Modulation of [gamma]-aminobutyric acid (GABA) type A receptor-mediated responses in spinal dorsal horn neurons by [mu]-opioid receptor agonists and Ca²+/calmodulin-dependent protein kinase and Monte Carlo simulation of the GABAergic synaptic transmission

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Wang, Rong
Major Professor
James L. Cornette
Mirjana Randic
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GABA is believed to be a major inhibitory neurotransmitter in the central nervous system. This work first used whole-cell voltage clamp technique the modulatory effects of [mu]-opioid receptor agonists and [alpha]-subunit of CaM-KII on GABA-induced currents in acutely isolated spinal dorsal horn (DH) neurons.;We found that a bicuculline-sensitive GABA-induced current was predominantly potentiated by [mu] agonists, DAGO and PLO17, in a dose dependent manner (in about 64% of the tested cells). The potentiating effect of DAGO was reduced or prevented by naloxone and the [mu]-opioid receptor selective antagonist [beta]-funaltrexamine. The potentiating effect of [mu]-opioids was mimicked by the Rp isomer of adenosine-3'5',-cyclic monophosphothioate (Rp-cAMPS), which is a potent inhibitor of protein kinase A (PKA), and blocked by intracellular dialysis of Sp isomer of the cAMPS, which activates PKA. Furthermore, the enhancing effect of PLO17 was also blocked in DH neurons which had been incubated for 5-8 hours with pertussis toxin.;Intracellularly applied, the [alpha]-CaM-KII enhanced the GABAA receptor-activated current. Calyculin A, an inhibitor of protein phosphatases 1 and 2A, also enhanced the GABA responses. Conventional intracellular recordings were made from hippocampal CA1 neurons in slices to determine the effect of intracellular application of CaM-KII on inhibitory synaptic potentials evoked by electrical stimulation of the stratum oriens/alveus. The inhibitory synaptic potential was enhanced by CaM-KII.;These findings suggest that in acutely isolated rat spinal DH neurons (and in hippocampal CA1 neurons), GABAA receptor-mediated responses can be modulated by [mu]-opioid agonists DAGO and PLO17 and endogenous and/or exogenous CaM-KII. The potentiating effect of the [mu]-opioids might have involved the cAMP-dependent second messenger system.;This study then used the Monte Carlo method to simulate the GABAA-mediated postsynaptic inhibitory current (IPSC) response in a model synapse. We tested the role of GABA release and receptor population in shaping IPSCs. Results of the simulations indicated that over 90% of GABA diffuse out of synaptic junction within tens of microseconds following their release. If there is no mechanism to keep a high concentration of GABA in the synaptic cleft, then increasing the number of postsynaptic receptors does not appear to be very efficient in enhancing the IPSCs. However, short persistence (1ms) of high concentration of GABA (in mM range) in the synaptic cleft is an efficient way to maintain a high receptor efficacy and to enhance the synaptic response through increasing the number of available receptors. In addition, the role of desensitized state D and intraburst closed states proposed in the kinetic model of GABAA receptors (Twyman, et al., 1992) was also examined.

Sun Jan 01 00:00:00 UTC 1995