Deficient endocannabinoid signaling in the central amygdala contributes to alcohol dependence-related anxiety-like behavior and excessive alcohol intake



Negative emotional states that are associated with excessive alcohol intake, particularly anxiety-like states, have been linked to opponent processes in the central nucleus of the amygdala (CeA), affecting stress-related transmitters and monoamines. This study extends these observations to include endocannabinoid signaling in alcohol-dependent animals. Rats and mice were exposed to chronic intermittent alcohol with vapor inhalation or liquid diet to induce dependence. In vivo microdialysis was used to estimate interstitial concentrations of endocannabinoids [N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG)] and amino acids (glutamate and GABA) in rat CeA. Additionally, we evaluated the inhibition of endocannabinoids clearance enzymes [monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase] on anxiety-like behavior and alcohol consumption in alcohol-dependent rats and mice. Results revealed that alcohol dependence produced decreases in baseline 2-AG dialysate levels and increases in baseline levels of glutamate and GABA. Acute alcohol abstinence induced an enhancement of these dependence-induced effects and the levels of 2-AG and GABA were restored upon alcohol re-exposure. Additional studies showed that the increased CeA 2-AG levels induced by restraint stress and alcohol self-administration were blunted in alcohol-dependent rats. Pharmacological studies in rats and mice showed that anxiety-like behavior and alcohol consumption were increased in alcohol-dependent animals, and these behavioral effects were attenuated mainly by MAGL inhibitors [MJN110 (10 and 20 mg/kg) in rats and JZL184 (1 and 3 mg/kg) in mice]. The present results suggest a key role for endocannabinoid signaling in motivational neuroadaptations during alcohol dependence, in which a deficiency in CeA 2-AG signaling in alcohol-dependent animals is linked to stress and excessive alcohol consumption.

  • Subscribe to Neuropsychopharmacology for full access:



Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

Additional information

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


  1. 1.

    Koob GF. Theoretical frameworks and mechanistic aspects of alcohol addiction: alcohol addiction as a reward deficit disorder. Curr Top Behav Neurosci. 2013;13:3–30.

  2. 2.

    Heilig M, Koob GF, Ekman R, Britton KT. Corticotropin-releasing factor and neuropeptide Y: role in emotional integration. Trends Neurosci. 1994;17:80–85.

  3. 3.

    Lindell SG, Schwandt ML, Sun H, Sparenborg JD, Bjork K, Kasckow JW, et al. Functional NPY variation as a factor in stress resilience and alcohol consumption in rhesus macaques. Arch Gen Psychiatry. 2010;67:423–31.

  4. 4.

    Caberlotto L, Rimondini R, Hansson A, Eriksson S, Heilig M. Corticotropin-releasing hormone (CRH) mRNA expression in rat central amygdala in cannabinoid tolerance and withdrawal: evidence for an allostatic shift? Neuropsychopharmacology. 2004;29:15–22.

  5. 5.

    Gilpin NW, Roberto M. Neuropeptide modulation of central amygdala neuroplasticity is a key mediator of alcohol dependence. Neurosci Biobehav Rev. 2012;36:873–88.

  6. 6.

    Koob GF, Volkow ND. Neurocircuitry of addiction. Neuropsychopharmacology. 2010;35:217–38.

  7. 7.

    Witkin JM, Statnick MA, Rorick-Kehn LM, Pintar JE, Ansonoff M, Chen Y, et al. The biology of nociceptin/orphanin FQ (N/OFQ) related to obesity, stress, anxiety, mood, and drug dependence. Pharmacol Ther. 2014;141:283–99.

  8. 8.

    Ciccocioppo R, Cippitelli A, Economidou D, Fedeli A, Massi M. Nociceptin/orphanin FQ acts as a functional antagonist of corticotropin-releasing factor to inhibit its anorectic effect. Physiol Behav. 2004;82:63–68.

  9. 9.

