Cocaine and amphetamine regulated transcript (CART) signalling in the central nucleus of the amygdala modulates stress-induced alcohol seeking

Abstract

The central nucleus of the amygdala (CeA) is a key hub of the neural circuitry regulating alcohol and stress interactions. However, the exact neuronal populations that govern this interaction are not well defined. Here we examined the role of the neuropeptide cocaine and amphetamine regulated transcript (CART) within the CeA in stress-induced alcohol seeking. We found that CART-containing neurons are predominantly expressed in the capsular/lateral division of the CeA and are a subpopulation of protein kinase Cδ (PKCδ) cells, distinct from corticotrophin releasing factor (CRF)-expressing cells. Both stress (yohimbine) and stress-induced alcohol seeking activated CART cells within the CeA, while neutralisation of endogenous CeA CART signalling (via antibody administration) attenuated stress-induced alcohol, but not sucrose seeking. Further, blocking CART signalling within the CeA did not alter the motivation to obtain and consume alcohol but did attenuate stressor-induced anxiety-like behaviour during abstinence from alcohol. Together, these data identify CeA CART cells as a subpopulation of PKCδ cells that influence stress × alcohol interactions and mediate stress-induced alcohol seeking behaviours.

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Fig. 1: The molecular phenotype of CeA CART cells.
Fig. 2: Yohimbine-induced reinstatement of alcohol seeking activates CART neurons within the CeA.
Fig. 3: CART signalling within the CeA in necessary, but not sufficient for reinstatement of alcohol seeking.
Fig. 4: CeA CART signalling does not alter the motivation to consume alcohol.
Fig. 5: CeA CART signalling mediates anxiety-like behaviour following alcohol consumption.

References

  1. 1.

    Koob GF, Buck CL, Cohen A, Edwards S, Park PE, Schlosburg JE, et al. Addiction as a stress surfeit disorder. Neuropharmacology. 2014;76:370–82.

    CAS  PubMed  Google Scholar 

  2. 2.

    George O, Koob GF, Vendruscolo LF. Negative reinforcement via motivational withdrawal is the driving force behind the transition to addiction. Psychopharmacology. 2014;231:3911–7.

    CAS  PubMed  Google Scholar 

  3. 3.

    Gilpin NW, Herman MA, Roberto M. The central amygdala as an integrative hub for anxiety and alcohol use disorders. Biol Psychiatry. 2015;77:859–69.

    PubMed  Google Scholar 

  4. 4.

    Babaev O, Chatain CP, Krueger-Burg D. Inhibition in the amygdala anxiety circuitry. Exp Mol Med. 2018;50:1–16.

    CAS  Google Scholar 

  5. 5.

    Cai H, Haubensak W, Anthony TE, Anderson DJ. Central amygdala PKC-δ+ neurons mediate the influence of multiple anorexigenic signals. Nat Neurosci. 2014;17:1240.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Kim J, Zhang X, Muralidhar S, LeBlanc SA, Tonegawa S. Basolateral to central amygdala neural circuits for appetitive behaviors. Neuron. 2017;93:1464–79.e5.

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    McCullough KM, Morrison FG, Hartmann J, Carlezon WA Jr, Ressler KJ Quantified coexpression analysis of central amygdala subpopulations. eNeuro 2018;5:ENEURO.0010-18.2018.

  8. 8.

    Sanford CA, Soden ME, Baird MA, Miller SM, Schulkin J, Palmiter RD, et al. A central amygdala CRF circuit facilitates learning about weak threats. Neuron. 2017;93:164–78.

    CAS  PubMed  Google Scholar 

  9. 9.

    Venniro M, Russell TI, Ramsey LA, Richie CT, Lesscher HM, Giovanetti SM, et al. Abstinence-dependent dissociable central amygdala microcircuits control drug craving. Proc Natl Acad Sci. 2020;117:8126–34.

    CAS  PubMed  Google Scholar 

  10. 10.

    Hunt S, Sun Y, Kucukdereli H, Klein R, Sah P. Intrinsic Circuits in the Lateral Central Amygdala. eNeuro. 2017;4:ENEURO.0367-16.2017.

  11. 11.

    Lee SC, Amir A, Haufler D, Pare D. Differential recruitment of competing valence-related amygdala networks during anxiety. Neuron. 2017;96:81–88.e5.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Schank JR, Nelson BS, Damadzic R, Tapocik JD, Yao M, King CE, et al. Neurokinin-1 receptor antagonism attenuates neuronal activity triggered by stress-induced reinstatement of alcohol seeking. Neuropharmacology. 2015;99:106–14.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Walker LC, Cornish LC, Lawrence AJ, Campbell EJ. The effect of acute or repeated stress on the corticotropin releasing factor system in the CRH-IRES-Cre mouse: A validation study. Neuropharmacology. 2019;154:96–106.

