Fig 1: Chronic binge alcohol drinking alters receptor-specific NPY modulation of GABAergic transmission in the BNST of mice and monkeys. (a) Experimental timeline for 3-cycle DID in mice. (b) BNST neurons from mice that drank ethanol (EtOH) had higher sIPSC frequency than water-drinking controls (CONs; unpaired t-test with Welch’s correction: t(11) = 2.32, *p = 0.040, CON n = 8, N = 5, EtOH n = 12, N = 5), but mIPSC frequency did not differ between groups (unpaired t-test: p > 0.95; CON n = 15, N= 7, EtOH n = 12, N = 6). (c) LeuPro NPY (300 nM) significantly increased mIPSC frequency in EtOH mice (paired t-test baseline vs. washout: t(5) = 3.58, p = 0.016; n = 6, N = 6) but not CONs (p > 0.60, n = 7, N = 7). (d) NPY 13–36 (300 nM) decreased mIPSC frequency in ethanol-drinking mice (t(5) = 2.97, p = 0.031; n = 6, N = 5) but not controls (p > 0.85, n = 7, N = 6). (e–g) Mean NPY-IR (average IR from 3–5 slices per mouse) was similar between groups (e; unpaired t-test: p > 0.75; CON N = 10, EtOH N = 7), but Y1R–IR (f; t(13) = 4.23, ***p = 0.001; CON N = 9, EtOH N = 6) and Y2R-IR (g; t(15) = 2.50, *p = 0.025; CON N = 10, EtOH N = 7) were higher in the BNST of EtOH mice than CONs. (h) NPY-IR was significantly decreased in the BNST of EtOH mice compared to water-drinking CONs immediately after the last binge ethanol drinking exposure in 1-cycle and 3-cycle DID (N’s = 10/group), but was not different between one and 3-cycle DID (one-way ANOVA: F(2,27) = 14.25, p < 0.0001; post-hoc Sidak’s multiple comparisons test: CON vs. 1-cycle: t(18) = 3.58,** p = 0.004; CON vs. 3-cycle DID: t(18) = 5.22, ***p < 0.001; 1-cycle DID vs. 3-cycle DID: p > 0.25), suggesting that NPY was similarly recruited acutely during each binge ethanol session across each cycle. (i) Experimental timeline for voluntary ethanol self-administration (ESA; access to 4% ethanol for 22 h/d, 7 d/wk for 12 mo) in adult male rhesus monkeys. (j) Representative traces of mIPSCs from ethanol self-administering rhesus monkey BNST neurons before and after bath application of LeuPro NPY (300 nM). (k) mIPSC frequency was unaltered by LeuPro NPY following one cycle of DID in EtOH mice (n = 8, N = 4) and water-drinking CONs (n = 5, N = 3; paired t-tests baseline vs. washout: p’s > 0.30), but it was increased in EtOH, but not CON, mice 1 d after the final binge session of 3-cycle EtOH DID, as shown in c, which could be blocked by intracellular inclusion of PKI (20 µM; p > 0.35; n = 3, N = 2). The adaptation in LeuPro NPY modulation of mIPSC frequency was still present 10 d after the final binge ethanol session in EtOH mice (t(5) = 3.09, *p = 0.027; n = 6, N = 3) but not CONs (p > 0.50, n = 5, N = 3) and was also observed in rhesus monkeys after 12 mo of continuous access to ethanol (t(8) = 4.21, **p = 0.003; n = 9, N = 5) but not control solution (p > 0.50; n = 4, N = 3). All data in b–h and k are presented as mean ± SEM.
