Nociceptin/orphanin FQ decreases glutamate transmission and blocks ethanol-induced effects in the central amygdala of naive and ethanol-dependent rats

Abstract

The central nucleus of the amygdala (CeA) mediates several addiction-related processes and nociceptin/orphanin FQ (nociceptin) regulates ethanol intake and anxiety-like behaviors. Glutamatergic synapses, in the CeA and throughout the brain, are very sensitive to ethanol and contribute to alcohol reinforcement, tolerance, and dependence. Previously, we reported that in the rat CeA, acute and chronic ethanol exposures significantly decrease glutamate transmission by both pre- and postsynaptic actions. In this study, using electrophysiological techniques in an in vitro CeA slice preparation, we investigated the effects of nociceptin on glutamatergic transmission and its interaction with acute ethanol in naive and ethanol-dependent rats. We found that nociceptin (100-1000 nM) diminished basal-evoked compound glutamatergic receptor-mediated excitatory postsynaptic potentials (EPSPs) and spontaneous and miniature EPSCs (s/mEPSCs) by mainly decreasing glutamate release in the CeA of naive rats. Notably, nociceptin blocked the inhibition induced by acute ethanol (44 mM) and ethanol blocked the nociceptin-induced inhibition of evoked EPSPs in CeA neurons of naive rats. In neurons from chronic ethanol-treated (ethanol-dependent) rats, the nociceptin-induced inhibition of evoked EPSP amplitude was not significantly different from that in naive rats. Application of [Nphe1]Nociceptin(1-13)NH2, a nociceptin receptor (NOP) antagonist, revealed tonic inhibitory activity of NOP on evoked CeA glutamatergic transmission only in ethanol-dependent rats. The antagonist also blocked nociceptin-induced decreases in glutamatergic responses, but did not affect ethanol-induced decreases in evoked EPSP amplitude. Taken together, these studies implicate a potential role for the nociceptin system in regulating glutamatergic transmission and a complex interaction with ethanol at CeA glutamatergic synapses.

Figure 1

Nociceptin decreases evoked excitatory postsynaptic potential (EPSP) amplitudes in the central nucleus of the amygdala (CeA) of naive rats. (a, left panel) Histograms representing percent peak decrease in evoked (at half-max stimulus intensity) EPSP amplitudes during superfusion of different concentrations (100, 250, 500, and 1000 nM) of nociceptin and washout. (Right panel) Representative recordings of evoked EPSPs in CeA neurons from naive rats recorded before, during, and after washout from nociceptin application. Statistical significance of nociceptin's effect compared with baseline by paired t-test is indicated by *P<0.05 and **P<0.001. (b) I–V curve showing that superfusion of nociceptin (500 nM) does not change the RMP in rat CeA neurons (n=15). (c) Nociceptin (250 and 500 nM) significantly increases the paired-pulse facilitation (PPF) ratio of evoked EPSPs using 50 and 100 ms interstimulus intervals. Significance of the effect of nociceptin compared with baseline by paired t-test (*P<0.05). (d) Representative recordings of PPF at both 50 (left traces) and 100 (right traces) ms in a CeA neuron from a naive rat before and during superfusion of 500 nM nociceptin.

Figure 2

Ethanol decreases evoked excitatory postsynaptic potential (EPSP) amplitude in central nucleus of the amygdala (CeA) neurons of naive rats and nociceptin blocks this effect. (a) Ethanol (44 mM) significantly (P<0.05; n=11 by paired t-test) and reversibly decreases EPSP amplitudes to ∼80% of control. (Inset) Representative EPSP recordings in a CeA neuron during baseline, ethanol, and washout. (b) Ethanol had no effect on 50 or 100 ms paired-pulse facilitation (PPF) ratios of EPSPs. (c) I–V curve showing that superfusion of ethanol (44 mM) does not change the RMP in rat CeA neurons (n=11). (d) Time course of changes in evoked EPSP amplitude induced by nociceptin (500 nM), concurrent application of ethanol, and washout (n=8). (Inset) Representative CeA EPSP recordings during baseline, nociceptin, nociceptin+ethanol, and washout. (e) Nociceptin significantly decreases the mean amplitudes of evoked EPSPs over the middle three stimulus strength intensities tested. *Significance for P<0.05 of nociceptin effect compared with baseline. Subsequent application of ethanol (44 mM) did not alter the EPSP amplitudes (n=8). (f) Time course of changes in EPSP amplitude evoked by ethanol, concurrent application of nociceptin (250 nM), and washout (n=6). (Inset) Representative CeA EPSP recordings during baseline, ethanol, ethanol+nociceptin, and washout. (g) Application of a NOP antagonist ([Nphe1]nociceptin(1–13)NH2) (N-Phe, 1 μM) does not alter the basal-evoked EPSP amplitudes (stimulus intensity equal to half-maximal EPSP amplitude) in 11 CeA neurons of control rats (left histogram). The NOP antagonist prevents the nociceptin-induced decrease of EPSP amplitudes (n=4, middle histograms), but does not prevent the ethanol-induced decrease of EPSP amplitudes (n=5, right histograms). *Significance P<0.05 of ethanol and NOP antagonist effects.

