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.
|Date of Award||2011|
|Sponsors||British Pharmacological Society|
|Supervisor||Delia Belelli (Supervisor) & Jeremy Lambert (Supervisor)|