Fig 2: The pyramidal neurons of PrL receive a direct inhibitory input from NPY+-GABAergic projection neurons of ipsilateral IL. (a) Schematic drawing of the position of stimulation and recording electrodes; (b and c) Sample traces (b), amplitude and frequency of spontaneous IPSCs in layer II/III pyramidal neurons of PrL (c); (d) Sample trace of an action potential in GABAergic interneuron elicited by 1-ms K+-application; (e) Spike train in a small unidentified neuron during 500-ms-long current injection in current-clamp mode; (f) iontophoretic activation of unidentified small neurons in layer V of IL evoked bicuculline-sensitive IPSC in pyramidal neurons of PrL; (g) Examples of IPSCs evoked in pyramidal neurons by iontophoretic activation of NPY–eGFP neurons in IL (red trace); absence of responses on stimulation of PV–eGFP neurons in IL (black trace); (h) Summary of all experimental recordings of eIPSCs in layer II pyramidal neurons of PrL. Note each data point illustrated an individual experiment, while the numbers over each column indicated the success rate of eliciting eIPSCs (n/N: n=successful experiments, N=total number of experiments). IL, infralimbic cortex; IPSC, inhibitory postsynaptic current; PrL, prelimbic cortex.
Fig 3: NPY regulates miR-216a and FoxO4. (A) The relative level of the miRNAs in CH. (B) The relative level of the FoxO4 in CH. (C) The relative level of the miRNAs in NPY treated cardiomyocytes. (D) The relative level of the miR-216b in NPY treated cardiomyocytes. (E) The relative level of the FoxO4 in NPY treated cardiomyocytes (n = 4). A&B:*P < 0.05, **P < 0.01 vs Sham; &P < 0.05 vs Sham; #P < 0.05, ##P < 0.01 vs CH. C-E: **P < 0.01 vs Control; #P<0.05, ##P < 0.01 vs NPY.
Fig 4: CCI induced a sprouting of sympathetic fibers into the upper dermis of the skin, and these fibers often expressed NPY. Sympathetic fibers labelled with DBH (in red), were rarely observed in the upper dermis of the skin in 6 week sham animals (a). 2 (b) and 6 (c) weeks after CCI, sympathetic fibers sprouted into the upper dermis, and many expressed NPY. The quantification of the number of DBH fibers per mm of upper dermis is shown in (d). The quantification of the percentage of DBH fibers that expressed NPY is shown in e and the values written on the graphs indicate the absolute number of sprouted sympathetic fibers counted across animals. The quantification of the percentage of NPY fibers that expressed DBH is shown in f and the values written on the graphs indicate the absolute number of NPY-positive fibers counted across animals. Dashed lines represent the border between the epidermis and the upper dermis. **p < 0.01 by 2 way ANOVA with Bonferroni post hoc (n = 6). Scale bar = 50 µm
Fig 5: Circadian regulation of receptor-specific NPY modulation of GABAergic transmission in the BNST. (a) Sample images representing mean NPY–IR in the BNST of mice sacrificed three hr into the light or dark phase of the light cycle (scale bars = 150 µM). Images shown were replicated for each individual data point shown in b. (b) Mean NPY–IR per mouse from five coronal slices (as in panel a) was not different between the light and dark phases of the light cycle (unpaired t-test: p > 0.40; N’s = 10/group). (c–d) Basal sIPSC and mIPSC frequency (c) and amplitude (d) were not different across the light cycle (unpaired t-tests: p’s > 0.15; CON n = 13, N = 7, EtOH n = 13, N = 7). (e) Bath application of LeuPro NPY (300 nM) increased, while the Y2R agonist NPY 13–36 (300 nM) decreased, mIPSC frequency during the light phase (LeuPro NPY: as shown in Fig. 1i, **p = 0.005; NPY 13–36: paired t-test baseline vs. washout: t(5) = 3.57, *p = 0.016, n = 6, N = 5) but not the dark phase (p’s > 0.15; LeuPro n = 5, N = 4, NPY 13–36 n = 6, N = 4), of the light cycle. (f) Bath application of the Y1R antagonist BIBP 3226 (1 µM; n = 11, N = 10) or the Y2R antagonist BIIE 0246 (1 µM; n = 4, N = 3) did not alter mIPSC frequency during the light phase (paired t-tests baseline vs. washout: p’s > 0.20); BIIE 0246 increased mIPSC frequency during the dark phase (t(3) = 8.45, **p = 0.004; n = 4, N = 3), while BIBP 3226 did not (p > 0.15; n = 5, N = 4). All data in b–l are presented as mean ± SEM.
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