Cellular and subcellular localization of γ-aminobutyric acidB receptors in the rat olfactory bulb

Neuroscience Letters 274 (1999) 195±198 www.elsevier.com/locate/neulet

Cellular and subcellular localization of g -aminobutyric acidB receptors in the rat olfactory bulb Michela Bonino, Dario Cantino, Marco SassoeÁ-Pognetto* Department of Anatomy, Pharmacology and Forensic Medicine, Corso Massimo d'Azeglio 52, I±10126 Turin, Italy Received 21 June 1999; received in revised form 30 August 1999; accepted 31 August 1999

Abstract Olfactory nerve axons terminate in rounded regions of the olfactory bulb, termed glomeruli, where they make excitatory synapses with the dendrites of second-order neurons. Neurotransmission from the olfactory nerve to the postsynaptic targets is negatively regulated by g -aminobutyric acid (GABA), and there is evidence that inhibition of sensory input is mediated, at least in part, by GABAB receptors. Using an antiserum that recognizes two GABAB receptor splice variants (GBR1a and GBR1b), we show here that GABAB receptors are located on the axon terminals of the olfactory nerve, where they are concentrated at sites of axodendritic apposition. Taken with previous data, these results indicate that GABAB receptors act presynaptically to regulate the release of glutamate from olfactory nerve terminals. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Olfactory glomeruli; Olfactory nerve; Presynaptic inhibition; Extrasynaptic receptors; g -Aminobutyric acid; Metabotropic receptor; Immunocytochemistry

The glomerular layer of the olfactory bulb is the ®rst stage of synaptic integration in the olfactory pathway. Olfactory nerve (ON) axons terminate in the glomerular neuropil, where they make excitatory synapses with the dendritic tufts of both the principal neurons (mitral and tufted cells) and the intrinsic neurons (periglomerular cells). Many periglomerular cells are GABAergic, and it is generally accepted that g -aminobutyric acid (GABA) exerts considerable control over sensory inputs to secondorder neurons [16]. There is evidence that intraglomerular inhibition is mediated, at least in part, by GABAB receptors. It has been shown by radioligand binding that GABAB receptors are highly expressed in the olfactory glomeruli [1], and activation of these receptors by baclofen inhibits transmission at the ®rst olfactory synapse [15]. Although experimental data indicate that GABAB receptors are probably involved in presynaptic inhibition of ON terminals (P. Duchamp, personal communication; and Ref. [11]), the precise localization and function of these receptors within the glomerular synaptic circuits have not been determined. Recent expression cloning of GABAB receptors from a rat cDNA library [8] has provided new molecular probes to * Corresponding author. Tel.: 139-011-670-7725; fax: 139-011670-7732. E-mail address: [email protected] (M. SassoeÁ-Pognetto)

analyze their distribution. GABAB receptors appear to be present in two subtypes (GBR1, further subdivided in two splice variants, GBR1a and GBR1b, and GBR2), that are probably associated as heterodimers [6,9,13,18]. In the present study, the cellular and subcellular localization of GABAB receptors was investigated in the rat olfactory bulb with a polyclonal antibody (Chemicon, AB1531) that recognizes the C-terminal sequence of both GBR1 isoforms. For light-microscopic immunocytochemistry, we used a sensitive protocol that enhances the detection of neurotransmitter receptors [4]. The antiserum AB1531 was diluted 1:8000. Double labeling was performed by using a mixture of AB1531 and a monoclonal antibody against tyrosine hydroxylase (TH; Incstar, 1:1000). For electron microscopy, pre-embedding immunocytochemistry was done as described elsewhere [4]. After perfusion with 4% paraformaldehyde, the olfactory bulbs were cut with a Vibratome, and the sections were incubated in the primary and secondary antibodies. GABAB receptor immunoreactivity was visualized with 3,3 0 -diaminobenzidine, and the reaction product was silver intensi®ed and gold toned. Immunolabel for GABAB receptors was most prominent in the glomerular neuropil, where it exhibited a typical patchy appearance (Fig. 1A). Careful examination of the sections revealed that the immunoreactivity was concentrated in small puncta, which were intensely labeled and

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Fig. 1. (A) Distribution of GABAB receptors in the rat olfactory bulb. Strong immunolabeling is visible in two glomeruli (GL), that are surrounded by GABAB receptor-positive periglomerular cells. Other labeled neurons are visible in the external plexiform layer (EPL) and in the granule cell layer (GCL). Some of the mitral cells (arrows) are labeled diffusely. Note that the olfactory nerve layer (ONL) is completely unstained. (B,C) Subcellular localization of GABAB receptors in the glomerular neuropil. Olfactory nerve axons (Ax), but not dendritic pro®les (D), are labeled. The immunoreactivity is concentrated at sites of axodendritic apposition (large arrows), but is not present where two axons are in contact with each other (small arrows). In (B), one of the labeled axons makes an asymmetric synapse (arrowhead) with a dendritic pro®le. Note that the dendrites contacted by the labeled nerve terminals contain numerous synaptic vesicles. Scale bars, (A) 80 mm; (B) 0.7 mm; (C) 0.4 mm.

