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NPY signaling inhibits extended amygdala CRF neurons to suppress binge alcohol drinking

Nature Neuroscience volume 18, pages 545–552 (2015)Cite this article

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Abstract

Binge alcohol drinking is a tremendous public health problem because it leads to the development of numerous pathologies, including alcohol abuse and anxiety. It is thought to do so by hijacking brain systems that regulate stress and reward, including neuropeptide Y (NPY) and corticotropin-releasing factor (CRF). The central actions of NPY and CRF have opposing functions in the regulation of emotional and reward-seeking behaviors; thus, dysfunctional interactions between these peptidergic systems could be involved in the development of these pathologies. We used converging physiological, pharmacological and chemogenetic approaches to identify a precise neural mechanism in the bed nucleus of the stria terminalis (BNST), a limbic brain region involved in pathological reward and anxiety behaviors, underlying the interactions between NPY and CRF in the regulation of binge alcohol drinking in both mice and monkeys. We found that NPY Y1 receptor (Y1R) activation in the BNST suppressed binge alcohol drinking by enhancing inhibitory synaptic transmission specifically in CRF neurons via a previously unknown Gi-mediated, PKA-dependent postsynaptic mechanism. Furthermore, chronic alcohol drinking led to persistent alterations in Y1R function in the BNST of both mice and monkeys, highlighting the enduring, conserved nature of this effect across mammalian species. Together, these data provide both a cellular locus and signaling framework for the development of new therapeutics for treatment of neuropsychiatric diseases, including alcohol use disorders.

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Figure 1: Activation of Gi-coupled Y1R in the BNST reduces binge ethanol drinking and enhances GABAergic transmission via a PKA-dependent, postsynaptic insertion of GABAA receptors.
Figure 2: Circadian regulation of receptor-specific NPY modulation of GABAergic transmission in the BNST.
Figure 3: Chronic binge alcohol drinking alters receptor-specific NPY modulation of GABAergic transmission in the BNST of mice and monkeys.
Figure 4: Y1R-mediated effects on inhibition in the BNST are specific to CRF neurons.
Figure 5: Direct in vivo chemogenetic activation of Gi signaling in BNST CRF neurons recapitulates, whereas chemogenetic activation of Gs signaling blocks the effect of Y1R activation on binge ethanol consumption.

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Acknowledgements

We thank S. Chien for assistance with histological verification of DREADD virus and cannulae placements, R. Thomas and G. Sprow for help with drinking behavior experiments, and B. Roth (UNC School of Medicine) for viral constructs and CNO. This work was supported by US National Institutes of Health grants AA021043 to K.E.P.; AA019454, AA020911 and AA011605 to T.L.K.; AA013573 and AA022048 to T.E.T.; AA013541 and AA109431 to K.A.G.; DK071561 to D.P.O.; and DK075632, DK096010, DK046200 and DK057521 to B.B.L., and by the Bowles Center for Alcohol Studies at the University of North Carolina School of Medicine.

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Author notes

  1. Jennifer A Rinker

    Present address: Present address: Department of Neurosciences, Medical University of South Carolina, Charleston, South Carolina, USA.,

  2. Nora M McCall

    Present address: Present address: Graduate Program in Neuroscience, University of Minnesota, Minneapolis, Minnesota, USA.,

  3. Alexis M Kendra

    Present address: Present address: Master of Biotechnology Program, Northwestern University, Evanston, Illinois, USA.,

  4. David P Olson

    Present address: Present address: Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan, USA.,

Authors and Affiliations

  1. Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA

    Kristen E Pleil, Emily G Lowery-Gionta, Nora M McCall, Alexis M Kendra & Thomas L Kash

  2. Bowles Center for Alcohol Studies, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA

    Kristen E Pleil, Jennifer A Rinker, Emily G Lowery-Gionta, Christopher M Mazzone, Nora M McCall, Alexis M Kendra, Todd E Thiele & Thomas L Kash

