Research Articles: Cellular/Molecular Alcohol dependence and withdrawal impair serotonergic regulation of GABA transmission in the rat central nucleus of the amygdala https://doi.org/10.1523/JNEUROSCI.0733-20.2020 Cite as: J. Neurosci 2020; 10.1523/JNEUROSCI.0733-20.2020 Received: 30 March 2020 Revised: 8 July 2020 Accepted: 14 July 2020 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2020 the authors 1 Alcohol dependence and withdrawal impair serotonergic regulation of GABA 2 transmission in the rat central nucleus of the amygdala 3 Abbreviated title: Alcohol dependence impairs CeA regulation by 5-HT 4 Sophia Khom, Sarah A. Wolfe, Reesha R. Patel, Dean Kirson, David M. Hedges, Florence P. 5 Varodayan, Michal Bajo, and Marisa Roberto$ 6 The Scripps Research Institute, Department of Molecular Medicine, 10550 N. Torrey Pines 7 Road, La Jolla CA 92307 8 $To whom correspondence should be addressed: 9 Dr. Marisa Roberto 10 Department of Molecular Medicine 11 The Scripps Research Institute 12 10550 N. Torrey Pines Road, La Jolla, CA 92037 13 Tel: (858) 784-7262 Fax: (858) 784-7405 14 Email: [email protected] 15 16 Number of pages: 30 17 Number of figures: 7 18 Number of tables: 2 19 Number of words (Abstract): 250 20 Number of words (Introduction): 650 21 Number of words (Discussion): 1500 22 23 The authors declare no conflict of interest. 24 25 Acknowledgments 26 This work was financially supported by grants from the NIH/NIAAA AA006420, AA013498, 27 AA015566, AA017477, AA027700, AA007456 and AA021491 (all to M.R.), AA026865 (R.R.P), 28 AA025262 and AA026638 (D.K.), AA025408 (F.P.V.), the Pearson Center for Alcoholism and 29 Addiction Research and the Austrian Science Fund (FWF Erwin Schrödinger Fellowship J-3942- 30 B30 to S.K.). This is Scripps manuscript number 29963. 31 Author contributions 32 S.K and M.R. designed research. S.K., S.A.W., R.R.P., D.K., D.M.H., F.P.V., and M.B. 33 performed research and analyzed data. S.K. wrote the manuscript. All authors edited and 34 approved the final version of the manuscript. 35 36 37 38 39 Abstract 40 Excessive serotonin (5-HT) signaling plays a critical role in the etiology of alcohol use disorder 41 (AUD). The central nucleus of the amygdala (CeA) is a key player in alcohol-dependence 42 associated behaviors. The CeA receives dense innervation from the dorsal raphe nucleus, the 43 major source of 5-HT, and expresses 5-HT-receptor subtypes (e.g., 5-HT2C and 5-HT1A) 44 critically linked to AUD. Notably, the role of 5-HT regulating rat CeA activity in alcohol 45 dependence is poorly investigated. 46 Here, we examined neuroadaptations of CeA 5-HT signaling in adult, male Sprague-Dawley rats 47 using an established model of alcohol dependence (chronic intermittent alcohol vapor 48 exposure), ex vivo slice electrophysiology and in situ hybridization. 5-HT increased frequency of 49 spontaneous inhibitory postsynaptic currents (sIPSCs) without affecting postsynaptic measures 50 suggesting increased CeA GABA release in naïve rats. In dependent rats, this 5-HT-induced 51 increase of GABA release was attenuated, suggesting blunted CeA 5-HT sensitivity which 52 partially recovered in protracted withdrawal (2 weeks). 5-HT increased vesicular GABA release 53 in naïve and dependent rats but had split effects (increase and decrease) after protracted 54 withdrawal indicative of neuroadaptations of presynaptic 5-HT receptors. Accordingly, 5-HT 55 abolished spontaneous neuronal firing in naïve and dependent rats but had bidirectional effects 56 in withdrawn. Alcohol dependence and protracted withdrawal did not alter either 5-HT1A- 57 mediated decrease of CeA GABA release or Htr1a expression but disrupted 5-HT2C-signaling 58 without affecting Htr2c expression. Collectively, our study provides detailed insights into 59 modulation of CeA activity by the 5-HT system and unravels the vulnerability of the CeA 5-HT 60 system to chronic alcohol and protracted withdrawal. 61 Significance statement 62 Elevated GABA signaling in the central nucleus of the amygdala (CeA) underlies key behaviors 63 associated with alcohol dependence. The CeA is reciprocally connected with the dorsal raphe 64 nucleus (DRN), the main source of serotonin (5-HT) in the mammalian brain, and excessive 5- 1 65 HT signaling is critically implicated in the etiology of alcohol use disorder (AUD). Our study 66 using a well-established rat model of alcohol dependence, ex vivo electrophysiology and in situ 67 hybridization provides mechanistic insights into how both chronic alcohol exposure and 68 protracted withdrawal dysregulate 5-HT signaling in the CeA. Thus, our study further expands 69 our understanding of CeA cellular mechanisms involved in the pathophysiology of alcohol 70 dependence and withdrawal. 