAb 62-3G1, which strongly labeled the hippocampus and dentate gyrus (Figs. 7B, 8D, respectively), the anti-p21L gave a weak reaction with these structures in tissue sections processed at the same time with the same reagents under identical conditions (Figs. 7A, 8B). In the hippocampus, the mAb 62-361 strongly labeled the neuropil of the stratum oriens (SO), stratum radiatum (SR), and stratum lacunosum (SL), in that order (Fig. 7B). In contrast, the anti-P21L antibody gave only a


Fig. 5. Immunocytochemistry with anti-pzIL (A,B,D,F) and mAb 62-3G1 (C,E,G) of cerebellum (A-C), cerebral cortex (D,E), and inferior colliculus (F,G). Note the similar and strong immunoreactivities of anti-&IL and mAb 62-3G1 in these brain regions. B is a control

showing displacement with purified PzIL peptide. Parasagittal sections. G, granule cell layer; M, molecular layer; P, Purkinje layer; WM, white matter. Scale bar = 77 pm.

faint reaction with the neuropil of these layers (Fig. 7A). Nevertheless, individual cells and processes were strongly labeled in these layers, including nonpyramidal neurons of the stratum pyramidale (Fig. 7A,C-E). Similar cells were also strongly labeled with mAb 62-3G1 (Fig. 7B; De Blas et al., 1988). Weaker labeling was also detected in pyramidal neurons, particularly the ones located next to the stratum oriens. In the dentate gyrus (Fig. 8A-C), the cells that were strongly labeled with anti-p21L were mainly interneurons of the molecular layer as well as the basket neurons that have the soma located in the boundary between the granular cell layer and the plexiform layer. The labeling was present in both cell bodies and processes (Fig. 8A,B). Cell bodies and processes of hilar neurons were also labeled (Fig. 8C). Similar cells were also labeled with mAb 62-3G1 in all the layers of the dentate gyrus, although they were more difficult to visualize in the molecular layer due to the strong neuropil staining of this layer with the mAb 62-301 (Fig. 8D; De Blas et al., 1988). Low levels of staining with anti-PzIL were detected on the granule cells (Fig. 8).

Identical immunocytochemical results through the brain were obtained when the brain sections and the anti-p21L antibody (0.11 p,g/ml) were incubated in the presence of a mixture (0.10 p,g/ml each) of plIL and p31L peptides (not shown). However, the binding of the antibody to all regions of the brain was blocked with 0.10 ygirnl of purified pzIL peptide.

DISCUSSION We and another group have previously made monoclonal

antibodies that recognize both p2 and p3 subunits of the GABAAR (Haring et al., 1985; Vitorica et al., 1988) after immunizing mice with affinity-purified GABAAR. Both monoclonal antibodies, bd 17 and 62-3G1, recognize a similar extracellular epitope located at the amino end of the protein that is present in both pz and p3 but not in (Ewert et al., 1990, 1992). The two mAbs immunoprecipitate the GABAARiBZDR complex (Haring et al., 1985; Vitorica et al., 1988, 1990; Khan et al., 1993) and have been

