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Title: Pharmacological characterisation of GABA receptors in potential spinal cord neural stem cells
Author: Shafin, Nazlahshaniza
ISNI:       0000 0004 8500 9695
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2019
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Modulation of GABA mediated inhibition is one of the most important approaches for the treatment of central nervous system (CNS) diseases. Precise targeting of such treatments depends on identification and characterisation of the different subunit complexes that exist. In the CNS, there are pools of neural stem cells (NSC), which can differentiate to become neurones, astrocytes or oligodendrocytes while progenitor cells have limited lineage. In addition to brain regions, where NSCs are now known to exist, the spinal cord area also has a neurogenic potential in the form of ependymal cells. Ependymal cells surrounding the central canal displayed typical properties of glial cells; lack of voltage-gated channels, and showed coupling indicative of the presence of gap junction. Ependymal cells showed responses to GABA as previously observed(Corns et al., 2013). The effects of GABA on ependymal cells were concentration dependant. Ependymal cells in our study showed atypical GABA receptors since it was not fully blocked by GABAA receptor antagonist; bicuculline. Hyperpolarising responses to low GABA were antagonised by the GABAρ receptor antagonist, (1, 2, 5, 6- Tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA). On further confirmation of this, Trans-4-aminocrotonic acid (TACA), a selective agonist of GABAρ1 elicited robust and reproducible hyperpolarising responses, similar to those observed with low concentrations of GABA. This provides evidence of a combination or coassembly of GABAA and ρ receptors in the ependymal cells or the presence of two different receptors. One potential site of modulation of GABA receptors is through diazepam binding inhibitor (DBI). DBI with its breakdown product, octadecaneurapeptide (ODN) acts as positive and negative modulator at central benzodiazepine receptor (CBR). The responses to high concentrations of GABA were modulated by midazolam suggesting a potential involvement of CBR and another receptor, Translocator Protein (TSPO). On testing TSPO, N, N-Dihexyl-2-(4-fluorophenyl) indole-3-acetamide (FGIN-1-27); selective agonist at TSPO, increased the responses in ependymal cells to the higher concentration of GABA. When combining FGIN-1-27 with 1-(2-Chlorophenyl)-Nmethyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK-11195); a selective TSPO antagonist, the augmented GABA responses were not observed, suggesting that this effect was due to activation of TSPO. These results indicate that modulation of GABA receptors by midazolam occurred at both CBR and TSPO. However, the responses of GABA in the presence of ODN (20 µM) did not show significant changes regardless of the GABA concentration used. Nanomolar ODN application alone causes fast hyperpolarisations similar to low GABA and TACA effects. Responses to nanomolar ODN were significantly but not fully blocked by cyclo1-8[dleu5] OP (cDLOP), an ODN metabotropic receptor antagonist. ODN is somehow directly activating the GABA receptors that contain rho subunits since the hyperpolarising effects of ODN were significantly blocked by TPMPA. So there is an evidence there are atypical GABA receptors responsible for this action with ODN. Baclofen (a selective GABAB receptor agonist) also hyperpolarised ependymal cells, an effect antagonised by CGP 55845 (GABAB antagonist), indicating a further role for GABAB receptors on ependymal cells. The numbers of proliferative cells in the central canal region were significantly lower in baclofen treated slices compared with control. Activation of the 5-HT receptor caused both depolarisation and hyperpolarisation contribute to the proliferative capabilities of ependymal cells. Blockade of 5-HT receptor mediated depolarisations with cinanserin showed the presence of 5-HT2 receptors in the spinal cord. Furthermore, hyperpolarisations with 8-OH-DPAT and cisapride indicate that 5-HT 1A and 5-HT4 receptors are also present in the central canal area of spinal cord. Overall, our study has shown a complex and varied response profile of ependymal cells to GABA and its modulators. This could have important implications in the proliferative capabilities of spinal cord neural stem cells and thus functional consequences. The modulation of proliferation and differentiation by GABA may be an interesting future therapeutic target for conditions where there is over proliferation such as in spinal cord ependymomas or in conditions such as in multiple sclerosis where replacement cells may be useful to aid recovery.
Supervisor: Deuchars, Susan ; Deuchars, Jim Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available