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Title: Mechanisms of GABAA and glycine receptor analgesia in the spinal dorsal horn : in vitro models as translational platforms for drug discovery
Author: Griffiths, Natalie Helen
ISNI:       0000 0004 5924 3226
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2015
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Available analgesics do not always provide adequate pain relief and are often associated with a significant side-effect profile. Following reports of a loss of inhibitory signalling in the spinal dorsal horn (DH) network in persistent pain conditions, γ-amino butyric acid type A (GABAA) and glycine receptors have been identified as promising targets. The present study assesses two in vitro models for their suitability for screening novel analgesics targeting GABAA and glycine receptors. Firstly, the embryonic cultured spinal DH cell model and secondly an acute rat spinal cord slice model. Immunofluorescence characterisation of the spinal DH culture illustrated that this model displays many similarities with the in vivo spinal DH. Immunofluorescence and RT-PCR demonstrated the presence of GABAA and glycine receptor subunits in the spinal DH culture. Calcium imaging and extracellular multi-electrode array (MEA) recording techniques were utilised to study the effect of GABAA, GABAB and glycine receptor drugs on the spinal DH culture network activity. All drugs tested significantly modulated the culture’s spontaneous firing. A further study assessed whether lentivirus and Accell siRNA mediated glycine receptor α subunit gene silencing modulates calcium responses in the DH culture model. The lentiviruses had low transfection efficiencies and caused cell death, however Accell siRNA transfection was successful and significantly decreased baseline spontaneous activity compared to untreated cultures. Single electrode and MEA extracellular recordings were performed with the acute spinal cord slice model. GABAA, GABAB and glycine receptor drugs modulated 4-aminopyridine-induced hyperexcitability in the substantia gelatinosa lamina of the slices. The MEA recordings illustrated that 4-aminopyridine-induced activity manifested more prominently in the DH than the ventral horn (VH) and that the DH network activity was highly synchronous. Taken together, these findings demonstrate that these in vitro models provide suitable platforms to test novel analgesics targeting GABAA and glycine receptors in the spinal DH network.
Supervisor: King, Anne E. ; Nickolls, Sarah Sponsor: BBSRC ; Pfizer
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available