Figure 6

ANTIBODIES TO GABAA RECEPTORS

used for the localization of GABAAR in the rat brain by immunocytochemistry (Richards et al., 1987; De Blas et al., 1988). More recently, antisera to PI, pz, and p3 synthetic peptides have been made by others (Buchstaller et al., 1991; Pollard et al., 1991; Endo and Olsen, 1992; Machu et al., 1993). The antipeptide antiserum to p1 also recognizes pz and p3 and did not immunoprecipitate the native GABAAR (Endo and Olsen, 1992). Two antipeptide antisera to p2 and p3, respectively (Buchstaller et al., 19911, did not precipitate well the native solubilized GABAAR (only 7-17%). A different anti-p3 peptide antiserum was able to precipitate 49% of the native receptors after two successive immunoprecipitations (Pollard et al., 1991). Moss et al. (1992) have made an antiserum to glutathione-S-transferase (GST)-PIIL fusion protein. Nevertheless, no immunoprecipitation activity of the native GABAAR with this antiserum or another anti-p2 peptide antiserum (Machu et al., 1993) has been reported. Furthermore, none of these anti-p subunit antibodies has been shown to be valuable reagents for immunocytochemistry with only one exception: Immunocytochemistry with an anti-pl antibody to a synthetic peptide has been reported recently (Gu et al., 1993). Nevertheless, no immunoprecipitation of the native receptor by this antibody was reported. In the present communication, we are presenting the development of an antibody to pzIL that 1) recognizes pzIL protein expressed in bacteria, 2) reacts with the pz subunit in immunoblots of both solubilized and purified GABAAR, 3) immunoprecipitates the native solubilized receptor, and 4) is a valuable reagent for the immunocytochemical localization of the p2 subunit in the brain. The specificity of the anti-pzIL antibodies for the pz subunit together with the specificity of the mAb 62-3G1 for pz and p3 has allowed us to reveal for the first time the immunocytochemical localization of the pz subunit and to resolve it from the localization of p3 in the rat brain. Antibodies to the IL of several GABAAR/BZDR subunits other than pz and p3 have been made by others, although no immunocytochemistry has been reported with these antibodies (McKernan et al., 1991; Moss et al., 1992).

Our anti-P21L antibody was able to precipitate around 90% of both [3H]FNZ and [3H]muscimol binding activity of the solubilized GABAA receptor from either rat cerebral cortex membranes or affinity-purified GABAAR from bovine cerebral cortex. Similar high immunoprecipitation values of [3H]FNZ and [3Hlmuscimol binding were obtained with mAb 62-3G1 (data not show) that were comparable to values reported elsewhere (Khan et al., 1993).

Immunoblots with anti-pzIL antibody and mAb 62-3G1 show a main peptide band(s) of 55-57 kDa reacting with both antibodies in two different receptor preparations (crude membranes and affinity-purified receptor). These results are consistent with our previous studies with mAb

267

Fig. 6. Olfactory bulb immunocytochemistry with anti-PzIL (A,B,D,E) and mAb 62-3G1 (C,F). The immunoreactivities of anti-P,IL and mAb 62-3G1 are similar except in the granular (GR) cell layer (Avs. C). D shows short axon cells, probably Blanes cells, whereas E shows the mitral cell layer separating the EP layer (left) and the GR layer (right). The latter also shows an immunoreactive short axon cell. The arrow in F shows a stained short axon cell on a background of stained granule cells. B is a control showing displacement with purified pzIL peptide. Parasagittal sections. EP, external plexiform layer; GL, glomerular layer; GR, granule cell layer; M, mitral cell layer. Scale bars = 77 bm in A-C, 19 km in D,E, 38 bm in F.

62-3G1 (Vitorica et al., 1988; Park and De Blas, 1991; Park et al. 1991).

The specificity of the anti-p21L antibody for the pz subunit was also demonstrated by 1) the low immunoreactivity of anti-pzIL with immobilized plIL and p31L peptides in ELISA and 2) immunocytochemistry (i.e., low immunoreactivity with the hippocampus, which is known to have high levels of p1 and p3 mRNAs but low levels of pz). The high specificity of the antibody (also shown by immunocytochemistry after incubating the anti-P21L with the brain tissue in the pressure of plIL, pzIL, or p31L peptides) was somewhat surprising, because there are amino acid sequences common to the IL of the three f3 subunits (Table 2). Neverthe- less, there are several possible explanations: The amino and carboxy ends (14 and 24 amino acids, respectively) of the IL near M3 and M4, respectively, are similar in the three p subunits. They occupy around 30% (11% and 19%, respectively) of the IL sequence. Therefore, the specificity that the anti-p21L antibody shows for pzIL indicates that these two common regions are not immunogenic and/or that they are inaccessible to the antibodies both in the native receptor (immunocytochemistry) as well as in the purified plIL, pzIL, and p31L peptides as shown by ELISA. These two regions of the IL, which contain amino acid sequences common to the three subunits, are located next to the membrane (M3 and M4), and they might not be accessible to the antibodies. Therefore, it is likely that the specificity of the anti-p21L for the native p2 subunit (vs. p1 and p3) in the immunocytochemistry assay is even larger than the specificity of this antibody for the bacterially expressed p21L peptide (vs. plIL and p31L) in ELISA. Fifty-seven percent of the IL is predicted to be antigen-specific, because it contains fewer than three amino acids in sequence that are common to PI, p2, and p3. A linear amino acid epitope is normally formed by five to seven consecutive amino acids. The subunit-specific region of &IL is formed by three specific sequences of 24, 25, and 22 amino acids (underscored in Table 2) separated by two common sequences of nine and six amino acids, respectively. These two centrally located regions, common to the three p subunits, together occupy only 12% of the IL. They might or might not be immunogenic and/or be hidden in the native receptor. In any case, they are small compared to the size of the Pz-specific regions (12% vs. 57% of the IL). This linear epitope analysis does not take into consideration conformational epitopes, which also might increase the immunological differences among the IL of the p subunits.

