The brain expresses many GABAaR subtypes, which exhibit distinct physiological/pharmacologi cal properties and influence discrete behaviours. It is an important clinical target for drugs eg diazepam, used to treat various psychiatric/ neurological conditions. However, as such drugs are permissive for GABAaR isoforms, their side effects limit clinical use. The project exploits an exciting Lilly discovery of drugs that selectively enhance alpha3-GABAaR function . This breakthrough provides a unique opportunity to understand their role in neuronal signalling/behaviour and to evaluate them as a target for treating anxiety/stress, depression, schizophrenia and drug addiction. In Dundee, the project will use voltage-clamp and mouse brain slices to characterise the role of alpha3-GABAaRs in areas that express the receptor and that are implicated in anxiety/stress, depression and addiction (ventral tegmental area- VfA and dorsal raphe- DR). At Lilly I will train in microdialysis of awake behaving rodents to measure changes of neurotransmitter
levels in specific brain regions after in vivo administration of alpha3-drugs. By simultaneously recording from electrodes in these areas, drug impact on neuronal activity can be determined , and by calculating levels of coherence between
regions, co-activation of different circuits assessed, during different behavioural states, or physiological conditions e.g. stress. Specifically: 1) VTA GABAergic and dopamine neurons primarily express alpha1- and alpha3-GABAaRs respectively (1). Using GAD67-GFP mice, biocytin-filled cells and posthoc tyrosine hydroxylase staining, the effect of an alpha3-drug will be compared with diazepam on the amplitude, kinetics and frequency of GABA and dopamine neuron spontaneous inhibitory postsynaptic currents (slPSCs) mediated by quantally-released GABA Diazepam, via alpha1-GABAaRs should reduce GABA release on to dopamine neurons and thereby via disinhibit ion increase dopamine release in the nucleus accumbens, despite an effect on alpha3-GABAaRs . By contrast, we predict the alpha3-selective drug will have no effect on GABA release, but by selectively prolonging dopamine neuron slPSCs, decrease dopamine release (Dundee mths. 0-18). The in vitro study predictions will be investigated by microdialysis measuring accumbal dopamine release and on accumbal activity by in vivo electrophysiology (Lilly mths. 37-40). As the diazepam increase of dopamine release underpins dependence liability, these studies will interrogate whether alpha3 drugs are likely to be devoid of such side effects. 2) DR immunohistochemistry reveals extrasynaptic and synaptic alpha3 subunit staining, which is increased by mild stress . Given the role of the DR in stress and anxiety, using a 5HT reporter mouse (THChrEYFP) and a selective ligand I will determine the role of alpha3-GABAARs in phasic (synaptic) and tonic (extrasynaptic) inhibition of DR 5HT neurons and their impact on the spontaneous firing of 5HT neurons. The investigations will be repeated in an established mouse model of early life adversity (Dundee mths. 19-36). The studies will be complemented by light activation of 5HT neurons using the channel rhodopsin expressing THChrEYFP mouse (established Dundee) and by in vivo electrophysiology and microdialysis assessment of 5HT release in prefrontat cortex and hippocampus (Lilly mths 41-44). Mths. 45-48-thesis production. (1) Rudolph, U. & Mohler, H. (2014). Ann. Rev. Pharmacol. Toxicol. 54: 483-507.