Current awarness

Multiple GABA_B receptors

γ-Hydroxybutyrate (GHB) has a complex array of neural actions that include effects on its own high-affinity GHB receptor, the release of neuroactive steroids, and agonist actions at GABA_A and GABA_B receptors. We previously reported partial overlap in the c-Fos expression patterns produced by GHB and the GABA_B agonist, baclofen in rats. The present study extends these earlier findings by examining the extent to which GHB Fos expression and behavioral sedation are prevented by (2S)-(+)-5,5-dimethyl-2-morpholineacetic acid (SCH 50911), a GABA_B antagonist, and NCS-382, a putative antagonist at the high-affinity GHB receptor. We also compare Fos expression caused by GHB and its precursor γ-butyrolactone (GBL), which is a pro-drug for GHB but lacks the high sodium content of the parent GHB molecule. Both GHB (1000 mg/kg) and GBL (600 mg/kg) induced rapid sedation in rats that lasted over 90 min and caused similar Fos expression patterns, albeit with GBL causing greater activation of the nucleus accumbens (core and shell) and dentate gyrus (granular layer). Pretreatment with SCH 50911 (100 mg/kg) partly reversed the sedative effects of GHB and significantly reduced GHB-induced Fos expression in only four regions: the tenia tecta, lateral habenula, dorsal raphe and laterodorsal tegmental nucleus. NCS-382 (50 mg/kg) had no effect on GHB-induced sedation or Fos expression. When given alone, both NCS-382 and SCH 50911 increased Fos expression in the bed nucleus of the stria terminalis, central amygdala, parasubthalamic nucleus and nucleus of the solitary tract. SCH 50911 alone affected the Islands of Calleja and the medial, central and paraventricular thalamic nuclei. Overall, this study shows a surprising lack of reversal of GHB-induced Fos expression by two relevant antagonists, both of which have marked intrinsic actions. This may reflect the limited doses tested but also suggests that GHB Fos expression reflects mechanisms independent of GHB and GABA_B receptors.

The functional role of presynaptic release-regulating metabotropic glutamate type 7 (mGlu7) receptors in hippocampal GABAergic terminals was investigated. Mouse hippocampal synaptosomes were preloaded with [³H]D-γ-aminobutyric acid ([³H]GABA) and then exposed in superfusion to 12 mM KCl. The K⁺-evoked [³H]GABA release was inhibited by the mGlu7 allosteric agonist N,N′-dibenzyhydryl-ethane-1,2-diamine dihydrochloride (AMN082, 0.001–10 μM), as well as by the group III mGlu receptor agonist l-(+)-2-amino-4-phosphonobutyric acid [(l)-AP4, 0.01–1 mM]. The mGlu8 receptor agonist (S)-3,4-dicarboxyphenylglycine [(S)-3,4-DCPG, 10–100 nM] was ineffective. AMN082 and (l)-AP4-induced effects were recovered by the mGlu7 negative allosteric modulator (NAM) 6-(4-methoxyphenyl)-5-methyl-3-(4-pyridinyl)-isoxazolo[4,5-c]pyridin-4(5H)-one hydrochloride (MMPIP). AMN082 also inhibited in a MMPIP-sensitive manner the K⁺-evoked release of endogenous GABA. AMN082 and the adenylyl cyclase (AC) inhibitor MDL-12,330A reduced [³H]GABA exocytosis in a 8-Br-cAMP-sensitive. AMN082-inhibitory effect was additive to that caused by (−)baclofen, but insensitive to the GABA_B antagonist 3-[[(3,4-Dichlorophenyl)methyl]amino]propyl] diethoxymethyl) phosphinic acid (CGP52432). Conversely, (−)baclofen-induced inhibition of GABA exocytosis was insensitive to MMPIP. Finally, the forskolin-evoked [³H]GABA release was reduced by AMN082 or (−)baclofen but abolished when the two agonists were added concomitantly. Mouse hippocampal synaptosomal plasmamembranes posses mGlu7 receptor proteins; confocal microscopy analysis unveiled that mGlu7 proteins colocalize with syntaxin-1A (Stx-1A), with vesicular GABA transporter (VGAT)-proteins and with GABA_B receptor subunit proteins. We propose that presynaptic inhibitory mGlu7 heteroreceptors, negatively coupled to AC-dependent intraterminal pathway, exist in mouse hippocampal GABA-containing terminals, where they colocalize, but do not functionally cross-talk, with GABA_B autoreceptors.

This article is part of a Special Issue entitled ‘Metabotropic Glutamate Receptors’.

