Immunocytochemical localization of GABAB receptors in mesencephalic trigeminal nucleus neurons in the rat

Neuroscience Letters 315 (2001) 93–97 www.elsevier.com/locate/neulet

Immunocytochemical localization of GABAB receptors in mesencephalic trigeminal nucleus neurons in the rat Jin-Lian Li a, Ryuichi Shigemoto b, Akos Kulik b, Peng Chen a, Sakashi Nomura c, Takeshi Kaneko d, Noboru Mizuno e,* a

Department of Anatomy and K. K. Leung Brain Research Center, The Fourth Military Medical University, Xi’an 710032, P.R. China b Division of Cerebral Structure, National Institute for Physiological Science, Okazaki, Aichi 444-8585, Japan c College of Medical Technology, Kyoto University, Kyoto 606-8507, Japan d Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan e Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo 183-8526, Japan Received 13 September 2001; accepted 20 September 2001

Abstract We examined immunoreactivities for g -aminobutyric acidB-receptor (GABABR) subtypes, GABABR1 and GABABR2, in the mesencephalic trigeminal nucleus neurons (MTN neurons) of the rat. Immunoreactivity for GABABR1 was prominent in cell bodies of MTN, whereas that for GABABR2 was very weak, if existed. For electron microscopy, the immunogoldsilver method for GABABR1 was combined with the immunoperoxidase method for glutamic acid decarboxylase (GAD: the synthetic enzyme of GABA). Immunogold-silver particles indicating GABABR1 immunoreactivity were distributed widely in the cytoplasm of the cell bodies postsynaptic to GAD-immunoreactive axon terminals, but were rarely associated with synaptic membrane specialization or extrasynaptic sites of plasma membrane. It has been indicated that GABABR1 may not be transported to plasma membrane when no GABABR2 exists. Thus, it was presumed that GABABR1 in the cell body of the rat MTN neurons might not be involved in the synaptic transmission. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Mesencephalic trigeminal nucleus; g -aminobutyric acidB-receptor; Glutamic acid decarboxylase; Immunohistochemistry; Electron microscopy; Rat

The neurons of the mesencephalic trigeminal nucleus (MTN) have been considered to be sensory ganglion neurons that are concerned chiefly with the muscle spindles of the jaw-closing muscles and the mechanoreceptors of the periodontal ligament. They are mostly pseudo-unipolar, though some bipolar or multipolar cells have been observed (for further review see Ref. [14]). A small number of synaptic terminals have also been observed on their cell bodies (for review, see Ref. [14,15]). In the rat, the presence of GABA (g -aminobutyric acid)ergic axon terminals on the cell bodies of MTN neurons has been indicated electron microscopically [2] as well as light microscopically (for review, see Ref. [14]). The cell bodies of the rat MTN neurons were also indicated to express receptors for GABA, both ionotropic GABA receptors * Corresponding author. Tel.: 181-42-335-3881; fax: 181-42321-8678. E-mail address: [email protected] (N. Mizuno).

(GABAAR) [7,9,18] and metabotropic GABA receptors (GABABR) [3,4,16]. On the other hand, however, the intracellular recording study in rat brain slice preparations indicated that the responses of the MTN neurons to application of GABA were mediated exclusively through GABAA receptors [9]. Thus, in the present study, we re-examined immunoreactivities for GABAB-receptor subtypes (GABABR1 and GABABR2) in the cell bodies of the rat MTN. All procedures of the experiments were approved by the Animal Care and Use Committees at the Graduate School of Medicine, Kyoto University. An antibody against a TrpE fusion protein containing a Cterminal sequence of GABABR2 was produced. A BamHIHindIII fragment encoding amino acid residues 844-892 of rat GABABR2 was prepared by polymerase chain reaction and subcloned into a bacterial expression vector, pATH3, as described previously [19]. The fusion protein was induced and purified by preparative SDA-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The excised

