Serge Bischoff

GABAB-receptor subtypes assemble into functional heteromeric complexes

B-type receptors for the neurotransmitter GABA (γ-aminobutyric acid) inhibit neuronal activity through G-protein-coupled second-messenger systems, which regulate the release of neurotransmitters and the activity of ion channels and adenylyl cyclase. Physiological and biochemical studies show that there are differences in drug efficiencies at different GABAB receptors, so it is expected that GABAB-receptor (GABABR) subtypes exist. Two GABAB-receptor splice variants have been cloned (GABABR1a and GABABR1b), but native GABAB receptors and recombinant receptors showed unexplained differences in agonist-binding potencies. Moreover, the activation of presumed effector ion channels in heterologous cells expressing the recombinant receptors proved difficult,. Here we describe a new GABAB receptor subtype, GABABR2, which does not bind available GABAB antagonists with measurable potency. GABABR1a, GABABR1b and GABABR2 alone do not activate Kir3-type potassium channels efficiently, but co-expression of these receptors yields a robust coupling to activation of Kir3 channels. We provide evidence for the assembly of heteromeric GABAB receptors in vivo and show that GABABR2 and GABABR1a/b proteins immunoprecipitate and localize together at dendritic spines. The heteromeric receptor complexes exhibit a significant increase in agonist- and partial-agonist-binding potencies as compared with individual receptors and probably represent the predominant native GABAB receptor. Heteromeric assembly among G-protein-coupled receptors has not, to our knowledge, been described before.

Epilepsy, Hyperalgesia, Impaired Memory, and Loss of Pre- and Postsynaptic GABA B Responses in Mice Lacking GABA B(1)

GABA(B) (gamma-aminobutyric acid type B) receptors are important for keeping neuronal excitability under control. Cloned GABA(B) receptors do not show the expected pharmacological diversity of native receptors and it is unknown whether they contribute to pre- as well as postsynaptic functions. Here, we demonstrate that Balb/c mice lacking the GABA(B(1)) subunit are viable, exhibit spontaneous seizures, hyperalgesia, hyperlocomotor activity, and memory impairment. Upon GABA(B) agonist application, null mutant mice show neither the typical muscle relaxation, hypothermia, or delta EEG waves. These behavioral findings are paralleled by a loss of all biochemical and electrophysiological GABA(B) responses in null mutant mice. This demonstrates that GABA(B(1)) is an essential component of pre- and postsynaptic GABA(B) receptors and casts doubt on the existence of proposed receptor subtypes.

The D-1 dopamine receptor antagonist SCH 23390 also interacts potently with brain serotonin (5-HT2) receptors

The interaction of SCH 23390 with central serotonin 5-HT2 receptors was studied in vivo on [3H]spiperone binding and in vitro on [3H]ketanserin binding. SCH 23390 inhibited [3H]spiperone binding in rat frontal cortex with an ID50 of 1.5 mg/kg i.p., thus being equipotent to the two 5-HT2 antagonists cinanserin and methysergide. In vitro, SCH 23390 competed with [3H]ketanserin with an IC50 of 30 nM. These data indicate that SCH 23390 also binds with high affinity to 5-HT2 receptors in rat brain.

Spatial distribution of GABABR1 receptor mRNA and binding sites in the rat brain

A γ‐aminobutyric acid (GABA)B receptor (named GABABR1) has been recently cloned in the rat and human brain and two variants generated by alternative RNA splicing were identified. In the present study, we addressed the question as to whether these variants contribute to the diversity of GABAB receptor‐mediated physiological responses and constitute real receptor subtypes with distinct functions. To this aim, we have mapped the GABABR1 (R1a) and GABABR1b (R1b) transcript distribution in the rat brain using in situ hybridization. We have compared the mRNA distribution with the distribution of [3H]CGP54626‐labeled binding GABABR1 receptor sites as assessed in adjacent cryosections by quantitative autoradiography. We found that GABAB receptor transcripts and binding sites are expressed in the brain in almost all neuronal cell populations. Expression in glial cells, if any, is marginal. We observed a good parallelism between GABABR1 mRNA transcripts and binding sites in broad neuroanatomical entities with highest densities in hippocampus, thalamic nuclei, and cerebellum. By contrast, R1a and R1b transcripts exhibit marked differences in their regional and cellular distribution pattern. A typical example is the cerebellum with an almost exclusive expression of R1b in the Purkinje cells and of R1a in the granule, stellate, and basket cells. Data pointing at a pre‐ versus postsynaptic localization for R1a and R1b, respectively, at some neuronal sites are presented. J. Comp. Neurol. 412:1–16, 1999.

