Driving the downward spiral: alcohol-induced dysregulation of extended amygdala circuits and negative affect

“The CeA exhibits heightened activation immediately after a threat is perceived (Ciocchi et al., 2010, LeDoux, 2000), while the BNST is chronically active during sustained periods of fear, depression, anxiety, and/or stress (Lebow and Chen, 2016, Ch’ng et al., 2018).”

“…by disturbances in three major neurocircuits: 1) basal ganglia-driven binge/intoxication stage, 2) extended amygdala (EA)-driven withdrawal/negative affect stage, and 3) prefrontal cortex/hippocampalregulated preoccupation/anticipation stage (Koob and Volkow, 2010, Koob and Volkow, 2016).”

“In addition, the CeA and BNST send and receive numerous cortical and subcortical projections synapsing onto cells in different subnuclei, and onto either GABAergic interneurons or output neurons (Dong et al., 2001, Janak and Tye, 2015).”

“The CeA exhibits heightened activation immediately after a threat is perceived (Ciocchi et al., 2010, LeDoux, 2000), while the BNST is chronically active during sustained periods of fear, depression, anxiety, and/or stress (Lebow and Chen, 2016, Ch’ng et al., 2018).”

“During withdrawal, patients often attribute stress and negative affective states to cravings and relapse (Grant et al., 2016) and the severity of symptoms is highly Alcohol Clin Exp Res.”

“CRF and its endogenous receptors are widely expressed in the BNST and are considered potent regulators of BNST neural activity and behavioral output (Walker et al., 2003).”

“The CeA exhibits heightened activation immediately after a threat is perceived (Ciocchi et al., 2010, LeDoux, 2000), while the BNST is chronically active during sustained periods of fear, depression, anxiety, and/or stress (Lebow and Chen, 2016, Ch’ng et al., 2018).”

“…in acute withdrawal came from Merlo Pich et al., demonstrating increased CRF levels in the CeA immediately after removing an ethanol diet after 2–3 weeks of continuous access (Merlo Pich et al., 1995), and this is associated with increases in the CRF precursor pre-proCRF mRNA (Lack et al., 2005).”

“…†Co-corresponding Authors: uthor M anuscript A uthor M anuscript A uthor M anuscript INTRODUCTION Alcohol misuse and related Alcohol Use Disorder (AUD) take an enormous toll, afflicting over 15 million adults, and resulting in 88,000 alcohol misuse-related deaths annually (Stahre et al., 2014).”

“In addition, the CeA and BNST send and receive numerous cortical and subcortical projections synapsing onto cells in different subnuclei, and onto either GABAergic interneurons or output neurons (Dong et al., 2001, Janak and Tye, 2015).”

“ACUTE ETHANOL EFFECTS CeA Numerous studies have focused on the CeA after ethanol intake (for detailed review see (Gilpin et al., 2015)).”

“The CeA and BNST are highly interconnected, sending and receiving dense, reciprocal GABAergic projections through the ventral amygdalofugal pathway and stria terminalis (Nauta, 1961).”

“CRF levels in the CeA are elevated after an acute intraperitoneal injection of ethanol (Lam and Gianoulakis, 2011), and bath applying CRF can augment CeA evoked IPSCs (Nie et al., 2009, Roberto et al., 2010).”

“Following CIE, CeA GABA transmission and tone are increased, and these changes are accompanied by increased CeA GABA levels (Roberto et al., 2004a, Repunte-Canonigo et al., 2015).”

“At the molecular level, increased ethanol intake following CIE or the limited access binge-drinking model drinking in the dark (DID), correlates with increased CeA CRF expression (Sommer et al., 2008, Lowery-Gionta et al., 2012).”

“CeA infusion of the GABAAR antagonist SR95531 reduced voluntary ethanol consumption (Hyytia and Koob, 1995), suggesting CeA GABAergic transmission is required for voluntary consumption.”

“Following CIE, CeA GABA transmission and tone are increased, and these changes are accompanied by increased CeA GABA levels (Roberto et al., 2004a, Repunte-Canonigo et al., 2015).”

“A majority of BNST neurons are GABAergic, interspersed with sparse glutamatergic neurons (Cassell et al., 1999, Poulin et al., 2009).”

“…differences in the BNST in ethanol withdrawal have not been widely examined to date, and given the known sex differences associated with the BNST (Allen and Gorski, 1990), more research in this area is required, as it could significantly change our understanding of the BNST in AUD and…”

“The negative emotional state that drives negative reinforcement in AUD is hypothesized to derive from decreased activity in brain reward circuits, and recruitment of stress response circuits in the EA (Koob, 2003, Koob, 2014).”