0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 32 1- 7

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gel band was crushed and homogenized with equal volume of complete Freund’s adjuvant. Rabbits were subcutaneously injected with 0.5 mg of the fusion protein, boosted every 4 weeks and bled 2 weeks after each boost. The IgG fraction purified from the collected serum with a protein A column was absorbed with an unfused TrpE protein to remove antibodies reactive to non-GABABR2 portions of the fusion protein. An antibody specific for the GABABR2 portion was then affinity purified with a Sepharose 4B column coupled with the TrpE-GABABR2 fusion protein. Immunoblot analysis was performed as described previously [19]. The crude membrane preparations from rat whole brain, and COS cells transfected with cDNAs for GABABR1a, GABABR1b and GABABR2, were separated by 7.5% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF; BioRad, Hercules, CA) membranes. The membranes were blocked with Block-Ace (Dainippon Pharmaceutical, Osaka, Japan) and then reacted with the affinitypurified GABABR2 antibody (1.0 mg/ml). An alkaline phosphatase-labeled secondary antibody (Chemicon, Temecula, CA) was used to visualize the reacted bands. Twelve adult male Wistar rats (Japan SLC, Shizuoka, Japan) weighing 200–250 g were used. The rats were anesthetized by intraperitoneal injection of an overdose of chloral hydrate (70 mg/100 g body weight). For light microscopic study, the anesthetized rats were perfused transcardially as described elsewhere [19]. The fixation solution was 0.1 M Na2HPO4 containing 4% (w/v) paraformaldehyde, 0.05% glutaraldehyde and 15% (v/v)-saturated picric acid, and was adjusted to pH 7.3 with NaOH. The lower brainstem parts containing the MTN were cut serially into frontal sections 40 mm thick on a freezing microtome after cryoprotection with 30% (w/v) sucrose in 0.05 M phosphate buffer (PB; pH 7.3) overnight at 48C. The sections were processed for GABABreceptors (GABABR1 or GABABR2) immunohistochemistry. Briefly,

Fig. 1. Immunoblot analysis of rat brain and receptor-expressing cells. Crude membrane preparations from the rat whole brain, and COS cells transfected with cDNAs for GABABR1a (COS/R1a), GABABR1b (COS/R1b), and GABABR2 (COS/R2), were subjected to 7.5% SDS-PAGE and transferred onto PVDF filters. The filters were reacted with an antibody to GABABR2 (B232). Positions of molecular mass markers (Bio-Rad) in kDa are indicated on the right.

the sections were incubated at room temperature sequentially with: (1) 1 mg/ml of affinity-purified rabbit antiGABABR1 [11], or the newly prepared rabbit GABABR2 antibody, overnight; (2) 10 mg/ml of biotinylated anti-rabbit goat IgG (Vector, Burlingame, CA) for 3 h; (3) avidin-biotinylated peroxidase complex (ABC-Elite; Vector Laboratories, Burlingame, CA), which was diluted 1:50 in the incubation medium, for 3 h. The incubation medium was prepared by using 0.05 M PBS containing 0.5% (v/v) Triton X-100, 0.25% (w/v) l-carrageenan, 0.05% (w/v) NaN3 and 5% (v/v) normal goat serum in step (1) and (2), and by using 0.05 M PBS containing 0.3% (v/v) Triton X-100 in step (3). Subsequently, the sections were reacted with 0.02% (w/v) 3,3 0 -diaminobenzidine tetrahydrochloride and 0.002% (v/v) H2O2 in 0.05 M Tris–HCl buffer (pH 7.6). For electron microscopic study, the anesthetized rats were perfused transcardially with 0.1 M PB (pH 7.4) containing 4% (w/v) paraformaldehyde, 0.1% glutaraldehyde and 15% (v/v)-saturated picric acid. The lower brainstem was removed immediately and placed in 0.1 M PB (pH 7.3).

Fig. 2. Photomicrographs of adjacent two sections through the MTN. The cell bodies of MTN neurons are GABABR1-immunopositive (a), but GABABR2-immunonegative (b). Scale bar, 50 mm.

J.-L. Li et al. / Neuroscience Letters 315 (2001) 93–97

The brainstem part containing the MTN were cut into frontal sections 50 mm thick on a vibratome (Microslicer; Dosaka, Kyoto, Japan), and placed in 0.05 M PB containing 25% sucrose and 10% (v/v) glycerol for 1 h for cryoprotection. Then, the section were freeze-thawed with liquid nitrogen for enhancement of penetration of antibodies in the following immunocytochemical reaction. Subsequently, the sections were placed in 50 mM Tris-buffered saline (TBS; pH 7.4) containing 20% normal goat serum for 1 h to block non-specific immunoreactivity. For electron microscopic double-immunocytochemistry, the immunogold-silver method for GABABR1 was combined with the immunoperoxidase method for glutamic acid decarboxylase (GAD). The sections were incubated with a mixture of affinity-purified rabbit anti-GABABR1 antibody [11] and mouse anti-GAD IgG (Chemicon), each was diluted to 1 mg/ml in TBS containing 2% (v/v) normal goat serum (TBS-G) for 24 h at room temperature. Then, the sections were washed in TBS and further incubated with a mixture of biotinylated anti-mouse IgG donkey antibody (Jackson) and anti-rabbit IgG goat antibody conjugated to 1.4 nm gold particles (Nanoprobes; Stony Brook, NY), each was diluted at 1:100 in the TBS-G, overnight at room

