GABAA receptors (GABAARs) mediate the majority of fast inhibition in the CNS and as such are crucial to neuronal function. Two distinct modes of GABAAR mediated inhibition exist: “phasic” involves the transient activation of postsynaptic GABAARs following presynaptic vesicular GABA release and “tonic” whereby high-affinity extrasynaptic GABAARs are persistently activated by ambient GABA. GABAARs exhibit a rich pharmacology and are the target for a number of clinically useful compounds including benzodiazepines, barbiturates and certain general anaesthetics. In addition, several naturally occurring steroids typified by the progesterone metabolite 5a-pregnan-3a-ol-20-one (5a3a) are potent positive allosteric modulators of GABAAR function - a property that endows these steroids with anxiolytic, sedative and anti-convulsant actions. Importantly, in addition to importing steroids from the periphery, the brain also harbours a steroidogenic capacity and can manufacture GABA-modulatory steroids, termed “neurosteroids”, from cholesterol. Although several studies have demonstrated neurosteroids to be implicated in a variety of physiological processes including neurodevelopment, important questions remain. In particular, are neurosteroids neurone selective? Do they discriminate between particular neuronal GABAARs subtypes? Under what conditions do endogenous neurosteroids influence neuronal function? And importantly, which cells synthesise neurosteroids in the central nervous system? Therefore, the principal aims of this study were: 1) to characterise the properties of GABAAR mediated inhibition in postnatal day (P) 17-24 neurones of the mouse ventrobasal (VB) nucleus of the thalamus – neurones which have a well defined GABAAR expression profile and which exhibit both synaptic and tonic GABAAR mediated inhibition. 2) To investigate the effects of 5a3a on synaptic and tonic GABAAR mediated inhibition in VB neurones. 3) To investigate whether there is a role for endogenous neurosteroids in regulating synaptic GABAAR function during postnatal development (P7-P20) in VB neurones and 4) cortical layer 2/3 neurones – a neuronal population with a more heterogenous, and less well defined GABAAR expression profile. To address these aims, whole-cell voltage clamp recordings were performed on acute brain slices derived from wild type and transgenic GABAAR subunit “knock-out” mice in conjunction with pharmacological approaches. Synaptic inhibition in WT VB neurones, as inferred by recording miniature inhibitory postsynaptic currents (mIPSCs) was characterised by relatively large amplitude and fast decaying mIPSCs. A large tonic conductance in WT VB neurones was inhibited following application of the competitive GABAAR antagonist bicuculline (30 µM). Deletion of the a4 subunit (a40/0) revealed alteration to both synaptic and tonic inhibition. Most notably, a40/0 VB neurones displayed a greatly diminished tonic conductance. These results are in agreement that the majority of extrasynaptic GABAARs in VB neurones contain the a4 subunit. Exogenous application of the neurosteroid 5a3a (100 nM) to WT VB neurones only modestly enhanced the tonic conductance and also gave rise to mIPSCs with prolonged decay kinetics. It is concluded 5a3a has a relatively low potency at the extrasynaptic GABAAR population in VB neurones and furthermore that this concentration does not discriminate between synaptic and tonic inhibition. Between P7 and P20, mIPSCs recorded from VB and L2/3 neurones became progressively faster decaying, a feature that has been associated with an increase in a1-GABAAR subunit expression during development. However, the developmental decrease in mIPSC decay time was still observed in recordings from a10/0 L2/3 pyramidal neurones indicating the contribution of additional factors. Here I show that blocking neurosteroid synthesis using the 5a-reductase inhibitor, finasteride, or treating the brain slice with the steroid-scavenger molecule, ?-cyclodextrin (?-CD), results in significantly faster mIPSC decay times between P7 and P10 in VB and L2/3 neurones. These results provide the first indication that an endogenous neurosteroid tone may influence the mIPSC decay kinetics of VB and L2/3 neurones during development. Moreover, compared to ?-CD pre-incubation, application of ?-CD to the intracellular compartment via the patch pipette was found to be equally effective thus suggesting a possible autocrine mechanism of neurosteroid action. For VB neurones at P20-24 (an age range at which the mIPSCs are insensitive to ?-CD treatment), incubation with the GABAAR-inactive neurosteroid precursor 5a-DHP resulted in a robust prolongation of the mIPSC decay time thus revealing the presence of functional neurosteroidogenic enzymes in the brain slice tissue. In summary, these results reveal for the first time that during the first ~2 weeks of life the developmental decrease in the duration of the mIPSCs recorded from VB and L2/3 neurones is largely due to a reduction in local neurosteroid synthesis. Moreover, P20-24 VB neurones are capable of reinitiating neurosteroid production thus giving rise to a mechanism that may, under certain conditions, locally influence neuronal inhibition. These results may have important physiological consequences as the developmental timing of the neurosteroid tone observed here coincides with a myriad of crucial neurodevelopmental processes including the transition of GABA from a depolarising to a hyperpolarising action.

