Research ArticleNEUROSCIENCE

Alcohol dependence potentiates substance P/neurokinin-1 receptor signaling in the rat central nucleus of amygdala

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Science Advances  18 Mar 2020:
Vol. 6, no. 12, eaaz1050
DOI: 10.1126/sciadv.aaz1050
  • Fig. 1 Subregion-specific expression of SP and NK-1R in rat CeA.

    (A) Scheme highlighting neuroanatomy of CeA and basolateral amygdala (BLA) in a rat coronal brain section as used for immunohistochemistry. (B) Representative images of naïve rat amygdala stained for SP (green), NK-1R (red), and counterstained with DAPI (4′,6-diamidino-2-phenylindole) (blue). Scale bar, 250 μm. (C) Quantification of NK-1R (left) and SP (right) expression in the CeA subregions normalized to CeM expression levels; n = 7. ***P < 0.001 and ****P < 0.0001, one-way ANOVA (Tukey post hoc mean comparison). n.s., not significant.

  • Fig. 2 SP increases action potential–dependent GABA release in the CeM of naïve rats.

    (A) Representative sIPSC recordings and (B) scaled averages before and during SP application. Bars represent means ± SEM of sIPSC properties (C to F; n = 6 to 14 cells). (G) Bars summarize effects of SP on mIPSCs (n = 11). Inset: Representative mIPSCs before and during SP (100 nM). (H) Percent change and representative traces of spontaneous action potential firing of CeM neurons under aCSF conditions (n = 10) and in the presence of blockers of synaptic glutamate transmission and GABAB receptors (n = 5) before and during SP. GABAB receptor, GABABR. (I) Bar graphs represent sIPSC frequencies and amplitudes in the presence of SP and SP + Tertiapin Q (TQ: 500 nM, n = 6, normalized to pre-SP + TQ baseline; note that TQ alone did not significantly alter sIPSC properties), respectively. *P < 0.05 and **P < 0.01, one-sample t tests compared to baseline conditions. #P < 0.01, unpaired t test between groups.

  • Fig. 3 Alcohol dependence decreases SP and NK-1R expression in the CeM.

    (A) Representative confocal images of SP (green) and NK-1R (red) of naïve (left) and dependent (right) rats. Scale bars, 10 μm. (B) Quantification of relative expression levels for SP (top) and NK-1R (bottom) of naïve (n = 7) and dependent animals (n = 6) expressed as arbitrary fluorescent units (a.u.). *P < 0.05, unpaired t test.

  • Fig. 4 SP effects on CeM GABA transmission are potentiated by alcohol dependence.

    (A) Representative sIPSCs during control and SP. (B) Time courses of SP effects in dependent (red lines) and naïve rats (gray lines) compared to baseline. Bars summarize sIPSC (C) frequencies, (D) amplitudes (n = 4 to 8), (E) mIPSC characteristics in the presence of SP (n = 11), and (F) representative mIPSCs before and during SP. *P < 0.05, **P < 0.01, and ***P < 0.001, one-sample t test compared to baseline conditions. #P < 0.05, multiple t tests between naïve and dependent (post hoc Holm-Sidak comparison).

  • Fig. 5 Acute alcohol interactions with SP/NK-1R signaling.

    (A) Representative sIPSCs before, during alcohol, and alcohol and SP, respectively. Bar graphs represent means ± SEM of sIPSC (B) frequencies, (C) amplitudes, and (D) current kinetics in the presence of alcohol and alcohol + SP, respectively [n = 11 (naïve) and n = 11 (dependent)] compared to baseline control (dashed lines). (E) Representative sIPSCs before and during the NK-1R antagonist L822429. Bar graphs represent means ± SEM of sIPSC in naïve (F) (n = 15) and dependent (G) (n = 10) rats. (H) Representative sIPSCs before, during the NK-1R antagonist, and NK-1R antagonist and alcohol, respectively. Bars summarize sIPSC (I) frequencies, (J) amplitudes, and (K) current kinetics in the presence of L822429 and L822429 + alcohol, respectively [n = 7 (naïve) and n = 8 (Dep.)] compared to baseline control (dashed lines). *P < 0.05, **P < 0.01, and ***P < 0.001, one-sample t test compared to baseline conditions. #P < 0.05 and ##P < 0.01, paired t test between groups.

  • Fig. 6 SP/NK-1R signaling remains elevated after withdrawal.

    (A) Representative sIPSCs before and during SP. (B) Time course of SP action. (C) Bar graphs represent means ± SEM of sIPSC characteristics in the presence of SP (n = 10). (D) Bars summarize sIPSC frequencies and amplitudes in the presence of alcohol and alcohol + SP, respectively. (E) Representative sIPSCs before, during alcohol, and alcohol + SP, respectively. (F) Representative sIPSCs before, during L822249, and L82249 + EtOH, respectively. Bars summarize in (G) sIPSC characteristics in the presence L822249 (n = 7). Bars show sIPSC (H) frequencies and (I) postsynaptic measures in the presence of L822249 and L822249 + EtOH, respectively (n = 5). *P < 0.05, **P < 0.01, and ***P < 0.001, one-sample t test compared to baseline conditions. ##P < 0.01, paired t test between groups.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/12/eaaz1050/DC1

    Fig. S1. Alcohol dependence induces long-lasting neuroadaptations of GABAA receptor–mediated transmission in the CeM.

    Fig. S2. The NK-1R antagonist L822429 blocks SP effects on CeM GABA transmission.

    Table S1. Summary of SP effects on sIPSC frequencies and amplitudes in the presence of blockers of potential SP/NK-1R intracellular signaling pathways in the CeM of naïve rats.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Alcohol dependence induces long-lasting neuroadaptations of GABAA receptor–mediated transmission in the CeM.
    • Fig. S2. The NK-1R antagonist L822429 blocks SP effects on CeM GABA transmission.
    • Table S1. Summary of SP effects on sIPSC frequencies and amplitudes in the presence of blockers of potential SP/NK-1R intracellular signaling pathways in the CeM of naïve rats.

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