MT-7716, a novel selective nonpeptidergic NOP receptor agonist, effectively blocks ethanol-induced increase in GABAergic transmission in the rat central amygdala

Abstract

The GABAergic system in the central amygdala (CeA) plays a major role in ethanol dependence and the anxiogenic-like response to ethanol withdrawal. A large body of evidence shows that Nociceptin/Orphanin FQ (N/OFQ) regulates ethanol intake and anxiety-like behavior. In the rat, ethanol significantly augments CeA GABA release, whereas N/OFQ diminishes it. Using electrophysiological techniques in an in vitro slice preparation, in this study we investigated the effects of a nonpeptidergic NOP receptor agonist, MT-7716 [(R)-2-3-[1-(Acenaphthen-1-yl)piperidin-4-yl]-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl-N-methylacetamide hydrochloride hydrate], and its interaction with ethanol on GABAergic transmission in CeA slices of naïve rats. We found that MT-7716 dose-dependently (100-1000 nM) diminished evoked GABAA receptor-mediated inhibitory postsynaptic potentials (IPSPs) and increased paired-pulse facilitation (PPF) ratio of these evoked IPSPs, suggesting a presynaptic site of action of the MT-7716 by decreasing GABA release at CeA synapses. The presynaptic action of MT-7716 was also supported by the significant decrease in the frequency of miniature inhibitory postsynaptic currents (mIPSCs) induced by the nociceptin receptor (NOP) agonist. Interestingly, MT-7716 prevented the ethanol-induced augmentation of evoked IPSPs. A putative selective NOP antagonist, [Nphe1]Nociceptin(1-13)NH2, totally prevented the MT-7716-induced inhibition of IPSP amplitudes indicating that MT-7716 exerts its effect through NOPs. These data provide support for an interaction between the nociceptin and GABAergic systems in the CeA and for the anti-alcohol properties of the NOP activation. The development of a synthetic nonpeptidergic NOP receptor agonist such as MT-7716 may represent a useful therapeutic target for alcoholism.

Keywords: GABA; NOP receptor; alcohol; amygdala; electrophysiology; nociceptin.

Figure 1

MT-7716 decreases evoked GABAergic transmission in CeA neurons. (A) Left panel: Representative recordings of evoked IPSPs in CeA neurons from naïve rats recorded before, during, and after washout from application of MT-7716 at all the concentrations tested. (B) Right Panel: Histograms representing the percent of the peak decrease in evoked (at half max stimulus intensity) IPSP amplitudes during superfusion of different concentrations (100, 250, 500, and 1000 nM) of MT-7716 and washout. Overall ANOVA revealed that MT-7716 decreased statistically significantly the IPSP amplitudes. Post hoc Newman-Keuls showed significant effect for all the doses at half max stimulus intensity. (*) Indicates p < 0.01.

Figure 2

The percentage effect of MT-7716 on the IPSP amplitude for the three middle stimulus intensities. (A) In the CeA of control rats, MT-7716 100 nM significantly (** p < 0.01) decreases the mean amplitude of evoked IPSP over the middle stimulus strength intensity tested (n = 11). (B) MT-7716 250 nM significantly decreases the mean amplitude of evoked IPSP over the three middle stimulus strength intensities tested (n = 10) (* p < 0.05) and (** p < 0.01). (C–D) MT-7716 500 and 1000 nM significantly decrease the mean amplitude of evoked IPSPs over the three middle stimulus strength intensities tested (n = 11/12) (** p < 0.01) and (*** p < 0.001). All data are expressed as % of control for three normalized stimulus strengths. Student t-test was used to analyze the percentage effect of MT-7716 on the IPSP amplitude.

Figure 3

MT-7716 decreases GABAergic transmission in CeA neurons by decreasing GABA release. (A) Representative recordings of PPF at both 50 (upper traces) and 100 (lower traces) ms in a CeA neuron from naïve rat before and during superfusion of 250 nM MT-7716. (B) Overall ANOVA revealed that MT-7716 (100 and 250 nM) significantly increases the PPF ratio of evoked IPSPs using 50 ms interstimulus intervals. MT-7716 (250 and 500 nM) significantly increases the PPF ratio of evoked IPSPs using 100 ms interstimulus intervals. (*) Indicates (p < 0.05) after appropriate Post-hoc Newman-Keuls test.

