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

Kallupi M, Oleata CS, Luu G, Teshima K, Ciccocioppo R, Roberto M - Front Integr Neurosci (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Committee on the Neurobiology of Addictive Disorders, The Scripps Research Institute, La Jolla CA, USA ; Pharmacology Unit, School of Pharmacy, University of Camerino Camerino, Italy.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: We have previously reported (Roberto et al., 2003) that 44 mM ethanol (a maximally effective concentration) increases evoked GABA IPSPs via increased GABA release in the CeA. We have also documented that N/OFQ blocks this ethanol-induced facilitation (Roberto and Siggins, 2006). In the present study we recapitulate that ethanol 44 mM significantly (p < 0.05) and reversibly increases by 36 ± 3% (n = 6) the amplitude of evoked IPSPs (Figures 6A–C). This ethanol-induced increase is associated with a significant (p < 0.05) decrease in the PPF ratio of IPSPs (both 50 and 100 ms intervals; data not shown). We then examined whether MT-7716 would block the ethanol-induced increase in evoked GABAergic responses. In separate groups of CeA cells, we applied MT-7716 at the doses of 100, 250 and 500 nM and then co-applied 44 mM ethanol on the top (Figures 6B, C). In Figures 6B the data are expressed as percent of control using the three middle stimulus intensities (1–3X) obtained from the I-O relationship. All three concentrations of MT-7716 used (100, 250 and 500 nM) significantly decreased IPSP amplitudes (half maximal intensity) and totally blocked the ethanol-induced facilitation of IPSPs. Specifically, MT-7716 (500 nM) significantly (p < 0.001; n = 7) reduced the amplitude of evoked IPSPs by 20% of control over all stimulus strengths in the CeA neurons (Figure 6B). After 15–20 min of MT-7716 superfusion, co-application of ethanol (44 mM) did not increase the evoked IPSP amplitude (72.9 ± 1.1% of control). MT-7716 effectively prevented the ethanol-induced enhancement of IPSPs, and GABA transmission returned to baseline levels upon washout (94 ± 10% of control; Figure 6D). Of note is the fact that MT-7716 in lower doses, 250 and 100 nM also decreased evoked IPSPs to 79 ± 8% (n = 6) and 100 nM to 90 ± 6% (n = 6) of control respectively and blocked ethanol-induced increase of IPSPs (the IPSPs amplitude remained the same 80 ± 10% and 83 ± 3% of control, respectively) with total recovery on washout. Interestingly, although the lowest concentration of 100 nM MT-7716 had no significant effect on evoked IPSP amplitudes (p > 0.05) (10% decrease compared to control), it still completely blocked the ethanol-induced increase of IPSPs with total recovery on washout, suggesting that the anti-ethanol actions of NOP activation were not due simply to a summation of opposing effects, but functionally independent effects on GABA transmission. We did not test the highest concentration of MT-7716 because although the inhibition induced by 1000 nM MT-7716 was comparable to the one obtained with 500 and 250 nM, this effect was only partially recovered upon washout, data not shown.


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

Kallupi M, Oleata CS, Luu G, Teshima K, Ciccocioppo R, Roberto M - Front Integr Neurosci (2014)

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.
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Related In: Results  -  Collection

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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.
Mentions: We have previously reported (Roberto et al., 2003) that 44 mM ethanol (a maximally effective concentration) increases evoked GABA IPSPs via increased GABA release in the CeA. We have also documented that N/OFQ blocks this ethanol-induced facilitation (Roberto and Siggins, 2006). In the present study we recapitulate that ethanol 44 mM significantly (p < 0.05) and reversibly increases by 36 ± 3% (n = 6) the amplitude of evoked IPSPs (Figures 6A–C). This ethanol-induced increase is associated with a significant (p < 0.05) decrease in the PPF ratio of IPSPs (both 50 and 100 ms intervals; data not shown). We then examined whether MT-7716 would block the ethanol-induced increase in evoked GABAergic responses. In separate groups of CeA cells, we applied MT-7716 at the doses of 100, 250 and 500 nM and then co-applied 44 mM ethanol on the top (Figures 6B, C). In Figures 6B the data are expressed as percent of control using the three middle stimulus intensities (1–3X) obtained from the I-O relationship. All three concentrations of MT-7716 used (100, 250 and 500 nM) significantly decreased IPSP amplitudes (half maximal intensity) and totally blocked the ethanol-induced facilitation of IPSPs. Specifically, MT-7716 (500 nM) significantly (p < 0.001; n = 7) reduced the amplitude of evoked IPSPs by 20% of control over all stimulus strengths in the CeA neurons (Figure 6B). After 15–20 min of MT-7716 superfusion, co-application of ethanol (44 mM) did not increase the evoked IPSP amplitude (72.9 ± 1.1% of control). MT-7716 effectively prevented the ethanol-induced enhancement of IPSPs, and GABA transmission returned to baseline levels upon washout (94 ± 10% of control; Figure 6D). Of note is the fact that MT-7716 in lower doses, 250 and 100 nM also decreased evoked IPSPs to 79 ± 8% (n = 6) and 100 nM to 90 ± 6% (n = 6) of control respectively and blocked ethanol-induced increase of IPSPs (the IPSPs amplitude remained the same 80 ± 10% and 83 ± 3% of control, respectively) with total recovery on washout. Interestingly, although the lowest concentration of 100 nM MT-7716 had no significant effect on evoked IPSP amplitudes (p > 0.05) (10% decrease compared to control), it still completely blocked the ethanol-induced increase of IPSPs with total recovery on washout, suggesting that the anti-ethanol actions of NOP activation were not due simply to a summation of opposing effects, but functionally independent effects on GABA transmission. We did not test the highest concentration of MT-7716 because although the inhibition induced by 1000 nM MT-7716 was comparable to the one obtained with 500 and 250 nM, this effect was only partially recovered upon washout, data not shown.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Committee on the Neurobiology of Addictive Disorders, The Scripps Research Institute, La Jolla CA, USA ; Pharmacology Unit, School of Pharmacy, University of Camerino Camerino, Italy.

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.

No MeSH data available.


Related in: MedlinePlus