superimposed over a more diffuse labeling. All the glomeruli appeared to be labeled, whereas the olfactory nerve layer was completely unstained. At the ultrastructural level, GBR1 immunolabeling was found in ON terminals, but was usually not present in dendritic pro®les (Fig. 1B). The immunoreactivity was associated with the cytoplasmic face of the plasma membrane, thus con®rming the proposed intracellular localization of the C-terminal domain [8]. Within ON terminals, GABAB receptor labeling was concentrated at sites of axodendritic apposition, but was not present where two axons were in contact with each other (Fig. 1C). The immunoreactivity was often associated with the extrasynaptic membrane, but labeling of presynaptic membrane specializations was also observed (Fig. 1B). In many cases, the dendrites

contacted by the labeled axons contained several vesicles and resembled periglomerular cell dendrites. In agreement with recent studies based on immunocytochemistry and in situ hybridization [9,14], moderate labeling was also found in the cell body of many neurons that were distributed throughout the olfactory bulb. In small interneurons, such as granule and periglomerular cells, the labeling was concentrated in a thin rim of cytoplasm surrounding the nucleus (Figs. 1A and 2A). Some of the principal neurons were labeled diffusely. As a step toward determining the identity of periglomerular cells expressing GABAB receptors, we performed double-label immuno¯uorescence with an antibody against TH. The results showed that GABAB receptor labeling was present in all the TH-positive cells, as well as in other types of periglo-

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Fig. 2. Double-immuno¯uorescence showing colocalization of GABAB receptor labeling ((A) CY3-coupled secondary antibodies) with TH-immunoreactivity ((B) FITC-coupled secondary antibodies) in periglomerular cells. Some of the double-labeled neurons are indicated by arrows. Triangles indicate neurons that are GABAB receptor-positive but are not labeled for TH. GABAB receptor immunolabeling of the olfactory glomeruli (A) was very strong, and there was some leakage of the CY3 ¯uorescence through the FITC ®lter (B). Scale bar, 50 mm.

merular neurons (Fig. 2A,B). Thus, GABAB receptor labeling is present in distinct types of GABAergic interneurons, including granule cells and TH-positive periglomerular cells [3,12]. The major ®nding of this study is that GABAB receptors are located on the axon terminals of the ON. It has been previously shown that activation of GABAB receptors dramatically attenuates the orthodromic stimulation of mitral cells [15]. In addition, paired-pulse depression of ON-evoked synaptic responses is reduced by application of a GABAB receptor antagonist [11]. Our results extend these observations and indicate that GABAB receptors act presynaptically to inhibit the release of glutamate from ON terminals, thereby limiting the excitation of second-order neurons. As ON axons do not receive chemical synapses [16], it must be assumed that GABAB receptors are activated by spillover of GABA released from periglomerular cell dendrites after ON stimulation, or that periglomerular dendrites form unconventional synapses with ON terminals. The observation that GABAB receptors are located in close proximity of dendritic pro®les, that contain numerous synaptic vesicles (Fig. 1C), is consistent with this idea. Another intriguing possibility is that GABAB receptors are activated by taurine [7], which is colocalized with glutamate in ON axons [2]. However, recent electrophysiological analyses indicate that, although taurine mediates presynap-

tic inhibition in the olfactory bulb, its effects are not blocked by a GABAB receptor antagonist (Belluzzi et al., submitted). The moderate GBR1 labeling of cell bodies suggests that GABAB receptors may have additional functions in the olfactory bulb, that may include an autoreceptor role. Recent studies, however, indicate that GABAB receptors in vivo are probably heterodimers composed of the GBR1 and GBR2 polypeptides [9,13]. In addition, GBR2 seems to be essential for the maturation and transport of GBR1 to the plasma membrane [18]. Olfactory bulb neurons express very low levels of GBR2 [9,13], and therefore they may not have functional GABAB receptors. In contrast, it is likely that GBR2 is present in ON axons, because olfactory glomeruli are labeled by an antibody against GBR2 [17]. The identity of the GBR1 isoforms that are present in the olfactory bulb cannot be inferred from the present study. However, an antiserum speci®c for the GBR1b isoform did not label the olfactory glomeruli (J.-M. Fritschy, personal communication; and Ref. [17]), suggesting that it is the GBR1a splice variant that is expressed in ON axons. This is consistent with recent observations in other brain regions, indicating that GBR1a is the predominant GABAB receptor isoform that is found in axon terminals and is involved in inhibition of neurotransmitter release [10,19]. In conclusion, the present results extend previous electrophysiological data [11,15] and provide conclusive evidence that GABAB receptors are involved in presynaptic inhibition

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