“A recent study from De Guglielmo et al., found that inactivating the neuronal ensembles activated in the CeA during ethanol withdrawal using the Daun02 inactivation method (Cruz et al., 2013) decreased the withdrawal phenotype and consumption after CIE withdrawal (de Guglielmo et al., 2016).”

“Olive et al., used in vivo microdialysis to measure CRF levels in the BNST after 2 weeks of ethanol diet (Olive et al., 2002).”

“CeA While initial CeA lesion studies reported markedly reduced ethanol intake (Dhaher et al., 2008, Moller et al., 1997), but no effect on escalated ethanol consumption after repeated cycles of ethanol vapor (CIE) (Dhaher et al., 2008), several subsequent studies have demonstrated a role for the…”

“The negative emotional state that drives negative reinforcement in AUD is hypothesized to derive from decreased activity in brain reward circuits, and recruitment of stress response circuits in the EA (Koob, 2003, Koob, 2014).”

“Local CeA infusion of the CRF antagonist D-Phe-CRF (Finn et al., 2007) or CRF1R antagonist antalarmin (Lowery-Gionta et al., 2012), and shRNA-induced downregulation of CRF binding protein (CRFBP) in the CeA (Haass-Koffler et al., 2016) are all sufficient to reduce ethanol consumption.”

“NPY and its endogenous receptors are highly expressed in the BNST. Activating NPY1 receptors in the BNST suppresses ethanol drinking by enhancing inhibitory synaptic transmission onto CRF neurons (Pleil et al., 2015b).”

“A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript of HDAC2 and HDAC4, which parallels increased anxiety-like behaviors 24h after bingeethanol exposure (Pandey et al., 2015).”

“…EXPOSURE CeA While individuals exposed to ethanol early in life (here defined as prenatal through adolescence) do not always enter into the addiction cycle, exposure dramatically shifts neurodevelopment and increases the likelihood of later addiction (Squeglia et al., 2014, Silveri et al., 2016).”

“…differences associated with the BNST (Allen and Gorski, 1990), more research in this area is required, as it could significantly change our understanding of the BNST in AUD and withdrawal, and potentially explain why many antidepressants are less effective in females (Keers and Aitchison, 2010).”

“CeA infusion of a 5-HT2A receptor antisense oligonucleotide, BDNF antisense, or nociceptin successfully blunts basal ethanol intake (Economidou et al., 2008, Blakley et al., 2001, Pandey et al., 2006).”

“Infusing a PKA activator into the CeA normalized CREB phosphorylation, while a PKA inhibitor provoked anxiety-like behavior in withdrawal (Pandey et al., 2003), suggesting a bidirectional role for CeA PKA in withdrawal.”

“NPY is another densely expressed neuropeptide in the CeA that has anxiolytic properties (Wu et al., 2011).”

“NPY and its endogenous receptors are highly expressed in the BNST. Activating NPY1 receptors in the BNST suppresses ethanol drinking by enhancing inhibitory synaptic transmission onto CRF neurons (Pleil et al., 2015b).”

“Epigenetically, histone deacetylase (HDAC) activity and HDAC2 protein levels are decreased after acute ethanol, which is associated with increased CeA expression of BDNF and Arc, and increased spine density (Moonat et al., 2013).”

“Infusion of a specific GABAAα2 siRNA into the CeA similarly blunts binge-consumption using the DID model (Liu et al., 2011).”

“[PubMed: 19593822] YOU C, ZHANG H, SAKHARKAR AJ, TEPPEN T & PANDEY SC 2014 Reversal of deficits in dendritic spines, BDNF and Arc expression in the amygdala during alcohol dependence by HDAC inhibitor treatment.”

“Local CeA infusion of the CRF antagonist D-Phe-CRF (Finn et al., 2007) or CRF1R antagonist antalarmin (Lowery-Gionta et al., 2012), and shRNA-induced downregulation of CRF binding protein (CRFBP) in the CeA (Haass-Koffler et al., 2016) are all sufficient to reduce ethanol consumption.”

“Interestingly, adult rats exposed to ethanol in adolescence have fewer CRF neurons in the CeA than naïve controls (Gilpin et al., 2012), and this effect is more pronounced in females than males (Karanikas et al., 2013), suggesting lasting effects of adolescent ethanol exposure on the CeA CRF system.”