    Ciccocioppo R, de Guglielmo G, Hansson AC, Ubaldi M, Kallupi M, Cruz MT, et al. Restraint stress alters nociceptin/orphanin FQ and CRF systems in the rat central amygdala: significance for anxiety-like behaviors. J Neurosci. 2014;34:363–72.

  10. 10.

    Morena M, Patel S, Bains JS, Hill MN. Neurobiological interactions between stress and the endocannabinoid system. Neuropsychopharmacology. 2016;41:80–102.

  11. 11.

    Dlugos A, Childs E, Stuhr KL, Hillard CJ, de Wit H. Acute stress increases circulating anandamide and other N-acylethanolamines in healthy humans. Neuropsychopharmacology. 2012;37:2416–27.

  12. 12.

    Hill MN, McLaughlin RJ, Morrish AC, Viau V, Floresco SB, Hillard CJ, et al. Suppression of amygdalar endocannabinoid signaling by stress contributes to activation of the hypothalamic-pituitary-adrenal axis. Neuropsychopharmacology. 2009;34:2733–45.

  13. 13.

    Patel S, Kingsley PJ, Mackie K, Marnett LJ, Winder DG. Repeated homotypic stress elevates 2-arachidonoylglycerol levels and enhances short-term endocannabinoid signaling at inhibitory synapses in basolateral amygdala. Neuropsychopharmacology. 2009;34:2699–709.

  14. 14.

    Bluett RJ, Gamble-George JC, Hermanson DJ, Hartley ND, Marnett LJ, Patel S. Central anandamide deficiency predicts stress-induced anxiety: behavioral reversal through endocannabinoid augmentation. Transl Psychiatry. 2014;4:e408.

  15. 15.

    Patel S, Roelke CT, Rademacher DJ, Cullinan WE, Hillard CJ. Endocannabinoid signaling negatively modulates stress-induced activation of the hypothalamic-pituitary-adrenal axis. Endocrinology. 2004;145:5431–8.

  16. 16.

    Sumislawski JJ, Ramikie TS, Patel S. Reversible gating of endocannabinoid plasticity in the amygdala by chronic stress: a potential role for monoacylglycerol lipase inhibition in the prevention of stress-induced behavioral adaptation. Neuropsychopharmacology. 2011;36:2750–61.

  17. 17.

    Haller J, Varga B, Ledent C, Freund TF. CB1 cannabinoid receptors mediate anxiolytic effects: convergent genetic and pharmacological evidence with CB1-specific agents. Behav Pharmacol. 2004;15:299–304.

  18. 18.

    Bellocchio L, Soria-Gomez E, Quarta C, Metna-Laurent M, Cardinal P, Binder E, et al. Activation of the sympathetic nervous system mediates hypophagic and anxiety-like effects of CB(1) receptor blockade. Proc Natl Acad Sci USA. 2013;110:4786–91.

  19. 19.

    Shonesy BC, Bluett RJ, Ramikie TS, Baldi R, Hermanson DJ, Kingsley PJ, et al. Genetic disruption of 2-arachidonoylglycerol synthesis reveals a key role for endocannabinoid signaling in anxiety modulation. Cell Rep. 2014;9:1644–53.

  20. 20.

    Dincheva I, Drysdale AT, Hartley CA, Johnson DC, Jing D, King EC, et al. FAAH genetic variation enhances fronto-amygdala function in mouse and human. Nat Commun. 2015;6:6395.

  21. 21.

    Hill MN, Miller GE, Ho WS, Gorzalka BB, Hillard CJ. Serum endocannabinoid content is altered in females with depressive disorders: a preliminary report. Pharmacopsychiatry. 2008;41:48–53.

  22. 22.

    Chouker A, Kaufmann I, Kreth S, Hauer D, Feuerecker M, Thieme D, et al. Motion sickness, stress and the endocannabinoid system. PLoS ONE. 2010;5:e10752.

  23. 23.

