Maurice Garret

Expression of GABA receptor Ρ subunits in rat brain

Abstract: The GABA receptor ρ1, ρ2, and ρ3 subunits are expressed in the retina where they form bicuculline‐insensitive GABAC receptors. We used northern blot, in situ hybridization, and RT‐PCR analysis to study the expression of ρ subunits in rat brains. In situ hybridization allowed us to detect ρ‐subunit expression in the superficial gray layer of the superior colliculus and in the cerebellar Purkinje cells. RT‐PCR experiments indicated that (a) in retina and in domains that may contain functional GABAC receptors, ρ2 and ρ1 subunits are expressed at similar levels; and (b) in domains and in tissues that are unlikely to contain GABAC receptors, ρ2 mRNA is enriched relative to ρ1 mRNA. These results suggest that both ρ1 and ρ2 subunits are necessary to form a functional GABAC receptor. The use of RT‐PCR also showed that, except in the superior colliculus, ρ3 is expressed along with ρ1 and ρ2 subunits. We also raised an antibody against a peptide sequence unique to the ρ1 subunit. The use of this antibody on cerebellum revealed the rat ρ1 subunit in the soma and dendrites of Purkinje neurons. The allocation of GABAC receptor subunits to identified neurons paves the way for future electrophysiological studies.

Brain-endocrine interactions: a microvascular route in the mediobasal hypothalamus.

Blood-borne hormones acting in the mediobasal hypothalamus, like those controlling food intake, require relatively direct access to target chemosensory neurons of the arcuate nucleus (ARC). An anatomical substrate for this is a permeable microvasculature with fenestrated endothelial cells in the ARC, a system that has awaited comprehensive documentation. Here, the immunofluorescent detection of endothelial fenestral diaphragms in the rat ARC allowed us to quantitate permeable microvessels throughout its rostrocaudal extent. We have determined that permeable microvessels are part of the subependymal plexus irrigating exclusively the ventromedial (vm) ARC from the subadjacent neuroendocrine median eminence. Unexpectedly, permeable microvessels were concentrated proximal to the pituitary stalk. This marked topography strongly supports the functional importance of retrograde blood flow from the pituitary to the vmARC, therefore making a functional relationship between peripheral long-loop, pituitary short-loop, and neuroendocrine ultra-short loop feedback, altogether converging for integration in the vmARC (formerly known as the hypophysiotrophic area), thereby so pivotal as a multicompetent brain endocrinostat.

Evidence for Inhibition Mediated by Coassembly of GABAA and GABAC Receptor Subunits in Native Central Neurons

Fast inhibition in the nervous system is commonly mediated by GABAA receptors comprised of 2α/2β/1γ subunits. In contrast, GABAC receptors containing onlyρ subunits (ρ1-ρ3) have been predominantly detected in the retina. However, here using reverse transcription-PCR and in situ hybridization we show that mRNA encoding the ρ1 subunit is highly expressed in brainstem neurons. Immunohistochemistry localized the ρ1 subunit to neurons at light and electron microscopic levels, where it was detected at synaptic junctions. Application of the GABAC receptor agonist cis-4-aminocrotonic acid (100-800 μM) requires the ρ1 subunit to elicit responses, which surprisingly are blocked independently by antagonists to GABAA (bicuculline, 10 μM) and GABAC [(1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA); 40-160 μM] receptors. Responses to GABAC agonists were also enhanced by the GABAA receptor modulator pentobarbitone (300 μM). Spontaneous and evoked IPSPs were reduced in amplitude but never abolished by TPMPA, but were completely blocked by bicuculline. We therefore tested the hypothesis that GABAA and GABAC subunits formed a heteromeric receptor. Immunohistochemistry indicated that ρ1 and α1 subunits were colocalized at light and electron microscopic levels. Electrophysiology revealed that responses to GABAC receptor agonists were enhanced by the GABAA receptor modulator zolpidem (500 nm), which acts on the α1 subunit when the γ2 subunit is also present. Finally, coimmunoprecipitation indicated that the ρ1 subunit formed complexes that also containedα1 and γ2 subunits. Taken together these separate lines of evidence suggest that the effects of GABA in central neurons can be mediated by heteromeric complexes of GABAA and GABAC receptor subunits.