“The authors concluded that the Dyn/KOR system in the CeA mediates ethanol selfadministration in withdrawal (Kissler et al., 2014).”

“…collectively support a general hyperactive state of the BNST in acute withdrawal following CIE, and furthermore, the respective effects are regulated by CRF (Silberman et al., 2013), 5HT2c (Marcinkiewcz et al., 2015), GluN2B (Kash et al., 2009, Wills et al., 2012), and KOR (Erikson et al., 2018).”

“…bath application of ethanol to an acute BNST slice decreases NMDA receptor-dependent EPSC amplitude early-phase LTP (Weitlauf et al., 2004, Wills et al., 2012, Kash et al., 2009), suggesting the actions of ethanol act in part through the modulation of the BNST neural activity via NMDA receptors.”

“Accordingly, silencing VTA-projecting GABAergic BNST neurons is sufficient to blunt binge-like ethanol intake (Companion and Thiele, 2018, Rinker et al., 2017).”

“PROTRACTED ABSTINENCE Protracted abstinence is an undefined period of time beginning after the initial physical withdrawal signs have subsided and lasting indefinitely (Perry, 2014).”

“Further, the neurons recruited during withdrawal in the CeA were identified as mostly CRF+, and optogenetically inhibiting these neurons alleviated somatic signs of withdrawal in mice (de Guglielmo et al., 2019).”

“In addition, Gabapentin decreased GABA release onto CeA neurons in dependent rats and reduced the dependence-induced escalation of ethanol consumption when infused directly into the CeA (Roberto et al., 2008).”

“A majority of BNST neurons are GABAergic, interspersed with sparse glutamatergic neurons (Cassell et al., 1999, Poulin et al., 2009).”

“CeA infusion of a 5-HT2A receptor antisense oligonucleotide, BDNF antisense, or nociceptin successfully blunts basal ethanol intake (Economidou et al., 2008, Blakley et al., 2001, Pandey et al., 2006).”

“NPY is another densely expressed neuropeptide in the CeA that has anxiolytic properties (Wu et al., 2011).”

“…EXPOSURE CeA While individuals exposed to ethanol early in life (here defined as prenatal through adolescence) do not always enter into the addiction cycle, exposure dramatically shifts neurodevelopment and increases the likelihood of later addiction (Squeglia et al., 2014, Silveri et al., 2016).”

“CeA infusion of an mGlu1 (JNJ-16259685), mGlu5 (MTEP), or PLC (U-73122) antagonist dose-dependently decreases binge drinking in mice with a 30-day history of DID, and co-infusing the PLC inhibitor with the JNJ-1625685, but not MTEP was additive, further decreasing binge ethanol consumption (Cozzoli et al., 2014).”

“…cycles of ethanol vapor (CIE) (Dhaher et al., 2008), several subsequent studies have demonstrated a role for the CeA in both basal and dependence-induced escalated drinking behavior (de Guglielmo et al., 2016, de Guglielmo et al., 2019, Kissler and Walker, 2016, Repunte-Canonigo et al., 2015).”

“Additionally, recent evidence from Avegno et al. implicate the CeA melanocortin system in acute withdrawal, as withdrawal-induced hyperalgesia correlates with reduced MC4R expression in the CeA (Avegno et al., 2018).”

“BDNF and Arc expression are increased, and this involves histone acetylation, as administering the HDAC inhibitor TSA in withdrawal normalizes this effect (You et al., 2014).”

“…ex vivo bath application of ethanol to an acute BNST slice decreases NMDA receptor-dependent EPSC amplitude early-phase LTP (Weitlauf et al., 2004, Wills et al., 2012, Kash et al., 2009), suggesting the actions of ethanol act in part through the modulation of the BNST neural activity via NMDA…”

“A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript behaviors, as a BNST microinjection of the dopamine D1 receptor antagonist SCH23390 dose-dependently decreased self-administration (Eiler et al., 2003).”

“CeA infusions of NPY 6h after the removal of ethanol significantly decreases operant responding for ethanol (Gilpin et al., 2008), whereas repeated NPY CeA infusions before each binge ethanol session do not alter responding in withdrawal (Henderson and Czachowski, 2012), highlighting the critical…”

“Intra-CeA infusion of the CRF2R agonist urocortin 3 reduced ethanol self-administration in dependent rats, but increased drinking in nondependent rats, highlighting a unique dichotomy for CRF2R regulation of ethanol drinking in dependent versus nondependent rats (Funk and Koob, 2007).”