No description available.

Exposure to chronic hypoxia (CH) leads to ventilatory acclimatisation to hypoxia (VAH) which is a time-dependent increase in breathing. This study examined the role of the GABAA receptor in establishing VAH. Rats were exposed to CH or control (normoxic) conditions for 10 days during which the GABAA receptor antagonist, bicuculline, was infused systemically or directly into the nucleus of the solitary tract (NTS). Acute breathing trials were then performed to measure resting ventilation and ventilatory chemoreflexes. Systemic administration of bicuculline caused reductions in breathing during acute hypoxia and acute hypercapnia in the control but not the CH animals. Continuous infusion of bicuculline in to the NTS caused a reduction in the acute hypoxic ventilatory response in animals exposed to CH but not in the control animals. The results indicate that exposure to CH alters the GABAA-mediated regulation of acute ventilatory chemoreflexes both in the NTS and elsewhere in the brain.

No description available.

Exposure to chronic hypoxia (CH) leads to ventilatory acclimatisation to hypoxia (VAH) which is a time-dependent increase in breathing. This study examined the role of the GABAA receptor in establishing VAH. Rats were exposed to CH or control (normoxic) conditions for 10 days during which the GABAA receptor antagonist, bicuculline, was infused systemically or directly into the nucleus of the solitary tract (NTS). Acute breathing trials were then performed to measure resting ventilation and ventilatory chemoreflexes. Systemic administration of bicuculline caused reductions in breathing during acute hypoxia and acute hypercapnia in the control but not the CH animals. Continuous infusion of bicuculline in to the NTS caused a reduction in the acute hypoxic ventilatory response in animals exposed to CH but not in the control animals. The results indicate that exposure to CH alters the GABAA-mediated regulation of acute ventilatory chemoreflexes both in the NTS and elsewhere in the brain.

Os efeitos dos canabinoides em algumas áreas encefálicas que expressam receptores endocanabinoides, como é o caso dos núcleos hipotalâmicos, não são ainda muito bem definidos. Vários estudos têm demonstrado o papel de alguns núcleos hipotalâmicos na organização das reações induzidas pelo medo inato e pelo pânico. As respostas de defesa induzidas pelo medo instintivo caracterizam-se por serem mais elaboradas e dirigidas para algum abrigo ou rota de fuga. O estado de pânico pode ser provocado experimentalmente em animais de laboratório através da diminuição da atividade do sistema GABAérgico. O objetivo deste trabalho foi estudar os padrões comportamentais de fuga elaborada induzidos pelo bloqueio de receptores GABAérgicos do tipo A, com microinjeções intra-hipotalâmicas de bicuculina (BIC), especificamente na divisão dorso-medial do hipotálamo ventro-medial (VMHDM), assim como estabelecer o envolvimento endocanabinoides e o papel do receptor canabinoide do tipo 1 (CB1) na modulação das respostas defensivas organizadas pelo hipotálamo medial. Os resultados mostraram que a administração prévia de doses intermediárias (5pmol) de anandamida (AEA) atenuaram as respostas defensivas induzidas pela microinjeção intra-VMHDM de bicuculina (40ng), efeito este prevenido pelo pré-tratamento intra-hipotalâmica com antagonista de receptores CB1. Os resultados indicam que a AEA pode modular os efeitos pró-aversivos da bicuculina no VMHDM por meio do recrutamento de receptores CB1. / The effects of cannabinoids in some brain areas that express endocannabinoid receptors, such as some hypothalamic nuclei, are not yet well known. Several studies have demonstrated a role of hypothalamic nuclei in the organisation of behavioural responses induced by innate fear and panic attacks. The defensive responses induced by instinctive fear are more elaborated and oriented toward a burrow or alternative route of escape. Panic-prone states are able to be experimentally induced in laboratory animals decreasing the GABAergic system activity. The aim of this work was to study panic-like elaborated defensive behaviour evoked by GABAA receptor blockade with bicuculline (BIC) in the dorsomedial division of the ventromedial hypothalamus (VMHDM), we also aimed to establish the involvement of endocannabinoids and the role of CB1 cannabinoid receptor in the modulation of elaborated defense behavioural responses organised by medial hypothalamus. The results showed that intra-hypothalamic administration of anandamide (AEA) at the intermediate dose (5pmol) attenuated defensive responses induced by intra-VMHDM microinjection of bicuculline (40ng). This effect, however, was prevented by the pre-treatment of VMHDM with the CB1 receptor antagonist AM251. These results indicate that AEA can modulate the pro-aversive effects of bicuculline into the VMHDM, recruiting CB1 receptors.