Localisation of GABAA receptor ϵ-subunit in cholinergic and aminergic neurones and evidence for co-distribution with the θ-subunit in rat brain

Abstract In situ hybridisation and immunohistochemical methodologies suggest the existence of a large diversity of GABA A receptor subtypes in the brain. These are hetero-oligomeric proteins modulated by a number of clinically important drugs, depending on their subunit composition. We recently cloned and localised the rat GABA A receptor ϵ-subunit by in situ hybridisation and immunohistochemical procedures. Here, in a dual-labelling immunohistochemical study in the rat brain, we used our affinity-purified antiserum to ϵ with antisera to markers of cholinergic, catecholaminergic, and serotonergic neurones. As far as cholinergic systems were concerned, ϵ-immunoreactivity was expressed in all forebrain cell-groups, as well as in the caudal lateral pontine tegmentum and dorsal motor nucleus of the vagus nerve. As far as dopaminergic systems were concerned, ϵ-immunoreactivity was found to be expressed in a great number of hypothalamic cell-groups (A15, A14 and A12) and in the substantia nigra pars compacta. The noradrenergic, and to a lesser extent, adrenergic cell-groups were all ϵ-immunoreactive. Also, ϵ-immunoreactivity was detected in all serotonergic cell-groups. We also revealed by in situ hybridisation in a monkey brain that ϵ mRNA was expressed in the locus coeruleus, as previously observed in rats. Finally, by using in situ hybridisation in rat brains, we compared the distribution of the mRNA of ϵ with that of the recently cloned θ-subunit of the GABA A receptor. Both subunits showed strikingly overlapping expression patterns throughout the brain, especially in the septum, preoptic areas, various hypothalamic nuclei, amygdala, and thalamus, as well as the aforementioned monoaminergic cell-groups. No θ-mRNA signals were detected in cholinergic cell-groups. Taken together with previously published evidence of the presence of the α3-subunit in monoamine- or acetylcholine-containing systems, our data suggest the existence of novel GABA A receptors comprising α3/ϵ in cholinergic and α3/θ/ϵ in monoaminergic cell-groups.

Pharmacological properties of GABAA receptors in rat hypothalamic neurons expressing the ∈-subunit

The pharmacological properties and functional role of native GABAA receptors (GABAARs) were investigated in rat hypothalamic neurons expressing the ϵ-subunit with the help of whole-cell patch-clamp recording and single-cell reverse transcription-PCR. Two cell groups were identified: histaminergic tuberomamillary and orexinergic/hypocretinergic neurons. Approximately 25% of histaminergic and 70% of orexinergic neurons contained mRNA encoding for the ϵ-subunit. Double-immunofluorescence staining revealed a somatic localization of this protein in these two neuronal groups. Constitutive activity, diazepam modulation, fast desensitization of maximal currents, and activation by propofol (6-98 μm) of GABAARs did not correlate withϵ-subunit expression. Propofol at 3-12 μm potentiated GABA-mediated currents similarly in all neurons. However, noise variance analysis of GABA-mediated currents enhanced by propofol revealed a significant difference between ϵ-positive and ϵ-negative neurons. The former displayed no difference between control and potentiated responses, and, in the latter, noise was decreased in the presence of propofol. Spontaneous IPSCs recorded in cultured hypothalamic neurons were prolonged in the presence of propofol in all ϵ-negative neurons, whereas propofol-resistant IPSCs were recorded in ϵ-positive cells. The infrequent expression of the ϵ-subunit may be a key factor in the recently discovered central role of the tuberomamillary nucleus in anesthesia.

GABAA receptor ε subunit expression in identified peptidergic neurons of the rat hypothalamus

Dual-labeling immunohistochemical or in situ hybridization studies for the recently cloned e-subunit and several neuropeptides were performed in the rat hypothalamus. We revealed an extensive co-expression (>90%) with hypocretin (Hcrt), oxytocin (OT), the gonadotropin-releasing hormone (GnRH), and the melanin-concentrating hormone (MCH) peptides, whereas occasional co-expression (<10%) with cocaine-amphetamine-regulated transcript (CART) was found. Our results suggest that novel GABAA receptor subtypes comprising e-subunit are important for metabolic and neuroendocrine functions.