71 2 72 Introduction 73 Alcohol use disorder (AUD) is a chronic, relapsing disease characterized by compulsive alcohol 74 seeking and taking, loss of control in limiting alcohol intake, and the emergence of negative 75 emotional states during absence from alcohol (Koob, 2008; Spanagel et al., 2014; Koob and 76 Volkow, 2016). These states are regulated by specific neurochemical systems modulating 77 neurocircuits involved in drug reward, negative affect, and executive control (Wise and Koob, 78 2014). Excessive serotonin (5-hydroxytryptamine, 5-HT) signaling has been implicated in the 79 etiology of AUD and can elicit craving in abstinent patients (Krystal et al., 1996; Pettinati et al., 80 2003; Bonkale et al., 2006; Kranzler et al., 2013; Marcinkiewcz et al., 2016). Specifically, 81 polymorphisms in genes encoding for distinct components of the 5-HT system are either 82 predisposing to the development of AUD or are linked to specific behaviors associated with 83 AUD (Lappalainen et al., 1998; Feinn et al., 2005; Yohros et al., 2018). Accordingly, drugs 84 modulating 5-HT signaling have been shown to decrease alcohol intake in both humans and 85 animal models (Lejoyeux, 1996; Lê et al., 1996; Belmer et al., 2016; Dos Santos et al., 2018). 86 Chronic alcohol exposure alters serotonergic synaptic transmission and causes potential 87 neuroadaptations in its receptors (Belmer et al., 2016; Marcinkiewcz et al., 2016). In rodents, 88 both acute alcohol and withdrawal increase excitability of dorsal raphe (DRN) neurons (Lowery- 89 Gionta et al., 2015) thereby likely increasing 5-HT release into DRN-projecting areas. The DRN 90 innervates all major players in the addiction cycle including the central nucleus of the amygdala 91 (CeA) (Vertes, 1991; Retson and Van Bockstaele, 2013; Linley et al., 2016). The CeA serves as 92 a integrative hub mediating key aspects of negative emotional states associated with alcohol 93 dependence including anxiety-like behaviors (Gilpin et al., 2015). The CeA is a mainly 94 GABAergic nucleus, particularly sensitive to both acute and chronic ethanol (Roberto et al., 95 2003, 2004, 2020). Thus, elevated CeA GABA transmission is a characteristic of alcohol 96 dependence across species including humans (Roberto et al., 2004; Herman et al., 2016; 3 97 Augier et al., 2018; Jimenez et al., 2019). Importantly, acute alcohol administration enhances 5- 98 HT release in the CeA (Yoshimoto et al., 2000). 99 The 5-HT receptor system comprises 14 different 5-HT activated either inhibitory (5-HT1 100 receptor subtypes) or excitatory (5-HT2, 4, 6, and 7 receptor subtypes) receptors coupled to G- 101 proteins and an excitatory ligand-gated ion channel (5-HT3) (McCorvy and Roth, 2015). 5-HT 102 receptor subtypes are abundantly expressed in the mammalian brain (Charnay and Léger, 103 2010) tightly regulating 5-HT signaling (Belmer et al., 2016). Multiple studies implicate a critical 104 role of 5-HT1A and 5-HT2C signaling in the extended amygdala in addictive behaviors including 105 AUD (Müller et al., 2007; Marcinkiewcz et al., 2016). For instance, site-specific injections of 5- 106 HT2C antagonists into the nucleus accumbens shell block escalation of alcohol intake in 107 rodents (Yoshimoto et al., 2012). Alcohol dependence elicits specific changes in 5HT2C 108 signaling conferring neuronal hyperexcitability in the ventral bed nucleus of the stria terminalis 109 (BNST) (Marcinkiewcz et al., 2015) and increased amygdalar 5-HT2C expression was observed 110 in genetic models of alcohol preference (Pandey et al., 1996) corroborating a critical role for 5- 111 HT2C in excessive alcohol intake. Lastly, 5-HT induces anxiety-like behavior in the dorsal BNST 112 via activation of 5-HT1A (Garcia-Garcia et al., 2018). 113 However, despite the strong serotonergic innervation of the CeA (Vertes, 1991; Retson and Van 114 Bockstaele, 2013; Linley et al., 2016) accompanied by dense expression of HT2C and 5-HT1A 115 (Asan et al., 2013), little is known about 5-HT regulation of CeA activity under physiological 116 (naïve) conditions and/or alcohol dependence and protracted withdrawal. Thus, we examined 117 potential neuroadaptations of the CeA 5-HT system induced by alcohol dependence using a rat 118 model of chronic intermittent vapor exposure, ex vivo slice electrophysiology and in situ 119 hybridization. Specifically, we studied 5-HT-modulation of CeA GABAA receptor mediated 120 synaptic transmission, 5-HT effects on spontaneous neuronal firing, and expression of Htr1a 121 and Htr2c mRNA accompanied by functional assessment of 5-HT1A and 5-HT2C signaling in 122 naïve, dependent and withdrawn rats. 4 123 Materials and Methods 124 All procedures were approved by the Scripps Research Institutional Animal Care and Use 125 Committee (IACUC) and are in line with the National Institutes of Health Guide for the Care and 126 Use of Laboratory Animals.