GABA_B receptors are the G-protein-coupled receptors (GPCRs) for γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the central nervous system. GABA_B receptors are implicated in the etiology of a variety of psychiatric disorders and are considered attractive drug targets. With the cloning of GABA_B receptor subunits 13 years ago, substantial progress was made in the understanding of the molecular structure, physiology, and pharmacology of these receptors. However, it remained puzzling that native studies demonstrated a heterogeneity of GABA_B responses that contrasted with a very limited diversity of cloned GABA_B receptor subunits. Until recently, the only firmly established molecular diversity consisted of two GABA_B1 subunit isoforms, GABA_B1a and GABA_B1b, which assemble with GABA_B2 subunits to generate heterodimeric GABA_B(1a,2) and GABA_B(1b,2) receptors. Using genetic, ultrastructural, biochemical, and electrophysiological approaches, it has been possible to identify functional properties that segregate with these two receptors. Moreover, receptor modifications and factors that can alter the receptor response have been identified. Most importantly, recent data reveal the existence of a family of auxiliary GABA_B receptor subunits that assemble as tetramers with the C-terminal domain of GABA_B2 subunits and drastically alter pharmacology and kinetics of the receptor response. The data are most consistent with native GABA_B receptors minimally forming dimeric assemblies of units composed of GABA_B1, GABA_B2, and a tetramer of auxiliary subunits. This represents a substantial departure from current structural concepts for GPCRs.

Glycine release and modulation of glycine exocytosis by presynaptic receptors have been rarely studied. We here investigate on the existence and the pharmacological profile of GABA_B receptors regulating glycine release in mouse spinal cord and hippocampus. Synaptosomes were preincubated with [³H]glycine in presence of the glycine transporter 1 inhibitor NFPS, in order to study release from glycinergic terminals selectively labelled through the glycine transporter 2, and depolarized in superfusion with concentrations of KCl (12 and 15 mM) previously found to elicit exocytosis of glycine. Exposure of synaptosomes to (−)-baclofen caused inhibition of [³H]glycine overflow: EC₅₀ = 0.62 ± 0.08 μM, $E_{\text{max}}\simeq 65\%$ in the spinal cord and EC₅₀ = 0.58 ± 0.07 μM, $E_{\text{max}}\simeq 55\%$ in the hippocampus. The effect of 3 μM (−)-baclofen in the spinal cord was prevented by the GABA_B receptor antagonists CGP 52432 (IC₅₀ = 22.6 ± 2.15 μM) and CGP 35348 (IC₅₀ = 5.99 ± 1.21 μM), whereas phaclofen was ineffective. In the hippocampus, the CGP antagonists were more potent than in the spinal cord, their IC₅₀ values amounting to 0.81 ± 0.08 μM and to 0.94 ± 0.09 μM, respectively; phaclofen (100–300 μM) was a weak antagonist. It is concluded that: (i) glycinergic nerve terminals in spinal cord and hippocampus possess GABA_B receptors mediating inhibition of the evoked glycine exocytosis; (ii) the pharmacological profiles of the receptors are qualitatively similar in the two regions; (iii) low concentrations of GABA_B antagonists appear able to preferentially inhibit the receptors located on hippocampal glycinergic nerve endings.

GHB is used therapeutically and recreationally, although the precise mechanism of action responsible for its different behavioral effects is not entirely clear. The purpose of this review is to summarize how behavioral procedures, especially drug discrimination procedures, have been used to study the mechanism of action of GHB. More specifically, we will review several different drug discrimination procedures and discuss how they have been used to qualitatively and quantitatively study different components of the complex mechanism of action of GHB. A growing number of studies have provided evidence that the behavioral effects of GHB are mediated predominantly by GABA_B receptors. However, there is also evidence that the mechanisms mediating the effects of GHB and the prototypical GABA_B receptor agonist baclofen are not identical, and that other mechanisms such as GHB receptors and subtypes of GABA_A and GABA_B receptors might contribute to the effects of GHB. These findings are consistent with the different behavioral profile, abuse liability, and therapeutic indications of GHB and baclofen. A better understanding of the similarities and differences between GHB and baclofen, as well as the pharmacological mechanisms of action underlying the recreational and therapeutic effects of GHB, could lead to more effective medications with fewer adverse effects.

Bone marrow stromal cells (BMSC) have the capability to undergo a change of morphology, reminiscent of neuronal cells, after exposure to an inductive medium. These induced BMSC-derived neuron-like (BDNL) cells express several neuronal markers, including Microtubule-Associated Protein Tau, Neurofilament M, and Nestin as revealed by immunocytochemistry analysis. To assess whether the induction process has possible functional relevance, we have focused our attention on the expression of neurotransmitter receptors. In particular, we show that the expression of GABA_A subunits α1, β2/3, ɛ and GABA_B1 mRNAs is greatly enhanced in BMSC by the induction treatment. Similar results were obtained from rat skin fibroblasts subjected to the same induction protocol, with the exception for the GABA_B2 transcript that was expressed only by BMSC and BDNL. The presence of both GABA_B1 and GABA_B2 subunits in BDNL cells suggests that functional GABA_B receptors might be assembled: we indeed found that a functional GABA_B receptor, negatively linked to cyclic AMP production, is expressed in BDNL. Therefore, we suggest that BMSC can be converted into cells equipped with appropriate receptors coupled to transduction mechanisms, potentially responding to a specific neurotransmitter.