“Moreover, antalarmin in the CeA of adult rats subjected to maternal separation prevented an increase in ethanol consumption (Gondre-Lewis et al., 2016), and optogenetically inhibiting CRF cells in the CeA reversed the escalation of ethanol drinking (de Guglielmo et al., 2019), implicating the CeA…”

“…days into abstinence following contingent ethanol exposure the number of c-fos+ BNST CRF neurons is increased, as is sEPSC frequency (Centanni et al., 2018), and further, administering a CRF1 receptor antagonist prevents abstinence-induced decreases in firing threshold (Francesconi et al., 2009).”

“…phase, as 15 days into abstinence following contingent ethanol exposure the number of c-fos+ BNST CRF neurons is increased, as is sEPSC frequency (Centanni et al., 2018), and further, administering a CRF1 receptor antagonist prevents abstinence-induced decreases in firing threshold (Francesconi…”

“Interestingly, a second dose increases BNST DNA methyltransferase mRNA expression, suggesting increased ethanol tolerance in adolescent mice (Sakharkar et al., 2014).”

“Signaling mechanisms involving nociceptin (Cruz et al., 2012, Kallupi et al., 2014a, Kallupi et al., 2014b, Roberto and Siggins, 2006), dynorphin (Dyn) (Kang-Park et al., 2007), and neuropeptide Y (NPY) (Gilpin et al., 2011) decrease basal GABA release and oppose the acute actions of ethanol by…”

“Furthermore, chronic exposure to ethanol in adolescence blunts LTP in the adult BNST, and is associated with increased anxiety in adulthood (Conrad and Winder, 2011).”

“…uthor M anuscript In accordance with the CRF literature, mRNA and protein expression of NPY and its receptor Y1R is decreased in acute withdrawal immediately after removal of ethanol diet and these effects were detected 24h later (Pandey et al., 2003, Sparrow et al., 2012, Zhang and Pandey, 2003).”

“Bath applying ethanol to CEA slices increases GABA miniature and spontaneous inhibitory postsynaptic current (mIPSC/sIPSC) frequency (Varodayan et al., 2017b, Varodayan et al., 2017a, Herman et al., 2013, Roberto et al., 2010, Roberto et al., 2004a, Kang-Park et al., 2009, Nie et al., 2009, Nie et al., 2004), sIPSC am…”

“Τhe proinflammatory cytokine IL-1ß increases basal mIPSCs in most CeA cells, but interestingly, IL-1ß prevents the ethanol-induced increase in mIPSC frequency (Bajo et al., 2015).”

“…in the CeA are less consistent and appear to vary between cell-types and rodent species and strains, with reports of both increased glutamate activity (Silberman et al., 2015, Herman et al., 2016b), and decreased glutamate activity (Roberto et al., 2004b, Zhu et al., 2007, Kirson et al., 2018).”

“Ethanol intake increases NPY expression in the CeA, and NPY levels in the CeA are positively correlated to ethanol consumption (Zhang et al., 2010, Primeaux et al., 2006), reinforcing an important role for CeA NPY in ethanol drinking behavior.”

“Signaling mechanisms involving nociceptin (Cruz et al., 2012, Kallupi et al., 2014a, Kallupi et al., 2014b, Roberto and Siggins, 2006), dynorphin (Dyn) (Kang-Park et al., 2007), and neuropeptide Y (NPY) (Gilpin et al., 2011) decrease basal GABA release and oppose the acute actions of ethanol by…”

“At the cellular level, withdrawal following CIE decreased phasic and tonic GABA transmission in CRF1 receptor-expressing CeA neurons, but increased GABA transmission in neurons not expressing CRF1R (Herman et al., 2016a), suggesting a withdrawal-induced shift from CeA inhibitory control over CRF1 neurons to non-CRF1 n…”

“Ethanol intake increases NPY expression in the CeA, and NPY levels in the CeA are positively correlated to ethanol consumption (Zhang et al., 2010, Primeaux et al., 2006), reinforcing an important role for CeA NPY in ethanol drinking behavior.”

“Binge intoxication also decreases expression of the extracellular matrix protein MMP9, which is thought to regulate spine density, synaptic transmission, and the generation of silent synapses (Stefaniuk et al., 2017).”

