The role of alpha3-GABAA receptors in mono-aminergic neuronal function

Lead Research Organisation: University of Dundee
Department Name: Neuroscience

Abstract

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.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M01620X/1 01/10/2015 30/09/2019
1721621 Studentship BB/M01620X/1 01/10/2015 30/09/2019 Grant David Phillips
 
Description My studies initially investigated the effects of an a3-GABAA receptor (a3-GABAAR) selective positive modulator of neuronal inhibition. The GABAAR is a major inhibitory receptor in the mammalian CNS and is a target for many clinically important drugs used to treat conditions such as anxiety, insomnia and epileptic seizures. However, such drugs do not discriminate between the various GABAAR subtypes and consequently are associated with serious side effects, limiting their clinical utility. Most neuronal synapses express multiple GABAAR subtypes, complicating interpretation. My host lab revealed mouse thalamic nRT neurons to primarily express synaptic a3-GABAARs, whilst thalamic VB neurons solely express a1-GABAARs at > P16, but synaptic a2-GABAARs at P8-10. Using whole-cell voltage-clamp techniques I have demonstrated the Lilly compound to selectively enhance synaptic a3-GABAARs with no effect on a2- or a1-GABAARs. Additionally, I demonstrated the drug to facilitate neuronal inhibition in the nucleus accumbens shell, which expresses a3-GABAARs, but to have little effect in the accumbal core, which expresses a2-GABAARs. As far as I am aware this is the first example of a truly a3-GABAAR selective drug.
My host lab developed a murine model of Early Life Adversity (ELA). Prior experience of ELA is now recognised as a major cause of future psychiatric disorders such as depression, anxiety and impaired cognition. As positive allosteric modulators (PAMS) of a3-GABAARs are putative anxiolytics and chronic stress is associated with changes in GABAAR expression I have extended my study to investigate how prior experience of ELA might influence neuronal inhibition and excitation in the mouse hippocampus. The hippocampus was chosen given studies have highlighted the role of hippocampal GABAARs in mediating the anxiolytic effect of diazepam.
This investigation revealed that the properties of hippocampal CA1 pyramidal synaptic GABAARs are perturbed by prior experience of ELA, as indicated by a decrease in the amplitude and frequency of miniature inhibitory postsynaptic currents (mIPSCs). Furthermore, the Lilly drug enhanced hippocampal neural inhibition, demonstrating for the first time the functional presence of synaptic a3-GABAARs in these hippocampal CA1 neurones. Although, GABA-ergic inhibition was perturbed by ELA, the effect of the a3-GABAAR PAM was not influenced by this prior adverse experience.
During the course of this investigation I additionally discovered that prior ELA greatly impaired hippocampal fast glutamatergic excitation, manifest as a decrease in the amplitude of miniature excitatory postsynaptic currents (mEPSCs). Previous studies from the Dundee lab had shown that the atypical antidepressant tianeptine acts to enhance glutamatergic excitation by selectively enhancing AMPARs composed of the GluA1 subunit. Remarkably this effect of the antidepressant, was nearly abolished in ELA mice, inferring this prior experience to have impaired synaptic GluA1-AMPAR expression. Given the critical role of GluA1-AMPARs in early development of neuronal circuits and in various forms of synaptic plasticity this impairment of excitatory transmission may contribute to the subsequent behavioural impairments evident in adult ELA mice. In translating to the clinical situation, it suggests that tianeptine may not be efficacious in the treatment of certain forms of depression associated with prior ELA. Recent studies have reported the ketamine metabolite 2R,6R-HNK to enhance AMPAR function and to exhibit rapid antidepressant-like actions in rodent models of mood disorders. By contrast to tianeptine, I found the AMPAR-enhancing effects of 2R,6R-HNK were not influenced by prior ELA, suggesting an alternative putative approach.
Given these exciting in vitro findings it was decided for my placement at Lilly to extend the study to profile the influence of prior ELA upon neuronal activity using EEG and implanted hippocampal/cortical electrodes in freely moving mice and to determine the effects of tianeptine and the rapid acting antidepressant ketamine. The ELA paradigm was established at Lilly. I became proficient at surgical implantation of electrodes to record hippocampal and cortical activity. Both tianeptine and ketamine produced clear changes in hippocampal activity, although the profile of the two antidepressant was quite distinct. The study has generated considerable and complex data and we are still engaged in their analysis having taken up a postdoctoral position in Dundee.
Exploitation Route Initially, I characterised the actions of a novel, selective PAM of a3-GABAARs. As far as I am aware the GABAAR isoform profile of the compound is unique. This data is held by the industrial placement holder (Eli Lilly) with the intention of publication in the near future in collaboration with the Dundee laboratory and other industrial collaborators not associated with the host laboratory in Dundee. It is anticipated that the publication will be of considerable interest to academic and industrial researchers in the field.
The in vitro studies on ELA and the antidepressants will be submitted soon for publication. The extensive analysis of the information obtained from the in vivo studies of freely moving mice (control and ELA) is ongoing prior to submission for publication.
My studies at Dundee University trained me in making high quality in vitro recordings from identified single neurons. This approach has been complemented by the invaluable skills obtained during my placement at Eli Lilly. In my current postdoctoral post funded by the MRC I am using these in vitro and in vivo skills to investigate the effects of novel GABAAR subtype-selective compounds, as part of an academic collaboration to develop novel anxiolytics.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Bright Club stand-up preformance 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Communicate Grant's research in the novel format of stand-up comedy, to engage the general public and other students of the university initially. Due to interest in Grant's research and his performance, Grant was asked to preform again at a ticketed public engagement event for the university of Dundee. Grant preformed a second stand-up set in front of 100 University administrators and members of the public and university donors about the research he was undertaking. This led to multiple conversations with university officials who were interested in his research and the possibility for more public engagement events.
Year(s) Of Engagement Activity 2017,2018