    Hill MN, Patel S. Translational evidence for the involvement of the endocannabinoid system in stress-related psychiatric illnesses. Biol Mood Anxiety Disord. 2013b;3:19.

  24. 24.

    Hillard CJ, Weinlander KM, Stuhr KL. Contributions of endocannabinoid signaling to psychiatric disorders in humans: genetic and biochemical evidence. Neuroscience. 2012;204:207–29.

  25. 25.

    Henricks AM, Berger AL, Lugo JM, Baxter-Potter LN, Bieniasz KV, Craft RM, et al. Sex differences in alcohol consumption and alterations in nucleus accumbens endocannabinoid mRNA in alcohol-dependent rats. Neuroscience. 2016;335:195–206.

  26. 26.

    Henricks AM, Berger AL, Lugo JM, Baxter-Potter LN, Bieniasz KV, Petrie G, et al. Sex- and hormone-dependent alterations in alcohol withdrawal-induced anxiety and corticolimbic endocannabinoid signaling. Neuropharmacology. 2017;124:121–33.

  27. 27.

    Serrano A, Rivera P, Pavon FJ, Decara J, Suarez J, Rodriguez de Fonseca F, et al. Differential effects of single versus repeated alcohol withdrawal on the expression of endocannabinoid system-related genes in the rat amygdala. Alcohol Clin Exp Res. 2012;36:984–94.

  28. 28.

    Vinod KY, Kassir SA, Hungund BL, Cooper TB, Mann JJ, Arango V. Selective alterations of the CB1 receptors and the fatty acid amide hydrolase in the ventral striatum of alcoholics and suicides. J Psychiatr Res. 2010;44:591–7.

  29. 29.

    Hirvonen J, Zanotti-Fregonara P, Umhau JC, George DT, Rallis-Frutos D, Lyoo CH, et al. Reduced cannabinoid CB1 receptor binding in alcohol dependence measured with positron emission tomography. Mol Psychiatry. 2013;18:916–21.

  30. 30.

    Ceccarini J, Hompes T, Verhaeghen A, Casteels C, Peuskens H, Bormans G, et al. Changes in cerebral CB1 receptor availability after acute and chronic alcohol abuse and monitored abstinence. J Neurosci. 2014;34:2822–31.

  31. 31.

    Varodayan FP, Soni N, Bajo M, Luu G, Madamba SG, Schweitzer P, et al. Chronic ethanol exposure decreases CB1 receptor function at GABAergic synapses in the rat central amygdala. Addict Biol. 2016;21:788–801.

  32. 32.

    Ahn K, Johnson DS, Mileni M, Beidler D, Long JZ, McKinney MK, et al. Discovery and characterization of a highly selective FAAH inhibitor that reduces inflammatory pain. Chem Biol. 2009;16:411–20.

  33. 33.

    Long JZ, Li W, Booker L, Burston JJ, Kinsey SG, Schlosburg JE, et al. Selective blockade of 2-arachidonoylglycerol hydrolysis produces cannabinoid behavioral effects. Nat Chem Biol. 2009;5:37–44.

  34. 34.

    Niphakis MJ, Cognetta AB 3rd, Chang JW, Buczynski MW, Parsons LH, Byrne F, et al. Evaluation of NHS carbamates as a potent and selective class of endocannabinoid hydrolase inhibitors. ACS Chem Neurosci. 2013;4:1322–32.

  35. 35.

    Roberto M, Madamba SG, Stouffer DG, Parsons LH, Siggins GR. Increased GABA release in the central amygdala of ethanol-dependent rats. J Neurosci. 2004a;24:10159–66.

  36. 36.

    Vendruscolo LF, Barbier E, Schlosburg JE, Misra KK, Whitfield TW Jr., Logrip ML, et al. Corticosteroid-dependent plasticity mediates compulsive alcohol drinking in rats. J Neurosci. 2012;32:7563–71.

  37. 37.