Catabolism of 3H-histamine in the rat brain after intracisternal administration☆

The fate of 3H-histamine of high specific activity has been studied after its administration into the cisterna magna or into the lateral ventricle. 3H-histamine metabolites were analyzed by a combination of methods involving ion-exchange and thin-layer chromatography, solvent extraction and isotope dilution. Histamine catabolism occurred mainly, if not solely, through ring-methylation followed by deamination into methylimidazole acetic acid which was found to be the main catabolite. This last step was inhibited in tranylcypromine-treated rats, resulting in a marked rise in brain 3H-methylhistamine. Partial inhibition of histamine methylation was achieved by administration of methylhistamine in high doses and resulted in a slower disappearance of 3H-histamine; it also induced a prompt increase in the level of endogenous histamine, suggesting a high turnover rate for the brain amine. Deamination of 3H-methylhistamine occurred more slowly in the newborn than in the adult rat brain, probably in relation with incomplete development of monoamine oxidase activity. If present, direct oxidative deamination of histamine was a minor pathway, as evidenced by the low 3H-imidazole acetic formation as well as by the slight effects of aminoguanidine treatment, both on 3H-histamine catabolism and on endogenous amine level. The efflux of 3H-deaminated metabolites from brain was not modified by treatment with probenecid.

Alteration of central alpha2- and beta-adrenergic receptors in the rat after DSP-4, a selective noradrenergic neurotoxin

A peripheral injection of DSP-4 [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine] produced a marked, selective, and lasting depletion of norepinephrine in certain regions of the rat central nervous system. This depletion at 10 days after injection was associated with regional alterations in some, but not all, adrenergic binding sites (receptors) as determined by in vitro [3H]prazosin (alpha 1), [3H]p-aminoclonidine (alpha 2), and [3H]dihydroalprenolol (beta) binding. The neocortical alpha 1-receptor was not changed. The alpha 2-receptor in several regions was altered as indicated by an increase in ligand affinity; additionally, the density of this receptor was slightly decreased in some regions. Depending on the region, the beta-receptor either increased in density or was unchanged. The increased density of this receptor in neocortex corresponded to an increased activity of isoproterenol-sensitive adenylate cyclase. These two changes were not affected by subchronic treatment with desipramine, a norepinephrine uptake inhibitor. The changes were, however, partially or completely reversed by subchronic administration of clenbuterol, a centrally-acting beta-receptor agonist. The dopaminergic receptor in various regions was unaltered as assessed by in vivo and/or in vitro binding of [3H]spiperone. The in vivo binding of this ligand also indicated that the serotoninergic receptor in frontal neocortex was unchanged. Assessment of adrenergic receptors in neocortex at 50 days after injection indicated only the above affinity change of the (presumably postsynaptic) alpha 2-receptor. The alpha 1-receptor remained unaltered. The density of the beta-receptor had normalized, as had the activity of isoproterenol-sensitive adenylate cyclase. Implicit in these findings is the following rank order of receptor sensitivity to chronic norepinephrine depletion: alpha 2 greater than beta greater than alpha 1. The use of DSP-4 has clear advantages over other methods of depleting central norepinephrine. This neurotoxin can be administered by intraperitoneal injection, the depletion of norepinephrine can be readily checked by absence of the post-decapitation reflex, and the changes in other neurotransmitter concentrations are relatively minor or nonexistent. The alteration of alpha 2- and beta-receptors, as a consequence of DSP-4 treatment, may form the basis of a new animal model of adrenergic receptor supersensitivity. Such a model may clarify the importance of these central receptors to physiological and behavioral processes.

Dual localization of histamine in an ascending neuronal pathway and in non-neuronal cells evidenced by lesions in the lateral hypothalamic area.