Figure 4

MT-7716 has no effect on voltage-current relationships on the CeA neurons. (A–D) I/V curves showing that MT-7716, in all doses superfused (100–1000 nM) Overall ANOVA indicates that MT-7716 does not modify the RMP of the CeA neurons (n = 6–11). (A) The mean RMPs for the neurons tested with 100 nM MT-7716 was −81 ± 1.2 mV and was −80 ± 0.5 mV for those tested 250 nM MT-7716 (B). Similarly the RMPs of the 10 and 6 CeA neurons tested with 500 nM and 1000 nM MT-7716 was −81.5 ± 0.9 mV (C) and −81 ± 1.2 mV (D). (E) Representative current clamp recordings of a CeA neuron (RMP = 80 mV; input resistance 113 M) during control and 500 nM MT-7716 superfusion (F). Overall, MT-7716 did not significantly affect the firing pattern or number of action potentials in our CeA neuronal population.

Figure 5

MT-7716 decreased spontaneous miniature inhibitory postsynaptic currents (mIPSCs) in CeA. (A) Representative CeA mIPSCs before, during the superfusion of 500 nM MT-7716 and washout. (B) Mean ± SEM frequency, amplitude, rise and decay of mIPSCs for CeA neurons from control rats. MT-7716 significantly (* p < 0.001) decreased the mean mIPSC frequencies and amplitude. Statistical significance * was set at p < 0.05 and calculated by Student’s t-test. (C) Cumulative fractions calculated by Kolmogorov-Smirnov sample test show that MT-7716 shifted the cumulative frequency to the right (in 11 out of 12 CeA neurons studied), indicating a longer inter-event interval during its application, suggesting decreased GABA release. (D) Cumulative fractions calculated by Kolmogorov-Smirnov sample test show that MT-7716 shifted the cumulative frequency to the right (in 10 out of 12 CeA neurons studied). MT-7716 shifted the cumulative amplitude to the left, indicating smaller mIPSC amplitudes, suggesting postsynaptic site of action.

Figure 6

Interactions of MT-7716 and ethanol at the CeA GABAergic synapses. (A) Overall ANOVA for the analyze of the time course of the % IPSP amplitude in CeA neurons during ethanol application per se shows that ethanol significantly increases the amplitude of evoked IPSPs. (B) Histograms representing the percent of the peak decrease in evoked (at half max stimulus intensity) IPSP amplitudes during superfusion of MT-7716 at the concentrations (100, 250 and 500 nM) alone, and in the presence of ethanol 44 mM on top. Newman-Keuls post-hoc test showed that MT-7716 decreased significantly the evoked IPSP amplitudes and blocked the ethanol-induced facilitation. (*) Indicates (p < 0.05) (**) indicates (p < 0.01). (C) Representative evoked IPSPs recorded before and during MT-7716 (100–500 nM) and co application with ethanol and washout. (D) Time course of the application of MT-7716 (500 nM) that reduces the amplitude of evoked IPSPs. After 15–20 min of MT-7716 superfusion, co-application of ethanol does not increase the evoked IPSP amplitude (72.9 ± 1.1% of control). MT-7716 effectively blocks the ethanol-induced enhancement of IPSPs, and GABA transmission returned to baseline levels upon 25 min of washout (94 ± 10% of control). (E) Ethanol significantly (p < 0.05) increased (137.1 ± 4.7% of control) the evoked IPSPs and 500 nM MT-7716 in the presence of ethanol significantly (** p < 0.01 by Newman-Keuls post-hoc test) decreased (91.3 ± 1.4%) the IPSPs and blocked the ethanol-induced facilitation. (F) Application of [Nphe1]Nociceptin(1–13)NH2 alone did not alter evoked IPSPs (105.1 ± 4.6% of control); n = 7; by paired t-test but blocked the MT-7716-induced decrease of IPSPs.