    Merlo Pich E, Lorang M, Yeganeh M, Rodriguez de Fonseca F, Raber J, Koob GF, et al. Increase of extracellular corticotropin-releasing factor-like immunoreactivity levels in the amygdala of awake rats during restraint stress and ethanol withdrawal as measured by microdialysis. J Neurosci. 1995;15:5439–47.

  38. 38.

    Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. New York: Academic Press, Spiral Bound; 1998.

  39. 39.

    Caille S, Alvarez-Jaimes L, Polis I, Stouffer DG, Parsons LH. Specific alterations of extracellular endocannabinoid levels in the nucleus accumbens by ethanol, heroin, and cocaine self-administration. J Neurosci. 2007;27:3695–702.

  40. 40.

    Zapata A, Chefer VI, Shippenberg TS, Denoroy L. Detection and quantification of neurotransmitters in dialysates. Curr Protoc Neurosci. 2009;4:1–30.

  41. 41.

    Haller J, Barna I, Barsvari B, Gyimesi Pelczer K, Yasar S, Panlilio LV, et al. Interactions between environmental aversiveness and the anxiolytic effects of enhanced cannabinoid signaling by FAAH inhibition in rats. Psychopharmacol (Berl). 2009;204:607–16.

  42. 42.

    Sciolino NR, Zhou W, Hohmann AG. Enhancement of endocannabinoid signaling with JZL184, an inhibitor of the 2-arachidonoylglycerol hydrolyzing enzyme monoacylglycerol lipase, produces anxiolytic effects under conditions of high environmental aversiveness in rats. Pharmacol Res. 2011;64:226–34.

  43. 43.

    Wiskerke J, Irimia C, Cravatt BF, De Vries TJ, Schoffelmeer AN, Pattij T, et al. Characterization of the effects of reuptake and hydrolysis inhibition on interstitial endocannabinoid levels in the brain: an in vivo microdialysis study. ACS Chem Neurosci. 2012;3:407–17.

  44. 44.

    Serrano A, Parsons LH. Endocannabinoid influence in drug reinforcement, dependence and addiction-related behaviors. Pharmacol Ther. 2011;132:215–41.

  45. 45.

    Hillard CJ. Biochemistry and pharmacology of the endocannabinoids arachidonylethanolamide and 2-arachidonylglycerol. Prostaglandins Other Lipid Mediat. 2000;61:3–18.

  46. 46.

    Sugiura T, Kobayashi Y, Oka S, Waku K. Biosynthesis and degradation of anandamide and 2-arachidonoylglycerol and their possible physiological significance. Prostaglandins Leukot Essent Fat Acids. 2002;66:173–92.

  47. 47.

    Kano M, Ohno-Shosaku T, Hashimotodani Y, Uchigashima M, Watanabe M. Endocannabinoid-mediated control of synaptic transmission. Physiol Rev. 2009;89:309–80.

  48. 48.

    Lovinger DM. Presynaptic modulation by endocannabinoids. Handb Exp Pharmacol. 2008;184:435–77.

  49. 49.

    Marsicano G, Lutz B. Neuromodulatory functions of the endocannabinoid system. J Endocrinol Invest. 2006;29:27–46.

  50. 50.

    Roberto M, Schweitzer P, Madamba SG, Stouffer DG, Parsons LH, Siggins GR. Acute and chronic ethanol alter glutamatergic transmission in rat central amygdala: an in vitro and in vivo analysis. J Neurosci. 2004;24:1594–603.

  51. 51.

    Roberto M, Cruz MT, Gilpin NW, Sabino V, Schweitzer P, Bajo M, et al. Corticotropin releasing factor-induced amygdala gamma-aminobutyric Acid release plays a key role in alcohol dependence. Biol Psychiatry. 2010;67:831–9.

  52. 52.

    Gerdeman G, Lovinger DM. CB1 cannabinoid receptor inhibits synaptic release of glutamate in rat dorsolateral striatum. J Neurophysiol. 2001;85:468–71.

