Al Hosni, Rumaitha Nasser Ali; (2020) Modulation of NMDA Receptor Currents in Rat Substantia Nigra. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Dopamine receptor signalling is essential for normal basal ganglia function but in Parkinson’s Disease (PD) substantia nigra (SNc) dopaminergic (DAergic) neurons degenerate with consequent loss of dopamine signalling. SNc DAergic neurons express D2 autoreceptors (D2Rs) that have been shown to mediate inhibition of NMDA responses in both hippocampus and striatum while Gascoupled adenosine A2A receptors (A2ARs) have the potential to counteract the action of Gai -coupled D2Rs. Here I tested whether D2R activation with ropinirole, a D2 receptor agonist currently used in PD therapy, modulates DAergic neuron NMDA responses in the SNc along with other proteins in the cell. Whole-cell patch clamp recordings were made from DAergic neurons in the SNc of acute midbrain slices from young (P7, P21 and P28) rats. DAergic neurons were identified by the presence of a prominent hyperpolarisation-activated inward current (in P7 rats, amplitude, 178 ± 5 pA; activation time constant, 797 ± 77 ms; mean ± SEM, N = 19) in response to a voltage step from -60 to -120 mV. In P7 and P28 rats, upon application of 200 nM ropinirole, the steady state NMDA current was not significantly changed suggesting D2-R activation may not modulate NMDARs in neonatal rat SNc. In addition, an A2AR agonist, CGS21680, and an A2AR antagonist, SCH 58621 were applied in the presence and absence of ropinirole to test for any A2AR – D2R interaction. Upon A2A-R activation, the NMDA-R current increased (P = 0.002, N = 16). Furthermore, to establish the effects of PKA on NMDA-R responses, 2.5µM Forskolin was introduced. It produced a statistically significant increase in NMDA-R current (NMDA: 419 ± 78pA; NMDA+ Forskolin: 515 ± 54pA, N=13). To determine whether the lack of effect of the D2-R agonist on NMDA-R response might be due to a low resting concentration of cAMP in the cell, forskolin was introduced to increase the levels of cAMP prior to introducing ropinirole. However, following addition of D2-R agonist after forskolin treatment, the NMDA-R current changed by only 11% (N=12). Intracellular tyrosine kinases, Src and Fyn have shown modulatory potential on NMDA-Receptors (NMDA-R) that is governed by the balance between kinase and phosphatase activity. Inhibiting Src kinase activity with PP2 and Src-I1 decreased the NMDA-R inward current however no such effect was seen in the presence of the interfering peptides suggesting a lack of direct interaction between Src/Fyn kinase and NMDA-Rs. Furthermore, ERK1/2 inhibitor, Ulixertinib, decreased the NMDA-R current suggesting an involvement in receptor modulation. Similar results were obtained in the presence of a CaMKII inhibitor CN21.

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