“At the cellular level, withdrawal following CIE decreased phasic and tonic GABA transmission in CRF1 receptor-expressing CeA neurons, but increased GABA transmission in neurons not expressing CRF1R (Herman et al., 2016a), suggesting a withdrawal-induced shift from CeA inhibitory control over CRF1 neurons to non-CRF1 n…”

“…uthor M anuscript In accordance with the CRF literature, mRNA and protein expression of NPY and its receptor Y1R is decreased in acute withdrawal immediately after removal of ethanol diet and these effects were detected 24h later (Pandey et al., 2003, Sparrow et al., 2012, Zhang and Pandey, 2003).”

“Under 10% of individuals diagnosed with AUD seek treatment, and even with treatment, relapse is still extremely common, even years into abstinence (Tuithof et al., 2014).”

“…collectively support a general hyperactive state of the BNST in acute withdrawal following CIE, and furthermore, the respective effects are regulated by CRF (Silberman et al., 2013), 5HT2c (Marcinkiewcz et al., 2015), GluN2B (Kash et al., 2009, Wills et al., 2012), and KOR (Erikson et al., 2018).”

“…in acute withdrawal came from Merlo Pich et al., demonstrating increased CRF levels in the CeA immediately after removing an ethanol diet after 2–3 weeks of continuous access (Merlo Pich et al., 1995), and this is associated with increases in the CRF precursor pre-proCRF mRNA (Lack et al., 2005).”

“In particular, TNFα increases firing rate and GABA release in the CeA and intra-CeA TNFα can sensitize mice to anxietylike behavior induced by subsequent withdrawal episodes (Knapp et al., 2011).”

“Early withdrawal results in increased CeA expression of several immune markers including CCL2, TNFα, NOS2, and CD74 (Harper et al., 2017, Freeman et al., 2012).”

“…models of the withdrawal/negative affect stage of AUD include repeated cycles of withdrawal following CIE, forced abstinence following continuous or limited access, or extinguishing behaviors associated with operant responding for ethanol (Holleran and Winder, 2016, Becker and Ron, 2014).”

“…increases GABA miniature and spontaneous inhibitory postsynaptic current (mIPSC/sIPSC) frequency (Varodayan et al., 2017b, Varodayan et al., 2017a, Herman et al., 2013, Roberto et al., 2010, Roberto et al., 2004a, Kang-Park et al., 2009, Nie et al., 2009, Nie et al., 2004), sIPSC amplitude…”

“Τhe proinflammatory cytokine IL-1ß increases basal mIPSCs in most CeA cells, but interestingly, IL-1ß prevents the ethanol-induced increase in mIPSC frequency (Bajo et al., 2015).”

“…models of the withdrawal/negative affect stage of AUD include repeated cycles of withdrawal following CIE, forced abstinence following continuous or limited access, or extinguishing behaviors associated with operant responding for ethanol (Holleran and Winder, 2016, Becker and Ron, 2014).”

“This study went on to implicate the CeA opioid system in responding, as intra-CeA infusion of naltrexone, a mu/kappa opioid receptor (MOR/KOR) antagonist, produced a similar reduction in operant ethanol responding (Foster et al., 2004).”

“A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript Park et al., 2013), inhibitory postsynaptic potential (IPSP) amplitude (Cruz et al., 2011, Bajo et al., 2014), and decreases paired-pulse facilitation (Cruz et al., 2011, Roberto and Siggins, 2006).”

“In many cases, high blood ethanol levels are achieved through noncontingent administration to induce dependence, and subsequent dependence-induced escalation in contingent ethanol drinking patterns can be measured (Lopez and Becker, 2014).”

“At the cellular level, withdrawal following CIE decreased phasic and tonic GABA transmission in CRF1 receptor-expressing CeA neurons, but increased GABA transmission in neurons not expressing CRF1R (Herman et al., 2016a), suggesting a withdrawal-induced shift from CeA inhibitory control over CRF1 neurons to non-CRF1 n…”

“Moreover, ethanol CPP increases neuronal excitability in the BNST (Pati et al., 2019), and inactivating the BNST (through electrolytic lesions, pharmacological lesions, and chemogenetic inhibition) disrupts expression of CPP (Pina et al., 2015).”

“For example, neuropeptide S expression is increased in the CeA one week into withdrawal (Ruggeri et al., 2010).”

“CRF is highly expressed in the CeA and is an integral component to the extrahypothalamic stress response system (Pomrenze et al., 2017).”

