Actions of the ORL1 receptor ligand nociceptin on membrane properties of rat periaqueductal gray neurons in vitro

Abstract

The actions of the endogenous ORL1-receptor ligand nociceptin on the membrane properties and synaptic currents in rat periaqueductal gray (PAG) neurons were examined by the use of whole-cell patch-clamp recording in brain slices. Nociceptin produced an outward current in all neurons tested, with an EC50 of 39 +/- 7 nM. The outward current was unaffected by naloxone. Outward currents reversed polarity at -110 +/- 3 mV in 2.5 mM extracellular potassium, and the reversal potential increased when the extracellular potassium concentration was raised (slope = 66.3 mV/log[K+]o mM). Thus, the nociceptin-induced outward current was attributable to an increased K+ conductance. Nociceptin inhibited evoked fast GABAergic (IP-SCs) and glutamatergic (EPSCs) postsynaptic currents and increased paired-pulse facilitation in a subpopulation of PAG neurons. Nociceptin inhibited evoked IPSCs and EPSCs in approximately 50% of neurons throughout the PAG, except in the ventrolateral PAG, where nociceptin inhibited evoked IPSCs in most neurons. Nociceptin decreased the frequency of spontaneous miniature postsynaptic currents (mIPSCs and mEPSCs) in a subpopulation of PAG neurons but had no effect on their amplitude distributions. Thus, nociceptin had a presynaptic inhibitory effect on transmitter release. These findings suggest that nociceptin, via its pre- and postsynaptic actions, has the potential to modulate the analgesic, behavioral, and autonomic functions of the PAG.

Fig. 1.

Nociceptin produces a dose-dependent outward current in all PAG neurons. Membrane current responses of PAG neurons in which A, met-enkephalin (ME 10 μm) had no effect, but nociceptin (Noc 300 nm) produced an outward current, andB, both met-enkephalin (ME 10 μm) and nociceptin (Noc 300 nm) produced an outward current. C, Concentration–response relationship for outward currents in PAG neurons produced by nociceptin. Each point shows the mean ± SEM of responses of several different neurons, with thenumber of cells indicated above thepoint. A logistic function was fit to determine the EC₅₀ (39 ± 7 nm). Neurons were clamped to a potential of −60 mV.

Fig. 2.

Nociceptin increases inwardly rectifying K⁺ conductance of PAG neurons. A, Voltage command steps of 250 msec duration were made in 10 mV increments from a holding potential of −60 to −140 mV. The resulting membrane currents in the absence (Control) and presence of nociceptin (Noc 300 nm) in a single neuron are shown. B, The current–voltage relationship for control (○), nociceptin (•), and wash (□) is plotted from the amplitudes of evoked currents shown in A.

Fig. 3.

Nociceptin inhibited evoked GABAergic IPSCs in a subpopulation of PAG neurons. Evoked IPSCs were produced by local stimulation in the presence of CNQX (10 μm). Shown is time course of the amplitude of evoked IPSCs (eIPSC) during the application of nociceptin (Noc; 300 nm) and then baclofen (Bacl; 10 μm) for PAG neurons in which nociceptin did (A) and did not (B) inhibit synaptic transmission. IPSCs were evoked every 30 sec. C, D, Averaged traces (n = 4) of evoked IPSCs before and then during application of nociceptin (Noc; 300 nm) and baclofen (Bacl; 10 μm) for the time plots of A and B, respectively.E, F, Histograms of the inhibition of the evoked IPSC produced by (E) nociceptin and (F) baclofen in all neurons. Neurons were clamped to a potential of −74 mV.

Fig. 4.

Nociceptin inhibited evoked glutamatergic EPSCs in a subpopulation of PAG neurons. Evoked EPSCs were produced by local stimulation in the presence of bicuculline (30 μm).A, Neuron in which nociceptin (300 nm) inhibited the evoked EPSC. B, Neuron in which nociceptin had no effect on the evoked EPSC. The subsequent application of baclofen (10 μm) inhibited the evoked EPSCs in bothA and B. Averaged traces (n = 4) are shown before, during nociceptin (Noc) and baclofen (Bacl), and after washout. Neurons were clamped to a potential of −74 mV.

Fig. 5.

Nociceptin decreases the frequency of spontaneous mIPSCs. Spontaneous mIPSCs were recorded in the presence of TTX (0.3 μm) and CNQX (10 μm). A, Five consecutive segments before, during, and after application of nociceptin (300 nm). B, C, Cumulative distribution plots of mIPSC inter-event interval and amplitude before, during (dashed line), and after application of nociceptin (number of events = 163, 177, and 112 for 80, 160, and 60 sec epochs of Control,Nociceptin, and Wash, respectively). Nociceptin had no effect on the amplitude distribution (p > 0.3, K–S statistic for control/nociceptin) but reversibly shifted the frequency distribution to longer inter-event intervals (p < 0.03, K–S statistic for control/nociceptin). All data are from the same neuron, which was clamped to a potential of −74 mV.

Fig. 6.

Nociceptin decreases the frequency, but has no effect on the amplitude, of both mIPSCs and mEPSCs in a subpopulation of PAG neurons. A, mIPSC andB, mEPSC frequency (filled bars) and mean amplitude (open bars) before, during, and after the application of nociceptin (300 nm) for those neurons in which there was a significant increase in inter-event interval (p < 0.05, K–S statistic for control/nociceptin). The pooled results of six neurons for mIPSCs and eight neurons for mEPSCs are shown. Error bars indicate ± SEM.

Fig. 7.

Nociceptin decreases the frequency of spontaneous mEPSCs. Spontaneous mEPSCs were recorded in the presence of TTX (0.3 μm) and bicuculline (30 μm).A, Shown are five consecutive segments before, during, and after application of nociceptin (300 nm).B, C, Shown are cumulative distribution plots of mEPSC inter-event interval and amplitude before, during (dashed line), and after application of nociceptin (number of events = 131, 105, and 168 for 50, 80, and 70 sec epochs of Control, Nociceptin, andWash, respectively). Nociceptin had no effect on the amplitude distribution (p > 0.3, K–S statistic for control/nociceptin) but reversibly shifted the frequency distribution to longer inter-event intervals (p < 0.05, K–S statistic for control/nociceptin). All data are from the same neuron, which was clamped to a potential of −74 mV.

Fig. 8.

Anatomical location of PAG neurons displaying nociceptin-sensitive and nociceptin-insensitive evoked IPSCs. Locations of PAG neurons in which evoked IPSCs were unaffected (○, inhibition <15%; n = 20) or inhibited (•;n = 44) by nociceptin (300 nm). Three dorsoventral levels of horizontal midbrain sections are shown (3.9, 4.4, and 4.9 mm dorsal to the interaural plane). Insetshows a coronal PAG section 0.7 mm rostral to the interaural plane, indicating the levels at which horizontal sections were taken.4V, Fourth ventricle; Aq, aqueduct;DL, dorsolateral PAG; DM, dorsomedial PAG; DR, dorsal raphe; L, lateral PAG;VL, ventrolateral PAG.