A γ2(R43Q) Mutation, Linked to Epilepsy in Humans, Alters GABAA Receptor Assembly and Modifies Subunit Composition on the Cell Surface

Genetic defects leading to epilepsy have been identified in γ2 GABAA receptor subunit. A γ2(R43Q) substitution is linked to childhood absence epilepsy and febrile seizure, and a γ2(K289M) mutation is associated with generalized epilepsy with febrile seizures plus. To understand the effect of these mutations, surface targeting of GABAA receptors was analyzed by subunit-specific immunofluorescent labeling of living cells. We first transfected hippocampal neurons in culture with recombinant γ2 constructs and showed that the γ 2(R43Q) mutation prevented surface expression of the subunit, unlike γ2(K289M) substitution. Several γ2-subunit constructs, bearing point mutations within the Arg-43 domain, were expressed in COS-7 cells with α3- and β3-subunits. R43Q and R43A substitutions dramatically reduced surface expression of the γ2-subunit, whereas R43K, P44A, and D39A substitutions had a lesser, but still significant, impact and K289M substitution had no effect. Whereas the mutant γ2(R43Q) was retained within intracellular compartments, αβ complexes were still targeted at the cell membrane. Coimmunoprecipitation experiments showed that γ2(R43Q) was able to associate with α3- or β3-subunits, although the stoichiometry of the complex with α3 was altered. Our data show that γ2(R43Q) is not a dominant negative and that the mutation leads to a modification of GABAA receptor subunit composition on the cell surface that impairs the synaptic targeting in neurons. This study reveals an involvement of the γ2-Arg-43 domain in the control of receptor assembly that may be relevant to the effect of the heterozygous γ2(R43Q) mutation leading to childhood absence epilepsy and febrile seizure.

Dopamine receptors set the pattern of activity generated in subthalamic neurons

Information processing in the brain requires adequate background neuronal activity. As Parkinsons disease progresses, patients typically become akinetic; the death of dopaminergic neurons leads to a dopamine‐depleted state, which disrupts information processing related to movement in a brain area called the basal ganglia. Using agonists of dopamine receptors in the D1 and D2 families on rat brain slices, we show that dopamine receptors in these two families govern the firing pattern of neurons in the subthalamic nucleus, a crucial part of the basal ganglia. We propose a conceptual frame, based on specific properties of dopamine receptors, to account for the dominance of different background firing patterns in normal and dopamine‐depleted states. Baufreton, J., Zhu, Z.‐T., Garret, M., Bioulac, B., Johnson, S. W., Taupignon, A. I. Dopamine receptors set the pattern of activity generated in subthalamic neurons. FASEB J. 19, 1771–1777 (2005)

Molecular and electrophysiological evidence for a GABAc receptor in thyrotropin-secreting cells.

In the pituitary, GABA regulates the release of several hormones via different receptors. GABAC receptors are heterooligomers that differ from GABAA receptors in that they containρ -subunits and are insensitive to bicuculline. However, molecular and functional evidence for the presence of GABAC receptors outside the retina has yet to be established. The present work was performed on guinea pig and rat pituitaries. Both Northern blot and RT-PCR analysis showed that, although ρ1- and ρ2-subunits were expressed at similar levels in the rat retina, ρ1 messenger RNA (mRNA) was enriched, relative to ρ2 mRNA in the rat pituitary. Northern blot experiments also showed that, in the pituitary, ρ1 andρ 2 mRNAs are shorter in size than those expressed in the retina. The use of a subunit-specific antibody revealed colocalization ofρ 1-subunit and anti-TSH labeling on rat pituitary sections. TSH guinea pig pituitary cells were also labeled with a ρ-subunit antiserum. Moreover, whole-cell patch clamp on single guinea pi...

An intracellular motif of P2X(3) receptors is required for functional cross-talk with GABA(A) receptors in nociceptive DRG neurons.

Functional cross‐talk between structurally unrelated P2X ATP receptors and members of the ‘cys‐loop’ receptor‐channel superfamily represents a recently‐discovered mechanism for rapid modulation of information processing. The extent and the mechanism of the inhibitory cross‐talks between these two classes of ionotropic receptors remain poorly understood, however. Both ionic and molecular coupling were proposed to explain cross‐inhibition between P2X subtypes and GABAA receptors, suggesting a P2X subunit‐dependent mechanism. We show here that cross‐inhibition between neuronal P2X3 or P2X2+3 and GABAA receptors does not depend on chloride and calcium ions. We identified an intracellular QST386–388 motif in P2X3 subunits which is required for the functional coupling with GABAA receptors. Moreover the cross‐inhibition between native P2X3 and GABA receptors in cultured rat dorsal root ganglia (DRG) neurons is abolished by infusion of a peptide containing the QST motif as well as by viral expression of the main intracellular loop of GABAAβ3 subunits. We provide evidence that P2X3 and GABAA receptors are colocalized in the soma and central processes of nociceptive DRG neurons, suggesting that specific intracellular P2X3‐GABAA subunit interactions underlie a pre‐synaptic cross‐talk that might contribute to the regulation of sensory synaptic transmission in the spinal cord.