“At the cellular level, withdrawal following CIE decreased phasic and tonic GABA transmission in CRF1 receptor-expressing CeA neurons, but increased GABA transmission in neurons not expressing CRF1R (Herman et al., 2016a), suggesting a withdrawal-induced shift from CeA inhibitory control over CRF1 neurons to non-CRF1 n…”

“Several other compounds infused into the CeA decrease ethanol responding including the neurokinin 1 receptor (NK1R) antagonist L822429 (Schank et al., 2013), FAAH inhibitor URB597 (Stopponi et al., 2018), and substance P (Yang et al., 2009).”

“In addition to NPY and CRF, the neuropeptides orexin and melanocortin are also implicated in modulating and regulating ethanol binge drinking (Aoun et al., 2018, Navarro et al., 2008, Olney et al., 2017).”

“Moreover, ethanol CPP increases neuronal excitability in the BNST (Pati et al., 2019), and inactivating the BNST (through electrolytic lesions, pharmacological lesions, and chemogenetic inhibition) disrupts expression of CPP (Pina et al., 2015).”

“Moreover, CRF may exert its effects by regulating neurotransmitter release via adenylate cyclase 7 (AC7) (Cruz et al., 2011) and/or P/Q-type voltage-gated calcium channels (Varodayan et al., 2017b).”

“Furthermore, rats on a chronic ethanol diet exhibit reduced levels of BNST alphamelanocyte stimulating hormone (alpha-MSH) (Navarro et al., 2008), a component of the melanocortin system.”

“Moreover, CRF may exert its effects by regulating neurotransmitter release via adenylate cyclase 7 (AC7) (Cruz et al., 2011) and/or P/Q-type voltage-gated calcium channels (Varodayan et al., 2017b).”

“Interestingly, adult rats exposed to ethanol in adolescence have fewer CRF neurons in the CeA than naïve controls (Gilpin et al., 2012), and this effect is more pronounced in females than males (Karanikas et al., 2013), suggesting lasting effects of adolescent ethanol exposure on the CeA CRF system.”

“Moreover, acute exposure to systemic ethanol (1 g/kg i.p.) increases expression of c-fos in the BNST in rodents (Saalfield and Spear, 2019, Sharko et al., 2016), and a recent study by Campbell et al., demonstrated that AAV-mediated knockdown of the scaffolding protein Homer2 in the BNST promotes…”

“BNST—Protracted abstinence has lasting impacts on BNST neural circuitry including decreased short-term plasticity (Vranjkovic et al., 2018), intrinsic firing threshold (Francesconi et al., 2009), and increased sEPSC frequency (Centanni et al., 2018) suggesting a shift in BNST homeostasis from a…”

“The number of activated microglia (marked by Iba-1) is reduced after CIE, and this effect is blocked by inhibiting TLR4 receptors (Bajo et al., 2016), highlighting a potential role for neuroimmune signaling in the CeA in binge drinking in a dependent population.”

“Moreover, ethanol CPP increases neuronal excitability in the BNST (Pati et al., 2019), and inactivating the BNST (through electrolytic lesions, pharmacological lesions, and chemogenetic inhibition) disrupts expression of CPP (Pina et al., 2015).”

“Furthermore, chronic exposure to ethanol in adolescence blunts LTP in the adult BNST, and is associated with increased anxiety in adulthood (Conrad and Winder, 2011).”

“…5-day bingedrinking bouts decreases CRF cell immuno-density in the CeA of adolescent, but not adult mice, which the authors attribute to increased CRF release resulting in depleted CRF cellular stores, suggesting adolescents may be more sensitive to stress-ethanol interactions (Wills et al., 2010).”

“Local CeA infusion of the CRF antagonist D-Phe-CRF (Finn et al., 2007) or CRF1R antagonist antalarmin (Lowery-Gionta et al., 2012), and shRNA-induced downregulation of CRF binding protein (CRFBP) in the CeA (Haass-Koffler et al., 2016) are all sufficient to reduce ethanol consumption.”

“CRF levels in the CeA are elevated after an acute intraperitoneal injection of ethanol (Lam and Gianoulakis, 2011), and bath applying CRF can augment CeA evoked IPSCs (Nie et al., 2009, Roberto et al., 2010).”

“NPY and its endogenous receptors are highly expressed in the BNST. Activating NPY1 receptors in the BNST suppresses ethanol drinking by enhancing inhibitory synaptic transmission onto CRF neurons (Pleil et al., 2015b).”