  3. Department of Psychology, University of North Carolina, Chapel Hill, North Carolina, USA

    Jennifer A Rinker, Todd E Thiele & Thomas L Kash

  4. Curriculum in Neurobiology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA

    Christopher M Mazzone

  5. Division of Endocrinology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA

    David P Olson & Bradford B Lowell

  6. Division of Neuroscience, Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, Oregon, USA

    Kathleen A Grant

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Contributions

K.E.P. wrote the manuscript, designed the study, performed all electrophysiological recordings, and performed in vivo behavioral pharmacology, in vivo DREADD DID experiments and immunohistochemistry. J.A.R. performed cannulation and DREADD viral injection surgeries and in vivo behavioral pharmacology and DREADD DID experiments. E.G.L.-G. performed cannulation surgeries and in vivo behavioral pharmacology experiments. C.M.M. performed cannulation surgeries, fluorescence and confocal microscope imaging of DREADD expression, and pilot experiments for DREADD constructs and CNO doses. N.M.M. performed RT-PCR and immunohistochemistry and bred CRF-Cre and CRF reporter mice. A.M.K. performed cannulation surgeries and bred CRF-Cre and CRF reporter mice. D.P.O. and B.B.L. generated the CRF-ires-Cre (Crh-ires-Cre) mice. K.A.G. oversaw rhesus monkey experiments and provided monkey brain tissue. T.E.T. aided in the design of and oversaw all behavioral experiments. T.L.K. helped to design the study and write the manuscript, and oversaw all molecular biology, microscopy and electrophysiology work. All of the authors edited the manuscript.

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Correspondence to Thomas L Kash.

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Integrated supplementary information

Supplementary Figure 1 Manipulation of Y1R and Y2R in the BNST alters binge-like ethanol drinking during 3-cycle DID and sucrose consumption.

(a) Standard Drinking-in-the-Dark (DID) voluntary ethanol consumption paradigm (1 cycle depicted), where mice are given 2 h of access to 20% ethanol three hours into the dark cycle on three consecutive days, followed by 4 h of access on the fourth, binge test day. (b) Bilateral infusion of the Y1R agonist LeuPro NPY (99 pmol/200 nL/side) into the BNST-adjacent dorsal striatum prior to ethanol access on cycle 3 Day 4 of DID did not alter binge ethanol consumption (unpaired t-test with Welch’s correction: p > 0.65; CON N = 6, LeuPro N = 7), suggesting the decrease in binge drinking shown in Figure 1b was specific to the BNST. (c−g) Bilateral intra-BNST infusion of the Y1R antagonist BIBP 3226 trifluoroacetate (BIBP 3226; 20 pmol/200 nL/side) prior to ethanol access on cycle 3 Day 4 of DID did not alter ethanol consumption during the first 2-h epoch of the binge session (c; unpaired t-test: p > 0.20; CON N = 7, BIBP N = 7) but did increase ethanol consumption during the second 2-hr epoch (d; t(12) = 3.94, **p = 0.002); it had no effect on the percent time in center (e) or locomotor behavior (f) in the OF (p’s > 0.45; CON N = 7, BIBP N = 5) or on sucrose drinking on Day 4 of sucrose DID (g; p > 0.75; CON N = 7, BIBP N = 6). (h−l) Bilateral intra-BNST infusion of the Y2R agonist neuropeptide Y 13−36 (NPY 13−36; 100 pmol/200 nL/side) did not alter ethanol consumption during the first 2−h epoch of the binge session (h; unpaired t-test: p > 0.20; CON N = 7, NPY 13-36 N = 8) but did increase ethanol consumption during the second 2−h epoch (i; t(13) = 2.24, *p = 0.044); it had no effect on the percent time in center (j) or locomotor behavior (k) in the OF (p’s > 0.65; CON N = 9, NPY 13-36 N = 7); NPY 13-36 decreased binge sucrose consumption on Day 4 of sucrose DID (l; t(14) = 2.25, *p = 0.041; CON N = 7, NPY 13-36 N = 9). (m−q) Bilateral intra-BNST infusion of the Y2R antagonist BIIE 0246 (120 pmol/200 nL/side) did not alter any measures in ethanol DID (CON N = 9, BIIE N = 8), OF (CON N = 8, BIIE N = 9), or sucrose DID (CON N = 7, BIIE N = 10; unpaired t-tests: p’s > 0.25). All data in b−q are presented as mean ± SEM.