  53. 53.

    Roberto M, Cruz M, Bajo M, Siggins GR, Parsons LH, Schweitzer P. The endocannabinoid system tonically regulates inhibitory transmission and depresses the effect of ethanol in central amygdala. Neuropsychopharmacology. 2010;35:1962–72.

  54. 54.

    Hill MN, McLaughlin RJ, Bingham B, Shrestha L, Lee TT, Gray JM, et al. Endogenous cannabinoid signaling is essential for stress adaptation. Proc Natl Acad Sci USA. 2010;107:9406–11.

  55. 55.

    Patel S, Roelke CT, Rademacher DJ, Hillard CJ. Inhibition of restraint stress-induced neural and behavioural activation by endogenous cannabinoid signalling. Eur J Neurosci. 2005;21:1057–69.

  56. 56.

    Hill MN, Kumar SA, Filipski SB, Iverson M, Stuhr KL, Keith JM, et al. Disruption of fatty acid amide hydrolase activity prevents the effects of chronic stress on anxiety and amygdalar microstructure. Mol Psychiatry. 2013;18:1125–35.

  57. 57.

    Gray JM, Vecchiarelli HA, Morena M, Lee TT, Hermanson DJ, Kim AB, et al. Corticotropin-releasing hormone drives anandamide hydrolysis in the amygdala to promote anxiety. J Neurosci. 2015;35:3879–92.

  58. 58.

    Natividad LA, Buczynski MW, Herman MA, Kirson D, Oleata CS, Irimia C, et al. Constitutive increases in amygdalar corticotropin-releasing factor and fatty acid amide hydrolase drive an anxious phenotype. Biol Psychiatry. 2017;82:500-510.

  59. 59.

    Bedse G, Hartley ND, Neale E, Gaulden AD, Patrick TA, Kingsley PJ, et al. Functional redundancy between canonical endocannabinoid signaling systems in the modulation of anxiety. Biol Psychiatry. 2017;82:488–99.

  60. 60.

    Di Marzo V, De Petrocellis L. Why do cannabinoid receptors have more than one endogenous ligand? Philos Trans R Soc Lond B Biol Sci. 2012;367:3216–28.

  61. 61.

    Richardson HN, Lee SY, O’Dell LE, Koob GF, Rivier CL. Alcohol self-administration acutely stimulates the hypothalamic-pituitary-adrenal axis, but alcohol dependence leads to a dampened neuroendocrine state. Eur J Neurosci. 2008;28:1641–53.

  62. 62.

    Di S, Itoga CA, Fisher MO, Solomonow J, Roltsch EA, Gilpin NW, et al. Acute stress suppresses synaptic inhibition and increases anxiety via endocannabinoid release in the basolateral amygdala. J Neurosci. 2016;36:8461–70.

  63. 63.

    Evanson NK, Tasker JG, Hill MN, Hillard CJ, Herman JP. Fast feedback inhibition of the HPA axis by glucocorticoids is mediated by endocannabinoid signaling. Endocrinology. 2010;151:4811–9.

  64. 64.

    Hill MN, McLaughlin RJ, Pan B, Fitzgerald ML, Roberts CJ, Lee TT, et al. Recruitment of prefrontal cortical endocannabinoid signaling by glucocorticoids contributes to termination of the stress response. J Neurosci. 2011;31:10506–15.

  65. 65.

    Wang M, Hill MN, Zhang L, Gorzalka BB, Hillard CJ, Alger BE. Acute restraint stress enhances hippocampal endocannabinoid function via glucocorticoid receptor activation. J Psychopharmacol. 2012;26:56–70.

  66. 66.

    Valdez GR, Sabino V, Koob GF. Increased anxiety-like behavior and ethanol self-administration in dependent rats: reversal via corticotropin-releasing factor-2 receptor activation. Alcohol Clin Exp Res. 2004;28:865–72.