The immunocytochemistry data presented in Figures 5-8 have revealed the regional and cellular localization of the p2 subunit of the GABAAR in the rat brain. The immunoreactivity was localized on neuronal somas and processes but not on glial cells. Our results are also consistent with the localization by in situ hybridization of the mRNAs encoding the three p subunits (Zhang et al., 1990, 1991; Laurie et al., 1992; Pershon et al., 1992; Wisden et al., 1992). The mAb 62-3G1 to pZl3, but not PI, and the anti-pzIL antibody show similar patterns of immunoreactivity in cerebellum, cerebral cortex, and inferior colliculus (Fig. 5). These findings are consistent with the abundant expression of pz and p3

mRNAs in cerebellar granule cells. The latter express low levels of p1 mRNA. The neurons of the cerebral cortex express high levels of 6 2 and p3 (and considerably less p1) mRNA through all layers. The neurons of the inferior colliculus express pz mRNA in abundance but very little p3

or pl. In the olfactory bulb, the mAb 62-3G1 and the


Fig. 7. Hippocampus immunocytochemistry with anti-P21L and SR. Parasagittal sections. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bars = 77 Frn in A,B, 38 Fm in C,D, and E.

(A,C,D,E) and mAb 62-3G1 (B). Note the low level of immunoreactivity of the hippocampus with the anti-pzIL vs that with mAb 62-3G1 under the same assay conditions. The latter is particularly strong in the SO


Fig. 8. Dentate gyrus immunocytochemistry with anti-P21L (A-C) and mAb 62-3G1 (D). Note the low immunoreactivity of the dentate gyrus with anti-p21L (A-C) compared to mAb 62-3G1 (D) under the same assay conditions. A detailed description of the mAb 62-3G1

immunocytochemistry in the dentate gyrus has been presented elsewhere (De Blas et al., 1988). Parasagittal sections. G, granule cell layer; H, hilus; M, molecular layer; P, plexiform layer. Scale bars = 38 pm in A-C, 77 p,m in D.

TABLE 2. Amino Acid Sequence of the Large Intracellular Loop of the Rat PI, p2, and p3 Subunits'

PiIL 303 NYIFFGKGPQ KKGASKQDQSANEKNKLEMNKVQVDAHGNILLSTLEI RNET S GSEVLT GVS DPKATMYSYDS PzIL 303 NYIFFGRGPQRQKKAAEKAANANNEKMRLDV NKM DPHENILLSTLEI KNEMA T SEAVMGLGDPRSTMLAYDA P,IL 303 NYIFFGRGPQRQKKLAEKTAKAKNDRS KS EI NRV DAHGNILLAPMDVHNEM NEVAGS VGDTRNSAI SFDN

p,IL 374 A S IQYRKPLS S R E G F G R GLDRHGVPGKGRI RRRAS QLKVKIPDLTDVNS I DK PzIL 374 PiIL 372

S S IQYRKAGL P RHS FGRNA LERHVAQKKS RL RRRAS QLKI TIPDLTDVNAI DR S GIQYRKQSMP KEGHGRY MGDRSI PHKKTHL RRRS S QLKI KIPDLTDVNAI DR

