Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.747542
Title: Diazepam-dependent modulation of GABAergic inhibitory synapses
Author: Nicholson, Martin William
ISNI:       0000 0004 7231 2951
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2018
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Abstract:
Diazepam is an allosteric modulator of GABAA receptors which potentiates GABAA receptor activity resulting in enhanced inhibitory synaptic transmission. Diazepam is used to treat anxiety, insomnia and seizures, however, its use is limited due to the development of tolerance. Here I show that prolonged treatment of cortical neurones with diazepam triggers endocytosis and subsequent downregulation of cell-surface GABAA receptors. Using pharmacological reagents, I have demonstrated that diazepam triggers PLC-dependent release of calcium from the endoplasmic reticulum which activates the phosphatase calcineurin resulting in dephosphorylation of the γ2 subunit of GABAA receptors and their endocytosis. This was elucidated using a combination of biochemical and cell biological approaches. In addition, I have developed HEK293 cell lines stably expressing various subtypes of GABAA receptors to investigate further diazepam and isoguvacine-dependent regulation of GABAA receptors. The same calcium-dependent signalling pathway that regulates cell-surface stability of GABAA receptors in neurones was found to operate in HEK293 cells. Subsequently, I focused on a key component of this signalling pathway; PLCδ1. Using biochemical techniques I have demonstrated that PLCδ1 binds directly to the GABAA receptor β3 subunit at two independent sites. This binding was confirmed by coimmunoprecipitation of PLCδ1 and GABAA receptors from cortical neuronal lysates. Interestingly, upon diazepam treatments, PLCδ1 was shown to dissociate from GABAA receptors, thus leading to mobilisation of calcium from the intracellular stores and activation of calcineurin. To assess how changes in cell-surface expression of GABAA receptors affect the stability of GABAergic synapses, I characterised the size and number of post-synaptic GABAA receptor clusters and the number of presynaptic GABA-releasing terminals following chronic diazepam treatment. I observed a reduction in the size and number of post-synaptic GABAA receptor clusters and a reduction in the number of GABA-releasing terminals. These data are consistent with the loss of cell-surface GABAA receptors following long-term treatments of cortical neurones with diazepam. These changes correlated with an increase in the expression of the early apoptosis marker, cleaved-caspase 3, in glutamatergic neurones suggesting indirect cytotoxic effects of diazepam treatments. The loss of inhibitory GABAergic synapses following chronic diazepam treatment may contribute to the well-known development of tolerance to these clinically important therapies for stress- and anxiety- related neurological disorders.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.747542  DOI: Not available
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