Supplementary Figure 2 Manipulation of Y1R, but not Y2R, in the BNST alters binge-like ethanol drinking during 2-cycle DID.

(a−b) Mice that received bilateral intra-BNST infusions of LeuPro NPY prior to access to EtOH on Cycle 2 Day 4 consumed significantly less ethanol than CON mice (a; unpaired t-test: t(12) = 2.49, *p = 0.029; CON N = 8, LeuPro N = 6), but locomotor behavior in the OF was unaffected (b; p > 0.90; CON N = 6, LeuPro N = 9). (c−d) Bilateral intra-BNST infusions of BIIE 0246 did not alter binge ethanol consumption in 2-cycle DID (c; CON N = 3, BIIE N = 5) or locomotor behavior in the OF (d; CON N = 3, BIIE N = 4; unpaired t-tests: p’s > 0.70). All data are presented as mean ± SEM.

Supplementary Figure 3 Y1R activation alters GABAergic transmission via a PKA-dependent mechanism without altering cell membrane or dendritic excitability.

(a) Bath pre-application of the PKA inhibitor Rp-cAMPs (10 µM; n = 5, N = 3), but not the PLC inhibitor U73122 (10 µM; n = 5, N = 3), blocked the ability of subsequently applied LeuPro NPY (300 nM) to increase mIPSC frequency (paired t-tests baseline vs. washout: p > 0.35 and t(4) = 4.72, **p = 0.009, respectively); LeuPro NPY did not alter mIPSC amplitude under either condition (p’s > 0.75). (b) Mean basal mIPSC frequency in BNST neurons is increased by pipette inclusion of NEM (t(31) = 3.63, **p < 0.003) and BAPTA (t(37) = 3.28, *p = 0.010), but no other drugs included in the recording pipette (p’s > 0.25; n’s are identical to Fig. 1k), when corrected for multiple comparisons with Sidak’s multiple comparisons tests. (c-d) Postsynaptic membrane resistance was not altered by bath application of LeuPro NPY (c; paired t-test baseline vs. washout: p > 0.60; n = 13, N = 11 as in Fig. 1i), and bath application of LeuPro NPY increased mIPSC frequency (t(5) = 3.15, p = 0.025; n = 6, N = 3) but not amplitude (p > 0.85) when a Cs-based intracellular solution was used, similar to the effect achieved using a K+-based solution (d), as in Fig. 1i, suggesting that effects of LeuPro NPY do not require K+ channels. All data are presented as mean ± SEM.

Supplementary Figure 4 mRNA for NPY and its receptors are unaltered following 3-cycle DID.

(a) Mice that underwent 3-cycle EtOH DID consistently consumed an average of 6.4−7.2 g/kg ethanol and achieved blood ethanol contents (BECs) of approximately 162 mg/dl (N = 9). (b−d) mRNA content of NPY (b; N’s = 10), Y1R (c; N’s = 10), and Y2R (d; CON n = 8, EtOH n = 9) in the BNST was not different between ethanol drinkers and controls one day following 3-cycle DID (unpaired t-tests: p’s > 0.65). All data are presented as mean ± SEM.

Supplementary Figure 5 Y1R-mediated inhibition of BNST CRF neurons occurs via postsynaptic Gi signaling.

(a) Model depiction of the mechanism of Y1R−mediated modulation of binge alcohol drinking, in which activation of postsynaptic Gi-coupled Y1R on CRF neurons in the BNST inhibits PKA to increase the surface expression of GABAAR, leading to postsynaptic inhibition of the CRF neurons. (b) Representative trace from a whole-cell current-clamp electrophysiological recording showing that bath application of CNO (10 µM) hyperpolarizes Gi-DREADD-expressing CRF-Cre neurons in the BNST. (c) Average magnitude of hyperpolarization of Gi-DREADD-expressing CRF−Cre neurons in the BNST after bath application of CNO (paired t-test baseline vs. CNO: t(8) = 4.03, **p = 0.004; n = 9, N = 4; data are presented as mean ± SEM).

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Pleil, K., Rinker, J., Lowery-Gionta, E. et al. NPY signaling inhibits extended amygdala CRF neurons to suppress binge alcohol drinking. Nat Neurosci 18, 545–552 (2015). https://doi.org/10.1038/nn.3972

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