“Intra-CeA infusion of a cDNA targeting the mGlu5 scaffolding protein Homer2 immediately after adolescent binge-drinking prevented negative affective behaviors 4 weeks into withdrawal, outlining the potential protective effect of decreasing Homer2 in the CeA (Lee et al., 2018).”

“Indeed, patients with AUD frequently report heightened anxiety, irritability, dysphoria, and negative emotional states as potent triggers of cravings and relapse, and the severity of symptoms is strongly correlated with the relapse susceptibility (Fox et al., 2017).”

“Moreover, administering the CRF1R antagonist CP376,395 directly into the BNST prior to testing reduced voluntary ethanol drinking (Albrechet-Souza et al., 2017).”

“…recently demonstrated that ethanol-induced decreases in BNST expression of Homer2, and ERK, the kinase responsible for crosslinking Homer2 and mGlu5, leads to increased ethanol consumption (Campbell et al., 2019), providing an intricate mechanism through which the BNST regulates binge drinking.”

“Early withdrawal results in increased CeA expression of several immune markers including CCL2, TNFα, NOS2, and CD74 (Harper et al., 2017, Freeman et al., 2012).”

“Moreover, CRF may exert its effects by regulating neurotransmitter release via adenylate cyclase 7 (AC7) (Cruz et al., 2011) and/or P/Q-type voltage-gated calcium channels (Varodayan et al., 2017b).”

“Similarly, inhibiting the ε splice variant of protein kinase C (PKCε) in the CeA prevented both basal and ethanol-induced increases in GABA release (Blasio et al., 2018).”

“Ghrelin, toll-like receptor 4 (TLR4), and anaplastic lymphoma kinase (ALK) increase basal GABA transmission and facilitate the ethanolinduced increase in GABA signaling (Bajo et al., 2016, Schweitzer et al., 2016).”

“Extracellular matrix protein (MMP2, 3, and 9) expression is also increased in acute withdrawal, and inhibiting MMP9 prevents CIE withdrawal-induced escalated ethanol consumption (Go et al., 2019).”

“Contet et al., identified numerous gene clusters in the rodent CeA, and Iauncu et al., recently reported similar findings in the non-human primate (Contet et al., 2011, Iancu et al., 2018).”

“…successfully reduced withdrawal-induced anxiety and increased CRF expression, and an infusion of CRF into the CeA after acupuncture treatment completely abolished these effects (Zhao et al., 2013), outlining a unique non-pharmacological treatment strategy for reducing negative affect in withdrawal.”

“Accordingly, silencing VTA-projecting GABAergic BNST neurons is sufficient to blunt binge-like ethanol intake (Companion and Thiele, 2018, Rinker et al., 2017).”

“Additionally, acute withdrawal decreases CeA expression of pERK, CREB, and CAMKIV (Bertotto et al., 2011, Pandey et al., 2003).”

“Furthermore, the NPY Y2R antagonist BIIE0246 reduces anxiety-like behavior, but not operant responding for ethanol (Kallupi et al., 2014c).”

“Moreover, acute exposure to systemic ethanol (1 g/kg i.p.) increases expression of c-fos in the BNST in rodents (Saalfield and Spear, 2019, Sharko et al., 2016), and a recent study by Campbell et al., demonstrated that AAV-mediated knockdown of the scaffolding protein Homer2 in the BNST promotes…”

“A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript an increase in caspase3/9 activity and expression in the CeA (Balaszczuk et al., 2011).”

“Bath applying the CRF1 antagonist antalarmin to CeA slices abolishes the acute ethanol-induced increase in GABA transmission (Roberto et al., 2010), ß-endorphin levels (Lam and Gianoulakis, 2011), and sEPSC frequency (Silberman et al., 2015).”

“Intra-CeA infusion of the NPY1 antagonist BIBP3226 before acupuncture abolished these effects (Zhao et al., 2014).”

“…models, activating mGlu2/3 decreases cue-induced reinstatement (Kufahl et al., 2011), while the glucocorticoid receptor antagonist mifepristone suppresses stressinduced reinstatement (Simms et al., 2012), and overexpressing CRF1R increases stressinduced reinstatement (Broccoli et al., 2018).”

“…significantly decreases operant responding for ethanol (Gilpin et al., 2008), whereas repeated NPY CeA infusions before each binge ethanol session do not alter responding in withdrawal (Henderson and Czachowski, 2012), highlighting the critical role for NPY in withdrawal-induced ethanol craving.”

