Effect And Mechanism Of Allopregnanolone On The Release Of Presynaptic Glutamate In Rat Medial Prefrontal Cortical Neurons

Allopregnanolone(Allo) is among the most active members of neurosteroids, which has an important influence on brain functions. A number of studies have indicated that Allo is a positive allosteric modulator of GABA_a receptors and can strengthen the inhibitory synaptic transmission by potentiating the function of GABA_a receptors to have sedative hypnotic, anxiolytic, anticonvulsant, antiepileptic action and to play important roles in learning and memory, cognition and psychoneurotic disease. However, there are fewer reports about the studies of the effects of allopregnanolone on excitatory synaptic transmission.In the present study, we investigated effect of Allo on the spontaneous and evoked excitatory postsynaptic currents in pyramidal cells of layers V-VI of the rat medial prefrontal cortex by using whole-cell patch-clamped recording method in slices. And we also investigated effect of Allo on the spontaneous and induced PKA activity in synaptosomes of the rat medial prefrontal cortex by using biochemistry technique . And then, we further explored the mechanism of the effect of Allo by using electrophysiological and biochemical methods combined with a pharmacological approach in slices or synaptosomes. The results showed that Allo had no effect on the frequency of spontaneous mEPSCs and sEPSCs in the pyramidal cells of the layers V-VI of the medial prefrontal cortex, but that Allo markedly inhibited depolarizing agent high K~+/veratridine-evoked increase in the frequency of mEPSCs/sEPSCs. Interestingly, Allo had a selective effect on neurotransmitter 5-HT /DA-evoked increase in the frequency of sEPSCs in the pyramidal cells of the layers V-VI of the medial prefrontal cortex, which didn't inhibit 5-HT-evoked effect but inhibited DA-evoked effect. To study the mechanism of the inhibitory effect of Allo on the high K~+/ veratridine/DA-evoked increase in the frequency of sEPSCs, we investigated the influence of PKA antagonist H89 on veratridine-evoked effect, the influence of Allo on adenyl cyclase(AC) agonist forskolin-evoked effect and on the intracellular calcium store releasing agent ryanodine-evoked effects and on the L-type calcium channel agonist Bay K-evoked effect, and the influence of the L-type calcium channel calcium channel antagonist verapamil and Gi protein blocker NEM on the inhibitory effect of Allo on veratridine. The result was that H89 significantly inhibitedveratridine-evoked effect; Allo didn't inhibit forskolin and ryanodine-evoked increase in the frequency of sEPSCs but notablely inhibited Bay k-evoked increase in the frequency of sEPSCs; NEM didn't block the inhibitory effect of Allo on evoked-increase in the frequency of sEPSCs, but Bay K blocked the inhibitory effect of Allo on veratridine.At one time, we also studied the influence of GABA on veratridine-induced increase in the PKA activity and the influence of Allo on PKA activity. The finding of the study were that GABA had no influence on veratridine-induced effect; Allo had no effect on basal PKA activity, but markedly inhibited increase in the PKA activity of high K+, veratridine, DA, 5-HT and Bay K-induced; Allo couldn't inhibit increase in the PKA activity of forskolin , D1 receptor agonist SKF, ryanodine-induced; NEM , D2 receptor antagonist sulpride, and N-type and T-type and P/Q-type calcium channel antagonist didn't blocked the effect of Allo on high K~+-evoked increase of PKA activity, but L-type calcium channel calcium channel antagonist verapamil and nimodipine both blocked the effect of Allo on high K~+.In the striatum, our study indicated that Allo and verapamil didn't inhibit veratridine-evoked increase in the PKA activity and the frequency of sEPSCs.The above result showed that Allo inhibit high K+, veratridine and DA-evoked increase in the PKA activity and in the frequency of sEPSCs, not through acting on these site of PKA, AC, Gi protein, dopamine D1 and D2 receptor, the intracellular calcium store release, N-type and T-type and P/Q-type calcium channel, but through acting on L-type calcium channel; Allo have different effect in the different brain region on evoked-increase in the PKA activity and the frequency of sEPSCs.