Os efeitos dos canabinoides em algumas áreas encefálicas que expressam receptores endocanabinoides, como é o caso dos núcleos hipotalâmicos, não são ainda muito bem definidos. Vários estudos têm demonstrado o papel de alguns núcleos hipotalâmicos na organização das reações induzidas pelo medo inato e pelo pânico. As respostas de defesa induzidas pelo medo instintivo caracterizam-se por serem mais elaboradas e dirigidas para algum abrigo ou rota de fuga. O estado de pânico pode ser provocado experimentalmente em animais de laboratório através da diminuição da atividade do sistema GABAérgico. O objetivo deste trabalho foi estudar os padrões comportamentais de fuga elaborada induzidos pelo bloqueio de receptores GABAérgicos do tipo A, com microinjeções intra-hipotalâmicas de bicuculina (BIC), especificamente na divisão dorso-medial do hipotálamo ventro-medial (VMHDM), assim como estabelecer o envolvimento endocanabinoides e o papel do receptor canabinoide do tipo 1 (CB1) na modulação das respostas defensivas organizadas pelo hipotálamo medial. Os resultados mostraram que a administração prévia de doses intermediárias (5pmol) de anandamida (AEA) atenuaram as respostas defensivas induzidas pela microinjeção intra-VMHDM de bicuculina (40ng), efeito este prevenido pelo pré-tratamento intra-hipotalâmica com antagonista de receptores CB1. Os resultados indicam que a AEA pode modular os efeitos pró-aversivos da bicuculina no VMHDM por meio do recrutamento de receptores CB1. / The effects of cannabinoids in some brain areas that express endocannabinoid receptors, such as some hypothalamic nuclei, are not yet well known. Several studies have demonstrated a role of hypothalamic nuclei in the organisation of behavioural responses induced by innate fear and panic attacks. The defensive responses induced by instinctive fear are more elaborated and oriented toward a burrow or alternative route of escape. Panic-prone states are able to be experimentally induced in laboratory animals decreasing the GABAergic system activity. The aim of this work was to study panic-like elaborated defensive behaviour evoked by GABAA receptor blockade with bicuculline (BIC) in the dorsomedial division of the ventromedial hypothalamus (VMHDM), we also aimed to establish the involvement of endocannabinoids and the role of CB1 cannabinoid receptor in the modulation of elaborated defense behavioural responses organised by medial hypothalamus. The results showed that intra-hypothalamic administration of anandamide (AEA) at the intermediate dose (5pmol) attenuated defensive responses induced by intra-VMHDM microinjection of bicuculline (40ng). This effect, however, was prevented by the pre-treatment of VMHDM with the CB1 receptor antagonist AM251. These results indicate that AEA can modulate the pro-aversive effects of bicuculline into the VMHDM, recruiting CB1 receptors.

Haemonchus contortus is a blood feeding parasitic nematode infecting ruminants causing anemia and poor health at great economic cost. The ability to pharmaceutically control infection has been challenged by the rapid development and spread of drug resistance. The discovery of new targets is therefore required for sustainable parasite control. UNC-49 is a nematode ligand-gated ion channel that plays an important role in muscle contraction required for normal locomotion. However, little is known regarding its sensitivity to different agonists and how they interact with the binding site. This thesis describes an investigation into the efficacy of a range of classical GABA receptor agonists on Hco-UNC-49 expressed in Xenopus oocytes. The results of our electrophysiological recordings indicate that there is a size requirement for full agonism of the Hco-UNC-49 binding site. Furthermore, a number of molecules that are known to act on vertebrate GABA receptors have no effect on Hco-UNC-49. This suggests that the binding site of nematode GABA receptors does exhibit some unique properties. These findings could possibly be exploited to develop new drugs that specifically target GABA receptors from parasitic nematodes. / UOIT