'Boldface letters represent amino acid residues that are only present in pJL. The regions of the PnlL that are predicted to be antigenically specific are underscored. In these regions, the amino acid sequences that are also present in p1 and p3 are shorter than four residues. The nonunderscored regions share common linear epitopes. The amino acid sequences are from Ymer et al. (1989).

anti-P21L show similar binding distribution in all layers except in the granule cell layer. In the latter, the anti-pzIL does not label granule cells but specifically labels short axon neurons. This distribution is also consistent with the in situ

hybridization studies that have revealed high levels of p2

and p3 mRNAs in periglomerular, tufted, and mitral cells. However, the granule cells do not express pz mRNA, although they do express high levels of p3 mRNA. The


mRNA is expressed in mitral cells but not in periglomerular, tufted, or granule cells. In addition, Zhang et al. (1990) and Laurie et al. (1992) have suggested that the scattered cell labeling observed by in situ hybridization in the granule cell layer with p2 probes probably reflects the presence of pz mRNA in short-axon neurons. Our results (Fig. 6D) con- firm this hypothesis. The immunocytochemical procedure has much higher resolution than the in situ hybridization technique for neuron identification. In addition, we have shown that the p2 subunit is present in both the soma and the spiny dendrites of the short axon Blanes neurons.

The weak reactivity of anti-&IL antibody with hippocampus and dentate gyrus is also consistent with the low level of the p2 mRNA expressed by the pyramidal and granule cell neurons (Persohn et al., 1992; Wisden et al., 1992). How- ever, the same in situ hybridization studies have shown that the p1 and p3 mRNAs are abundantly expressed in these two cell types, which shows once more the specificity of the anti-P21L antibody for the p2 subunit in immunocytochemistry. In addition, the high expression of p3 subunits in the hippocampus and dentate gyrus explains the high immunoreactivity of these structures with the mAb 62- 3G1. Therefore, the low levels of p2 mRNA found in pyramidal and granule cells is translated in low levels of pz protein. Strong anti-P21L immunoreactivity was found on nonpyramidal cells of the stratum pyramidale of the hippocampus as well as in interneurons of the stratum oriens and stratum moleculare (Fig. 7A,C-E). In situ hybridization studies have shown that cells in striatum oriens express p2 but neither PI nor p3 (Persohn et al., 1992). The interneurons of the molecular layer of the dentate gyrus as well as the ones in the plexiform layer and the basket cells (located in the boundary between molecular layer and plexiform layer) were also strongly immunoreactive with anti-p21L antibody as well as the hilar neurons (Fig. 8C,D). Most likely, the strongly labeled processes revealed with anti- pzIL in the stratum oriens, radiatum and moleculare of the hippocampus as well as in the molecular and plexiform layers of the dentate gyrus and in the hilus are processes from these interneurons. These interneurons were also labeled with mAb 62-3G1 (Figs. 7B, 8D). We and others have previously shown that these interneurons are GABAer- gic (Ribak et al., 1978, 1986; Seress and Ribak, 1983; Somogyi et al., 1983; Gamrani et al., 1986; De Blas et al., 1988).

We have taken advantage of the recent cloning of the GABAAR subunits and molecular biology techniques for the preparation of a novel and specific anti-Pz antibody that is valuable for GABAAR immunocytochemistry. This anti-Pz antibody was obtained by immunizing rabbits with SPA- p21L fusion protein followed by affinity purification on immobilized p21L. To the best of our knowledge, this is the first time that this approach has been used for raising antibodies aiming to localize GABAAR subunits by immunocytochemistry. The subunit specificity shown by the anti- PZIL antibody in immunocytochemistry suggests that a similar procedure could be used for obtaining other subunit- specific antibodies that are valuable for GABAAR immunocytochemistry. We are currently using this approach for the preparation of other GABAAR subunit-specific antibodies.

ACKNOWLEDGMENTS We thank Dr. Peter Seeburg for his gift of rat pz and p3

cDNAs. This research was supported by grant NS17708

from the National Institute of Neurobiological Disorders and Stroke.

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