Figure 3

The effects of nociceptin on evoked EPSPs are unchanged in ethanol-dependent rats. (a) Central nucleus of the amygdala (CeA) baseline compound glutamatergic transmission is equivalent in ethanol-dependent rats compared with control naive rats. Input/output curves of mean evoked EPSP amplitudes. Note that the EPSPs are superimposable in the two groups using five equivalent stimulus intensities. (b) In the CeA of ethanol-dependent rats, both 250 and 500 nM nociceptin significantly decreased the mean amplitudes of evoked EPSP over the middle three stimulus strength intensities tested. *Significance of the nociceptin effect compared with baseline for P<0.05. (Inset) Representative evoked CeA EPSPs from ethanol-dependent rats during the control, nociceptin application, and washout. (c) Nociceptin (250 and 500 nM) significantly increased the paired-pulse facilitation (PPF) ratio of evoked EPSPs using 50 and 100 ms interstimulus intervals. *Significance of nociceptin compared with baseline for P<0.05 by paired t-test. (d) Application of the NOP antagonist ([Nphe1]nociceptin(1–13)NH2) (1 μM) significantly increases basal-evoked EPSP amplitudes (stimulus intensity equal to half-maximal EPSP amplitude) in seven CeA neurons of ethanol-dependent rats. **Significance for P<0.001 of NOP antagonist compared with baseline. (e) Ethanol (44 mM) significantly (P<0.05; n=9) and reversibly decreases EPSP amplitudes to ∼80% of control over the stimulus intensities in CeA of ethanol-dependent rats. (Inset) Representative EPSP recordings in a CeA neuron during baseline, ethanol (44 mM), and washout. (f) Ethanol significantly decreases the 50 and 100 ms PPF ratio of EPSPs. *Significance for P<0.05.

Figure 4

Nociceptin prevents the ethanol-induced decrease in evoked EPSPs in ethanol-dependent rats. (a) Time course of changes in evoked EPSP amplitude induced by nociceptin (500 nM), concurrent application of ethanol, and washout in seven central nucleus of the amygdala (CeA) neurons of ethanol-dependent rats. (Inset) Representative CeA EPSP recordings during baseline, nociceptin, nociceptin+ethanol, and washout. (b) Nociceptin (500 nM) significantly decreases the mean amplitudes of evoked EPSPs compared with baseline over the middle three stimulus strength intensities tested (*significance for P<0.05). Subsequent application of 44 mM ethanol did not alter the EPSPs (n=7). (c, Inset) Representative recordings of paired-pulse facilitation (PPF) at 50 ms in a CeA neuron from an ethanol-dependent rat during baseline, nociceptin, nociceptin+ethanol, and washout. In ethanol-dependent rats, 500 nM nociceptin significantly increases the 50 and 100 ms PPF ratios of evoked CeA EPSPs (*significance at P<0.05). Ethanol in the presence of nociceptin does not alter these PPF ratios.

Figure 5

Nociceptin decreases spontaneous glutamatergic transmission mainly via decrease in glutamate release. (a) Representative spontaneous EPSCs (sEPSCs) and miniature EPSCs (mEPSCs) recordings in rat central nucleus of the amygdala (CeA) neurons before and during 250 nM nociceptin application. (b) Mean±SEM frequency, amplitude, rise, and decay time of sEPSCs and mEPSCs for CeA neurons from control-naive rats. Nociceptin significantly decreased the sEPSC and mEPSC frequencies indicating presynaptic inhibition of both spontaneous and vesicular release of glutamate in CeA. *Significance of nociceptin effect compared with baseline by paired t-test for P<0.05. Nociceptin also significantly decreased the sEPSC amplitude (*significance at P<0.05), suggesting also postsynaptic effects. (c) Representative sEPSC and mEPSC recordings in rat CeA neurons before and during 1 μM [Nphe1]nociceptin(1–13)NH2 in CeA neurons of control rats. (d) Mean±SEM frequency, amplitude, rise, and decay time of sEPSCs and mEPSCs for CeA neurons from control-naive rats. The NOP antagonist significantly increased the mean sEPSC and mEPSC frequencies (*significance at P<0.05), indicating presynaptic enhancement of both spontaneous and vesicular release of basal glutamate in CeA. (e) Mean±SEM frequency, amplitude, rise, and decay of sEPSCs for CeA neurons from ethanol-dependent rats. Nociceptin significantly decreased (*significance at P<0.05) and the NOP antagonist significantly increased (*significance at P<0.05) the mean sEPSC frequencies, indicating presynaptic actions of the nociceptin system on spontaneous glutamate release in CeA.