Nous avons étudié les récepteurs GABAA dans un modèle de la maladie de Huntington. En combinant des approches biochimiques, moléculaires, électrophysiologiques et de l’imagerie haute résolution, nous avons montré une modification de la neurotransmission GABAergique chez des animaux à des stades pre- et post-symptomatiques. Nos études montrent une diminution de de la neurotransmission GABAergique dans le globus pallidus des souris Huntington qui pourrait conduire à une modification des noyaux de sortie des ganglions de la base et de l’activité motrice. L’ensemble de nos résultats permet de définir le rôle de différents types de récepteurs GABAA dans le cerveau dans des conditions physiologiques et pathologiques. / We explored GABAergic neurotransmission in a mouse model of Huntington's disease. Combining molecular, imaging and electrophysiologicaltechniques, we showed changes of GABAergic neurotransmission in presymptomatic and symptomatic R6/1 mice. Our data demonstrated a decreased GABAergic inhibition in the globus pallidus of R6/1 mice, which could result in an alteration of basal ganglia output nuclei and motor activity. Taken together, our results will help to define the contribution of receptor subtypes to inhibitory transmission throughout the brain in physiological and pathophysiological states.

Introduction: Neuroactive steroids represent a class of both synthetic and naturally occurring steroids that have an effect on neural function. In addition to classical genomic mechanism by the hormones progesterone, deoxycorticosterone and testosterone 3α-OH metabolites of these hormones enhance GABAA receptor through rapid non-genomic mechanism. The site(s) of action of these neuroactive steroids namely 3α-OH-5α-pregnan-20 one, (3α,5α)-3,21-deoxycorticosterone(3α5α-THDOC) and 5α androstane-3α,17β-diol on GABAA receptor are distinct from that of benzodiazepines and barbiturate binding sites. The modulation site(s) has a well-defined structure activity relationship with a 3α-hydroxy and a 20-ketone configuration in the pregnane molecule required for agonistic action. Pregnenolone sulfate is a noncompetitive GABAA receptor antagonist and inhibit GABA activated Cl- current in an activation dependant manner. 3β-hydroxy A-ring reduced pregnane steroids are also GABAA receptor antagonist and inhibit GABAA receptor function and its potentiation induced by their 3α-diesteromers in a noncompetitive manner. Aim: The aim was to investigate if the effect of GABA, pentobarbital antagonism by bicuculline and if the effect of GABA-agonist and antagonist neuroactive steroids including pregnenolone sulfate is dependant on the α-subunits of GABAA receptor. Furthermore, the studies aimed at investigating the binding site of pregnenolone sulfate and if its effect is dependent on γ-subunit. In addition, the inhibitory effect of pregnenolone sulfate and 3β-hydroxy steroids has been characterized. We also wanted to investigate if the neuroactive steroids effect vary between the human and rat recombinant α1β2γ2L receptors and between the long (L) and short (S) variants of γ2-subunit. Method: Experiments were performed by the two electrodes voltage-clamp technique using oocytes of Xenopus laevis expressed with recombinant GABAA receptors containing α1, α4 or α5, β2, γ2L and γ2S-subunits. Results: There was no difference between the α1, α4 and α5-containing subunits regarding GABA and pentobarbital inhibition by bicuculline. GABA-activated current in the binary αβ was potent than that of ternary αβγ receptor. Unlike Zn2+ effect, inhibition by pregnenolone sulfate on the GABAA receptor is not dependant on the γ-subunit. It is likely that the 2’ residue closest to the N-terminus of the protein at M2 helix on both α1 and β2 subunit are critical to the inhibitory actions of PS and the function of Cl- channels. Point mutation at M2 helix of the β2-subunit (b2A252S) can dramatically reduce the inhibitory effect of PS on the GABAA receptors without affecting the inhibitory properties of 3β-hydroxysteroids. Agonist and antagonist steroids also varied in their efficacy between the human and rat α1β2γ2L receptor. Neuroactive steroids also showed difference between human γ2L and γ2S-containing receptor. Conclusions: GABA and pentobarbital antagonism by bicuculline is not dependant on α-subunit. Pregnenolone sulfate binding site is different from that of Zn2+. 3β-hydroxysteroids and pregnenolone sulfate inhibit GABAA receptor through different mechanisms. Neuroactive steroids also differ between species and between the long and short variant of γ- subunit.