    CAS  PubMed  Google Scholar 

  14. 14.

    Zhao Y, Dayas CV, Aujla H, Baptista MA, Martin-Fardon R, Weiss F. Activation of group II metabotropic glutamate receptors attenuates both stress and cue-induced ethanol-seeking and modulates c-fos expression in the hippocampus and amygdala. J Neurosci. 2006;26:9967–74.

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Walker LC, Kastman HE, Krstew EV, Gundlach AL, Lawrence AJ. Central amygdala relaxin-3/relaxin family peptide receptor 3?signalling modulates alcohol seeking in rats. Br J Pharmacol. 2017;174:3359–69.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Douglass J, Daoud S. Characterization of the human cDNA and genomic DNA encoding CART: a cocaine-and amphetamine-regulated transcript. Gene. 1996;169:241–5.

    CAS  PubMed  Google Scholar 

  17. 17.

    Ong ZY, McNally GP. CART in energy balance and drug addiction: current insights and mechanisms. Brain Res. 2020;1740:146852.

    CAS  PubMed  Google Scholar 

  18. 18.

    Dayas CV, McGranahan TM, Martin-Fardon R, Weiss F. Stimuli linked to ethanol availability activate hypothalamic CART and orexin neurons in a reinstatement model of relapse. Biol Psychiatry. 2008;63:152–7.

    CAS  PubMed  Google Scholar 

  19. 19.

    James MH, Charnley JL, Jones E, Levi EM, Yeoh JW, Flynn JR, et al. Cocaine-and amphetamine-regulated transcript (CART) signaling within the paraventricular thalamus modulates cocaine-seeking behaviour. PLoS ONE. 2010;5:e12980.

    PubMed  PubMed Central  Google Scholar 

  20. 20.

    King BJ, Furlong TM, McNally GP. Cocaine and amphetamine related transcript (CART) inhibits context induced reinstatement of reward seeking. Behav Neurosci. 2010;124:423.

    CAS  PubMed  Google Scholar 

  21. 21.

    Stanek LM. Cocaine-and amphetamine related transcript (CART) and anxiety. Peptides. 2006;27:2005–11.

    CAS  PubMed  Google Scholar 

  22. 22.

    Upadhya MA, Kokare DM, Subhedar NK. Cocaine-and amphetamine-regulated transcript peptide (CART) in the central nucleus of amygdala potentiates behavioral and hormonal responses of the rat exposed to its predator. Behavioural Brain Res. 2013;243:129–37.

    CAS  Google Scholar 

  23. 23.

    Millan EZ, McNally GP. Cocaine-and amphetamine-regulated transcript in the nucleus accumbens shell attenuates context-induced reinstatement of alcohol seeking. Behav Neurosci. 2012;126:690.

    CAS  PubMed  Google Scholar 

  24. 24.

    Salinas AG, Nguyen CT, Ahmadi-Tehrani D, Morrisett RA. Reduced ethanol consumption and preference in cocaine-and amphetamine-regulated transcript (CART) knockout mice. Addiction Biol. 2014;19:175–84.

    CAS  Google Scholar 

  25. 25.

    Koylu EO, Couceyro PR, Lambert PD, Kuhar MJ. Cocaine-and amphetamine-regulated transcript peptide immunohistochemical localization in the rat brain. J Comp Neurol. 1998;391:115–32.

    CAS  PubMed  Google Scholar 

  26. 26.

    Balkan B, Gozen O, Yararbas G, Koylu EO, Akinturk S, Kuhar MJ, et al. CART expression in limbic regions of rat brain following forced swim stress: sex differences. Neuropeptides. 2006;40:185–93.

    CAS  PubMed  Google Scholar 

  27. 27.

    Hunter RG, Bellani R, Bloss E, Costa A, Romeo RD, McEwen BS. Regulation of CART mRNA by stress and corticosteroids in the hippocampus and amygdala. Brain Res. 2007;1152:234–40.

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Dandekar MP, Singru PS, Kokare DM, Lechan RM, Thim L, Clausen JT, et al. Importance of cocaine-and amphetamine-regulated transcript peptide in the central nucleus of amygdala in anxiogenic responses induced by ethanol withdrawal. Neuropsychopharmacology. 2008;33:1127–36.

    CAS  PubMed  Google Scholar 

  29. 29.

    Ch'ng SS, Fu J, Brown RM, Smith CM, Hossain MA, McDougall SJ, et al. Characterization of the relaxin family peptide receptor 3 system in the mouse bed nucleus of the stria terminalis. J Comp Neurol. 2019;527:2615–33.

    CAS  PubMed  Google Scholar 

  30. 30.

