Inhibition by adenosine receptor agonists of synaptic transmission in rat periaqueductal grey neurons

Abstract

1. The actions of selective adenosine A1 and A2 receptor agonists were examined on synaptic currents in periaqueductal grey (PAG) neurons using patch-clamp recordings in brain slices. 2. The A1 receptor agonist 2-chloro-N-cyclopentyladenosine (CCPA), but not the A2 agonist, 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS21680), inhibited both electrically evoked inhibitory (eIPSCs) and excitatory (eEPSCs) postsynaptic currents. The actions of CCPA were reversed by the A1 receptor antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX). 3. In the absence or presence of forskolin, DPCPX had no effect on eIPSCs, suggesting that concentrations of tonically released adenosine are not sufficient to inhibit synaptic transmission in the PAG. 4. CCPA decreased the frequency of spontaneous miniature action potential-independent IPSCs (mIPSCs) but had no effect on their amplitude distributions. Inhibition persisted in nominally Ca2+-free, high Mg2+ solutions and in 4-aminopyridine. 5. The CCPA-induced decrease in mIPSC frequency was partially blocked by the non-selective protein kinase inhibitor staurosporine, the specific protein kinase A inhibitor 8-para-chlorophenylthioadenosine-3',5'-cyclic monophosphorothioate (Rp-8-CPT-cAMPS), and by 8-bromoadenosine cyclic 3',5' monophosphate (8-Br-cAMP). 6. These results suggest that A1 adenosine receptor agonists inhibit both GABAergic and glutamatergic synaptic transmission in the PAG. Inhibition of GABAergic transmission is mediated by presynaptic mechanisms that partly involve protein kinase A.

Figure 1. A₁ adenosine receptor agonist inhibits GABAergic IPSCs in PAG neurons

eIPSCs produced by local stimulation in the presence of CNQX (10 μM) at a holding potential of -75 mV. A, time course of the amplitude of eIPSCs during the application of CCPA (1 μM) and then CCPA plus DPCPX (1 μM). B, averaged traces (4 for each trace) of eIPSCs before and during application of CCPA (1 μM), and CCPA plus DPCPX (1 μM) from the cell shown in the time plot (A). C, concentration-response relationship for inhibition of eIPSC amplitude by CCPA. Each point shows the mean ±s.e.m. of responses of 6-12 neurons. The fitted logistic function is shown.

Figure 2. A₁ adenosine receptor agonist inhibits glutamatergic EPSCs in PAG neurons

eEPSCs produced by local stimulation in the presence of bicuculline (30 μM) at a holding potential of -75 mV. A, time course of the amplitude of eEPSCs during the application of CCPA (1 μM) and then CCPA plus DPCPX (1 μM). B, averaged traces (4 for each trace) of eEPSCs before and during application of CCPA (1 μM), and CCPA plus DPCPX (1 μM) for the cell shown in the time plot (A).

Figure 3. A₁ adenosine receptor agonist decreases the frequency of mIPSCs in PAG neurons

mIPSCs were recorded in the presence of TTX (0.3 μM) and CNQX (3 μM). All data shown are from the same neuron, which was clamped at -75 mV. A, 3 consecutive sweeps before and during application of CCPA (1 μM), and CCPA plus DPCPX (1 μM). B, time course of the frequency of mIPSCs before and during application of CCPA (1 μM), and CCPA plus DPCPX (1 μM). C, cumulative distribution plots of mIPSC amplitude and inter-event interval before and during application of CCPA (dashed line), and CCPA plus DPCPX. CCPA had no effect on the amplitude distribution (P > 0.3, Kolmogorov-Smirnov statistics for control - CCPA), but reversibly shifted the frequency distribution to longer inter-event intervals (P < 0.01, Kolmogorov-Smirnov statistics for control - CCPA).

Figure 4. Inhibition of mIPSC frequency is partially mediated by adenylyl cyclase and protein kinase A

Inhibition of mIPSC frequency by CCPA (1 μM; ▪) and methionine enkephalin (10 μM; □) during superfusion of the indicated drugs is presented. 8Br, 8-Br-cAMP (1 mM); Staur, staurosporine (1 μM); RP, Rp-8-CPT-cAMPS (100 μM); Ca²⁺ free, 0 mM Ca²⁺ and 10 mM Mg²⁺; 4-AP, 4-aminopyridine (0.1-1 mM). Asterisks signify statistical significance for comparisons between inhibition of mIPSC frequency in control and in the specified treatment (P < 0.01, Student's unpaired t tests).