  67. 67.

    Valdez GR, Zorrilla EP, Roberts AJ, Koob GF. Antagonism of corticotropin-releasing factor attenuates the enhanced responsiveness to stress observed during protracted ethanol abstinence. Alcohol. 2003;29:55–60.

  68. 68.

    Cippitelli A, Cannella N, Braconi S, Duranti A, Tontini A, Bilbao A, et al. Increase of brain endocannabinoid anandamide levels by FAAH inhibition and alcohol abuse behaviours in the rat. Psychopharmacol (Berl). 2008;198:449–60.

Download references


We thank Dr. George F. Koob for insightful comments on the manuscript and Mr. Michael Arends for editing the manuscript. We dedicate this manuscript in loving memory to our departed friend, colleague, and mentor, LHP. He was an extraordinary researcher in the field of endocannabinoid signaling, stress, and drug addiction.

Funding and disclosure

This research was supported by the following grants: National Institute on Alcohol Abuse and Alcoholism (AA020404, AA022249, and AA024146 to RMF; AA006420 to RMF and MR; AA017447 and AA015566 to MR; and K99-AA025393 to LAN) and National Institutes of Health (K99/R00DA037344 to JS). This research was also supported by the Pearson Center for Alcoholism and Addiction Research, Instituto de Salud Carlos III (ISCIII) and European Regional Development Funds-European Union (ERDF-EU; Subprograma RETICS Red de Trastornos Adictivos RD12/0028/0001), Ministerio de Economía y Competitividad and ISCIII (PI16/01953 and PI17/02026), Ministerio de Sanidad, Servicios Sociales e Igualdad and Plan Nacional sobre Drogas (PND2017/043), and Consejería de Economía, Innovación y Ciencia, Junta de Andalucía, and ERDF-EU (CTS-433). AS and FJP hold a “Miguel Servet” research contract funded by ISCIII and ERDF-EU (CP14/00173 and CP14/00212, respectively). This is article number 29530 from The Scripps Research Institute.

Author information

Author notes

  1. Deceased: Loren H. Parsons.


  1. Department of Neuroscience, The Scripps Research Institute, La Jolla, CA, USA

    • Antonia Serrano
    • , Francisco J. Pavon
    • , Matthew W. Buczynski
    • , Luis A. Natividad
    • , Ilham Y. Polis
    • , David G. Stouffer
    • , Eric P. Zorrilla
    • , Marisa Roberto
    • , Rémi Martin-Fardon
    •  & Loren H. Parsons
  2. Unidad de Gestión Clínica de Salud Mental, Instituto de Investigación Biomédica de Málaga, Hospital Regional Universitario de Málaga, Málaga, Spain

    • Antonia Serrano
    • , Francisco J. Pavon
    •  & Fernando Rodriguez de Fonseca
  3. Department of Pharmacology & Toxicology, Virginia Commonwealth University, Richmond, VA, USA

    • Joel Schlosburg
  4. Department of Chemical Physiology, Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, USA

    • Benjamin F. Cravatt


  1. Search for Antonia Serrano in:

  2. Search for Francisco J. Pavon in:

  3. Search for Matthew W. Buczynski in:

  4. Search for Joel Schlosburg in:

  5. Search for Luis A. Natividad in:

  6. Search for Ilham Y. Polis in:

  7. Search for David G. Stouffer in:

  8. Search for Eric P. Zorrilla in:

  9. Search for Marisa Roberto in:

  10. Search for Benjamin F. Cravatt in:

  11. Search for Rémi Martin-Fardon in:

  12. Search for Fernando Rodriguez de Fonseca in:

  13. Search for Loren H. Parsons in:

Conflict of interest

The authors declare that they have no conflict of interest.

Corresponding authors

Correspondence to Antonia Serrano or Rémi Martin-Fardon or Fernando Rodriguez de Fonseca.

Electronic supplementary material