    Wang F, Flanagan J, Su N, Wang LC, Bui S, Nielson A, et al. RNAscope: a novel in?situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J Mol Diagn. 2012;14:22–29.

    CAS  Article  Google Scholar 

  31. 31.

    Kastman HE, Blasiak A, Walker L, Siwiec M, Krstew EV, Gundlach AL, et al. Nucleus incertus Orexin2 receptors mediate alcohol seeking in rats. Neuropharmacology. 2016;110:82–91.

    CAS  PubMed  Google Scholar 

  32. 32.

    Walker LC, Kastman HE, Koeleman JA, Smith CM, Perry CJ, Krstew EV, et al. Nucleus incertus corticotrophin-releasing factor 1 receptor signalling regulates alcohol seeking in rats. Addict Biol. 2017;22:1641–54.

    CAS  PubMed  Google Scholar 

  33. 33.

    Walker LC, Kastman HE, Lawrence AJ. Pattern of neural activation following yohimbine-induced reinstatement of alcohol seeking in rats. Eur J Neurosci. 2019;51:706–20.

    PubMed  Google Scholar 

  34. 34.

    Walker LC, Berizzi AE, Chen NA, Rueda P, Perreau VM, Huckstep K, et al. Acetylcholine muscarinic M4 receptors as a therapeutic target for alcohol use disorder: converging evidence from humans and rodents. Biol Psychiatry. 2020. [Epub ahead of print] https://doi.org/10.1016/j.biopsych.2020.02.019.

  35. 35.

    Farid WO, Lawrence AJ, Krstew EV, Tait RJ, Hulse GK, Dunlop SA. Maternally administered sustained-release naltrexone in rats affects offspring neurochemistry and behaviour in adulthood. PLoS ONE. 2012;7:e52812.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Koob GF, Schulkin J. Addiction and stress: an allostatic view. Neurosci Biobehav Rev. 2019;106:245–62.

    PubMed  Google Scholar 

  37. 37.

    Viviani D, Charlet A, van den Burg E, Robinet C, Hurni N, Abatis M, et al. Oxytocin selectively gates fear responses through distinct outputs from the central amygdala. Science. 2011;333:104–7.

    CAS  PubMed  Google Scholar 

  38. 38.

    Han S, Soleiman MT, Soden ME, Zweifel LS, Palmiter RD. Elucidating an affective pain circuit that creates a threat memory. Cell. 2015;162:363–74.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    McCullough KM, Daskalakis NP, Gafford G, Morrison FG, Ressler KJ. Cell-type-specific interrogation of CeA Drd2 neurons to identify targets for pharmacological modulation of fear extinction. Transl Psychiatry. 2018;8:1–16.

    CAS  Google Scholar 

  40. 40.

    Farzi A, Lau J, Ip CK, Qi Y, Shi YC, Zhang L, et al. Arcuate nucleus and lateral hypothalamic CART neurons in the mouse brain exert opposing effects on energy expenditure. Elife. 2018;7:e36494.

    PubMed  PubMed Central  Google Scholar 

  41. 41.

    Lau J, Farzi A, Qi Y, Heilbronn R, Mietzsch M, Shi YC, et al. CART neurons in the arcuate nucleus and lateral hypothalamic area exert differential controls on energy homeostasis. Mol Metab. 2018;7:102–18.

    CAS  PubMed  Google Scholar 

  42. 42.

    Lau J, Shi YC, Herzog H. Temperature dependence of the control of energy homeostasis requires CART signaling. Neuropeptides. 2016;59:97–109.

    CAS  PubMed  Google Scholar 

  43. 43.

    Bajo M, Cruz MT, Siggins GR, Messing R, Roberto M. Protein kinase C epsilon mediation of CRF-and ethanol-induced GABA release in central amygdala. Proc Natl Acad Sci. 2008;105:8410–5.

    CAS  PubMed  Google Scholar 

  44. 44.

    Lesscher HM, Wallace MJ, Zeng L, Wang V, Deitchman JK, McMahon T, et al. Amygdala protein kinase C epsilon controls alcohol consumption. Genes Brain Behav. 2009;8:493–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Cui Y, Lv G, Jin S, Peng J, Yuan J, He X, et al. A central amygdala-substantia innominata neural circuitry encodes aversive reinforcement signals. Cell Rep. 2017;21:1770–82.

    CAS  PubMed  Google Scholar 

  46. 46.

    Botta P, Demmou L, Kasugai Y, Markovic M, Xu C, Fadok JP, et al. Regulating anxiety with extrasynaptic inhibition. Nat Neurosci. 2015;18:1493.

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Tanaka M, Yoshida M, Emoto H, Ishii H. Noradrenaline systems in the hypothalamus, amygdala and locus coeruleus are involved in the provocation of anxiety: basic studies. Eur J Pharmacol. 2000;405:397–406.