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temperature. Subsequently the sections were processed for: (1) postfixation with 1% glutaraldehyde in 0.1 M PB for 10 min; (2) silver enhancement with HQ Silver Kit (Nanoprobes); (3) incubation with ABC Kit (Vector) diluted at 1:50 in 50 mM TBS for 3 h at room temperature; (4) visualization of GAD-immunoreactivity by incubation with 0.05 M Tris–HCl (pH 7.6) containing 0.02% diaminobenzidine tetrahydrochloride and 0.003% H2O2 for 20–30 min at room temperature; (5) osmification with 1% OsO4 in 0.1 M PB for 1 h; (6) counterstaining with 1% (w/v) urany1 acetate for 1 h; and (7) flat-embedding in Durcupan (Fluka, Buchs, Switzerland). Ultrathin sections containing the MTN were prepared and examined electron microscopically as described elsewhere [2,15]. The reactivity and specificity of the affinity-purified antibody to GABABR2 (B232) was verified by immunoblot analysis (Fig. 1). One immunoreactive band with the estimated molecular weight of 110 kDa [11] was detected with the B232 antibody in the brain. In membrane fractions prepared from receptor-expressing COS cells, the B232 antibody reacted specifically with GABABR2, showing no cross-reactivity to GABABR1a or GABABR1b. GABABR1 immunoreactivity was seen in large, medium-

Fig. 3. Electron micrographs showing GABABR1 immunoreactivity (immunogold silver grains) of a cell body of a MTN neuron, with which two GAD immunopositive axon terminals showing GAD immunoreactivity (electron-dense, peroxidase immunoreaction product) are in symmetric synaptic contact. No immunogold silver grains are associated with synaptic specialization. The rectangles in (a) is enlarged in (b) and (c); arrowheads indicate the synaptic zones in the postsynaptic cell body. Scale bar, 0.6 mm for (a); 0.25 mm for (b) and (c).

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sized and small cell bodies of MTN neurons; a substantial number of MTN neurons appeared to be GABABR1-immunopositive (Fig. 2a). On the other hand, no definite immunoreactivity for GABABR2 was detected in the cell bodies of the MTN neurons (Fig. 2b). Weak immunoreactivities for both GABABR1 and GABABR2 were also seen diffusely within neuropil of the MTN (Fig. 2). In the control experiments, when the primary antibodies were omitted or replaced with normal IgG, no immunoreactivity for the omitted or replaced antibody was found. In the electron microscopic experiments, somatic profiles of MTN neurons were labeled with immunogold-silver grains indicating GABABR1 immunoreactivity; many of them were in synaptic contact with axon terminals labeled with electron-dense reaction products (flocculent precipitates) of the GAD immunocytochemistry (Fig. 3). The GABABR1 immunogold-silver particles were distributed widely in the cytoplasm of the MTN neurons; most of them were associated with membranous structures including the endoplasmic reticulum. However, these particles were rarely associated with synaptic specialization or extrasynaptic sites of plasma membrane (Fig. 3). According to the study in brain slice preparations, the depolarizations observed in the rat MTN neurons in response to GABA application were mediated exclusively through GABAAR [9]. On the other hand, however, the MTN neurons of the rat were reported to show intense immunoreactivity for GABABR1 [16]. This was confirmed in the present study. In the present study, GABABR1 were localized electron microscopically in the cytoplasm of the perikarya, as reported in the monkey striatum [1] and the rat visual cortex [8]. However, the present results further indicated that GABABR1 were rarely associated with synaptic specialization or extrasynaptic sites of plasma membrane, although the previous reports indicated the localization of GABABR1 on the extrasynaptic sites of the plasma membrane in the other brain regions of the rat [6,12]. It has been indicated that co-assembly of GABABR1 and GABABR2 subunits is required to provide functional GABABR receptor; when GABABR2 does not exist, GABABR1 may not be transported to the plasma membrane [5,10,11,13,20] (for further review, see Ref. [17]). The presence of GABABR2 mRNA in the rat MTN has also been reported in the in situ hybridization study [4]. However, in the present study, GABABR2 immunoreactivity in the cell bodies of the MTN neurons was very weak, if it existed. GABABR2 in the MTN neurons might be expressed mainly in axon terminals. GABABR1 in the cell bodies without GABABR2 subunit might not be transported to the plasma membrane and therefore may not be involved in the synaptic transmission. This work was supported in part by Grants-in-Aid from the National Natural Science Foundation of China (39870262, 39970239), from the Foundation for University

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