“…CeA, relatively few studies have examined the effects of early life ethanol exposure on the BNST. Bath applying ethanol to BNST slices from adolescent mice results in greater inhibition of NMDA-EPSC and increased sensitivity of GABAergic transmission compared to adult slices (Wills et al., 2013).”

“Following CIE, CeA GABA transmission and tone are increased, and these changes are accompanied by increased CeA GABA levels (Roberto et al., 2004a, Repunte-Canonigo et al., 2015).”

“Several other compounds infused into the CeA decrease ethanol responding including the neurokinin 1 receptor (NK1R) antagonist L822429 (Schank et al., 2013), FAAH inhibitor URB597 (Stopponi et al., 2018), and substance P (Yang et al., 2009).”

“Prenatal ethanol exposure increases expression of fos-related antigen (Fra) in the CeA in adolescence (PND 30) (Godino et al., 2015), underscoring the lasting effects of prenatal ethanol exposure in the CeA.”

“Interestingly, GABAAα2 expression in the BNST is decreased after 4 weeks of intermittent ethanol access in female, but not male mice, and this effect appears to be dependent on βendorphins in the BNST (Rhinehart et al., 2018).”

“elements of negative affect and hyperkatifeia (Centanni et al., 2019).”

“Finally, activation of the BST is required for stress-related relapse of drug and alcohol self-administration (see reviews of (Centanni et al., 2019; Goode and Maren, 2019)).”

“Higher substance abuse, and lower life satisfaction, are common in such downward spirals (Centanni et al., 2019; Garland et al., 2013; Oishi et al., 2009).”

“The IC likely influences behavior via its projections to the NAcore (Rogers-Carter et al., 2019) and to the extended amygdala (Centanni et al., 2019; Venniro et al., 2017).”

“The full relationship between impairments/alterations in anxiety and fear behaviors and underlying circuitry in substance abuse disorders has been explored in detail in several recent reviews (Agoglia and Herman, 2018; Centanni et al., 2019; Gimeno et al., 2017; Pleil and Skelly, 2018).”

“The BNST receives numerous glutamatergic and GABAergic afferents, with the CeA known to exert significant control over BNST stress-related signaling (for review see [42,49]).”

“Although DO34 effectively mitigates reinstatement of alcohol drinking after abstinence, affective disturbances are particularly problematic during protracted abstinence from alcohol drinking (35, 36).”

“comprehensive review summarizes contributions of the CeA and BNST to negative affect during alcohol abstinence.(30) The NAccshell is involved in reward processing and motivated behaviors, which contribute to its role in aversion behaviors,(31,32) and drives compulsive drug seeking during abstinence.”

“This may be explained by dissociation and alcohol consumption serving similar emotion regulatory functions for responding to intense emotions and stressors (Cardeña & Carlson, 2011; Centanni et al., 2019).”

“…associated with stress, craving, or habitual behavior such as central amygdala, bed nucleus of the stria terminalis, lateral hypothalamus, noradrenergic nuclei, dorsal striatum, or other structures implicated in ethanol use (Koob, 2014, Corbit and Janak, 2016, Hopf, 2020, Centanni et al., 2019).”

“…with the dysregulation of executive functions that are mediated by the medial prefrontal cortex (mPFC) (Abernathy et al., 2010) and by dysfunctional stress systems that are modulated by the central nucleus of the amygdala (CeA) (Centanni et al., 2019; Roberto et al., 2021; Tunstall et al., 2017).”

“Alcohol consumption in particular has been shown to alter stress processing via druginduced changes to PVN projecting limbic regions, including alterations to BNST and subiculum receptor signalling (Bach et al., 2021; Centanni et al., 2019; Haun et al., 2020; Mira et al., 2020).”

“Forced abstinence precipitates phenotype-speci fi c ethanol seeking linked to BNST activation Abstinence following ethanol exposure represents a critical window for behavioral and neurophysiological changes observed in both humans and rodents [14, 15, 61].”

“Further, not only is it shown that stress affects AUD [59], but also that AUD has an effect on stress pathways, notably on the extended amygdala [60].”

“…similarly critical roles of disparate brain regions in withdrawal-induced negative affect, including to name just a few, extended amygdala (Centanni et al., 2019), prefrontal cortex (Patel et al., 2022), and striatum (Borrego et al., 2022; Lepreux et al., 2024), which raises the question…”

“Most notably, the BNST has a prominent role in modulating responses to threatening or stressful events (e.g Davis et al., 2010; Centanni et al., 2019).”