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    See RE, Waters RP. Pharmacologically-induced stress: a cross-species probe for translational research in drug addiction and relapse. Am J Transl Res. 2011;3:81.

    CAS  Google Scholar 

  49. 49.

    McDougle CJ, Price LH, Heninger GR, Krystal JH, Charney DS. Noradrenergic response to acute ethanol administration in heathly subjects: comparison with intravenous yohimbine. Psychopharmacology. 1995;118:127–35.

    CAS  PubMed  Google Scholar 

  50. 50.

    Umhau JC, Schwandt ML, Usala J, Geyer C, Singley E, George DT, et al. Pharmacologically induced alcohol craving in treatment seeking alcoholics correlates with alcoholism severity, but is insensitive to acamprosate. Neuropsychopharmacology. 2011;36:1178–86.

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Cippitelli A, Damadzic R, Hansson AC, Singley E, Sommer WH, Eskay R, et al. Neuropeptide Y (NPY) suppresses yohimbine-induced reinstatement of alcohol seeking. Psychopharmacology. 2010;208:417–26.

    CAS  PubMed  Google Scholar 

  52. 52.

    Funk D, Coen K, Tamadon S, Li Z, Loughlin A, Lê AD. Effects of prazosin and doxazosin on yohimbine-induced reinstatement of alcohol seeking in rats. Psychopharmacology. 2016;233:2197–207.

    CAS  PubMed  Google Scholar 

  53. 53.

    Lê AD, Funk D, Harding S, Juzytsch W, Fletcher PJ. The role of noradrenaline and 5-hydroxytryptamine in yohimbine-induced increases in alcohol-seeking in rats. Psychopharmacology. 2009;204:477–88.

    PubMed Central  Google Scholar 

  54. 54.

    Marinelli PW, Funk D, Juzytsch W, Harding S, Rice KC, Shaham Y, et al. The CRF 1 receptor antagonist antalarmin attenuates yohimbine-induced increases in operant alcohol self-administration and reinstatement of alcohol seeking in rats. Psychopharmacology. 2007;195:345–55.

    CAS  PubMed  Google Scholar 

  55. 55.

    Schmeichel BE, Herman MA, Roberto M, Koob GF. Hypocretin neurotransmission within the central amygdala mediates escalated cocaine self-administration and stress-induced reinstatement in rats. Biol Psychiatry. 2017;81:606–15.

    CAS  PubMed  Google Scholar 

  56. 56.

    Chen YW, Fiscella KA, Bacharach SZ, Tanda G, Shaham Y, Calu DJ. Effect of yohimbine on reinstatement of operant responding in rats is dependent on cue contingency but not food reward history. Addiction Biol. 2015;20:690–700.

    CAS  Google Scholar 

  57. 57.

    Funk D, Li Z, Coen K, Lê AD. Effects of pharmacological stressors on c-fos and CRF mRNA in mouse brain: relationship to alcohol seeking. Neurosci Lett. 2008;444:254–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Funk D, Li Z, Le A. Effects of environmental and pharmacological stressors on c-fos and corticotropin-releasing factor mRNA in rat brain: relationship to the reinstatement of alcohol seeking. Neuroscience. 2006;138:235–43.

    CAS  PubMed  Google Scholar 

  59. 59.

    Torruella-Suárez ML, Vandenberg JR, Cogan ES, Tipton GJ, Teklezghi A, Dange K, et al. Manipulations of central amygdala neurotensin neurons alter the consumption of ethanol and sweet fluids in mice. J Neurosci. 2020;40:632–47.

    PubMed  PubMed Central  Google Scholar 

  60. 60.

    Yosten GL, Harada CM, Haddock C, Giancotti LA, Kolar GR, Patel R, et al. GPR160 de-orphanization reveals critical roles in neuropathic pain in rodents. J Clin Investig. 2020;130:2587–92.

    CAS  PubMed  Google Scholar 

  61. 61.

    Foster SR, Hauser AS, Vedel L, Strachan RT, Huang XP, Gavin AC, et al. Discovery of human signaling systems: pairing peptides to G protein-coupled receptors. Cell. 2019;179:895–908.e21.

    CAS  PubMed  PubMed Central  Google Scholar 

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LCW, AH, BL, KH & EJC conducted experiments, LCW and AH performed analysis. LCW & AJL conceived experiments and wrote the paper with input from all authors.

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Correspondence to Leigh C. Walker or Andrew J. Lawrence.

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Walker, L.C., Hand, L.J., Letherby, B. et al. Cocaine and amphetamine regulated transcript (CART) signalling in the central nucleus of the amygdala modulates stress-induced alcohol seeking. Neuropsychopharmacol. (2020). https://doi.org/10.1038/s41386-020-00807-4

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