The effects of lesions placed in the lateral hypothalamic area, i.e., interrupting the MFB (as evidenced by a 65% decrease in cortical noradrenaline and serotonin) suggest a pluricompartmentation of brain histamine (HA). The existence of an ascending histaminergic system is indicated by the reductions in l-histidine decarboxylase (H.D.) activity, in [3H]histamine synthesis and in HA content, in the cortex of lesioned rats. Moreover, the decrease in H.D. activity was restricted to the regions rostral to the lesion, without modification caudally. The time-course of the alterations in H.D. activity and in HA content is compatible with a process of anterograde degeneration. In subcellular fractionation studies, the reduction in cortical HA content was found to be mainly confined to the P2 fraction, which contains the synaptosomes. Although the widespread ipsilateral distribution of HA synthesizing terminals resembles that of monoaminergic ones, the absence of reduction in H.D. activity after selective destruction of catecholaminergic and serotoninergic neurons, by 6-hydroxydopamine or 5,6-dihydroxytryptamine respectively, renders likely the existence of specific HA-containing neurons. That the release of the amine from these neurons might be related to the nerve impulse flow is suggested by the transient effects of the lesions which preceded the degenerative process (elevated endogenous HA level and slowed rate of [3H]HA synthesis). In addition, the discrepancy between the reduction in H.D. activity and in HA level after the lesions could be explained by the presence of the amine in another neuronal system and/or in non-neuronal cells, not affected by the lesion. This additional compartment is characterized by a high HA content and a low H.D. activity and could therefore be localized in mast-cells. The respective sizes of these two compartments, estimated by several methods, appear to be approximately the same.

Spatial distribution of GABA(B)R1 receptor mRNA and binding sites in the rat brain

A gamma-aminobutyric acid (GABA)(B) receptor (named GABA(B)R1) has been recently cloned in the rat and human brain and two variants generated by alternative RNA splicing were identified. In the present study, we addressed the question as to whether these variants contribute to the diversity of GABA(B) receptor-mediated physiological responses and constitute real receptor subtypes with distinct functions. To this aim, we have mapped the GABA(B)R1 (R1a) and GABA(B)R1b (R1b) transcript distribution in the rat brain using in situ hybridization. We have compared the mRNA distribution with the distribution of [(3)H]CGP54626-labeled binding GABA(B)R1 receptor sites as assessed in adjacent cryosections by quantitative autoradiography. We found that GABA(B) receptor transcripts and binding sites are expressed in the brain in almost all neuronal cell populations. Expression in glial cells, if any, is marginal. We observed a good parallelism between GABA(B)R1 mRNA transcripts and binding sites in broad neuroanatomical entities with highest densities in hippocampus, thalamic nuclei, and cerebellum. By contrast, R1a and R1b transcripts exhibit marked differences in their regional and cellular distribution pattern. A typical example is the cerebellum with an almost exclusive expression of R1b in the Purkinje cells and of R1a in the granule, stellate, and basket cells. Data pointing at a pre- versus postsynaptic localization for R1a and R1b, respectively, at some neuronal sites are presented.

Neuron-specific transgene expression of Bcl-XL but not Bcl-2 genes reduced lesion size after permanent middle cerebral artery occlusion in mice

Protective effects after focal cerebral ischemia were assessed in transgenic mice that overexpress in a neuron-specific fashion mouse Bcl-XL or human Bcl-2. Both Bcl genes were under the control of the same mouse Thy-1 regulatory sequences resulting in very similar expression patterns in cortical neurons. Furthermore, these sequences direct lateonset (i.e. around birth) expression in brain, thus minimizing effects of transgene expression during brain development. Effects on infarct volume were measured using MRI after permanent occlusion of the middle cerebral artery (MCA). When compared to their non-transgenic littermates, Thy1mbcl-XL mice showed a significant 21% reduction in infarct size whereas Thy1hbcl-2 mice did not reveal any reduction. These findings suggest a selective protective advantage of Bcl-XL as compared with Bcl-2 in this mouse model for human stroke.

Correlations between different measures of antiserotonin activity of drugs

SummaryThe antiserotonin properties of a series of neuroleptics, 5-HT-receptor blockers and some adrenoceptor antagonists were investigated in several in vivo test systems (l-5-HTP syndrome and 5-HT-paw edema in the rat) and in an in vitro test (isolated rat uterus preparation). The results were compared to the results obtained with these drugs in an in vivo 3H-spiperone binding assay in the rat. The computations of the relative ED50 (or IC50) values obtained in different test procedures showed that the ability of drugs to bind to 5-HT receptors labelled by 3H-spiperone in the rat frontal cortex correlates fairly well with their potencies to inhibit the l-5-HTP syndrome or 5-HT-induced rat pawedema (Spearman rank correlation coefficient, r=0.80 and 0.79 respectively, n=22). In an in vitro test (rat uterus) the estimated 5-HT-receptor blocking potency of the tested drugs did not, however, correlate with any of the in vivo measures used for this purpose. The results suggest, therefore, that for the determination of central antiserotonin effects of drugs in the rat, functional in vivo tests (l-5-HTP syndrome or 5-HT-induced rat paw-edema) could yield about the same information as the specific, in vivo 3H-spiperone binding assay. The 5-HT-receptor type mediating the behavioral responses to l-5-HTP is tentatively defined as a 5-HT2 receptor.