Lateral hypothalamus-projecting noradrenergic locus coeruleus pathway modulates binge-like ethanol drinking in male and female TH-ires-cre mice
Introduction
Alcohol abuse poses significant health risks and economic costs to individuals around the world (Lim et al., 2012; Rehm et al., 2009). Repeated bouts of binge drinking, defined as a pattern of drinking yielding blood ethanol concentrations (BECs) achieving the U.S. legal limit of 80 mg/dl within a short (2-h) time window (NIAAA, 2004), are thought to contribute to the development of later-life alcohol dependence (Hingson et al., 2006). Recent estimates indicate that nearly 90% of U.S. adults that drink excessively consume alcohol in the form of a binge (Esser et al., 2014). Thus, understanding the neurochemical signaling systems that modulates binge-like ethanol consumption is critical for identification of potential therapies for reducing this dangerous pattern of behavior. To this end, the 4-day “drinking-in-the-dark” (DID) paradigm and its variants are among the more popular paradigms for modeling voluntary binge-like ethanol intake in mice (Rhodes et al., 2005; Thiele and Navarro, 2014) and rats (Bell et al., 2011; Holgate et al., 2017), promoting ingestion of large quantities of ethanol and reliably generating BECs exceeding 80 mg/dl. Using DID procedures, researchers have begun to examine roles for neurochemical signaling systems and circuitry that modulate binge-like ethanol intake (Sprow and Thiele, 2012). Interestingly, there is evidence that overlapping neurochemical pathways in the brain modulate both alcohol use disorders and eating disorders (Thiele et al., 2003), suggesting that at least in some circumstances common therapeutic strategies may be identified.
Norepinephrine (NE) is centrally synthesized in brainstem nuclei, with the majority of its synthesis occurring via the locus coeruleus (LC) (Sawchenko and Swanson, 1982), and has long been implicated in ethanol ingestion (Arango et al., 1994; Gilpin and Koob, 2010; Lu et al., 1997; O'Neil et al., 2013; Rasmussen et al., 2014a; Rasmussen et al., 2009; Rasmussen et al., 2014b; Simpson et al., 2009; Verplaetse et al., 2012) as well as feeding behavior (Bello et al., 2019). In fact, the NE/dopamine (DA) reuptake inhibitor bupropion (BUP), when combined with the non-selective opioid antagonist, naltrexone (NAL), has been used successfully to treat binge eating in human (Halseth et al., 2018), and we have recently shown that BUP alone and in combination with NAL blunts binge-like ethanol intake in mice (Navarro et al., 2019). Further, in a preliminary open-label study we recently reported that BUP + NAL therapy blunts the frequency of binge ethanol drinking in humans (Walter et al., 2020). These observations suggest that blunted NE tone contributes to binge behavior, and increasing NE tone is protective. However, there is a gap in the literature on the specific NE neurocircuitry that modulates binge ethanol drinking.
Insight into the NE mechanisms involved in modulating binge behavior comes from studies that have used c-Fos immunoreactivity (IR) as a marker of neuronal activation. It has been shown that the LC is activated following voluntary binge-like ethanol consumption (Burnham and Thiele, 2017). Interestingly, i. p. injection of ethanol into rats bred for high versus low ethanol consumption revealed reduced ethanol-induced c-Fos IR in the LC among rats bred for high levels of intake (Thiele et al., 1997). More recently, we showed that an inbred line of mice that was selectively bred to achieve binge-like blood ethanol levels (the iHDID-1 line) failed to exhibit ethanol-induced c-Fos expression in the LC at doses that induced c-Fos expression in the control HS/Npt line (Robinson et al., 2020). These observations suggest that LC activity is triggered by ethanol, and the observation that this activity is blunted in high drinking lines of rats and mice supports a role for ethanol-induced LC activity as a protective mechanism to limit ethanol intake.
In rodents, the lateral hypothalamus (LH) has been implicated in modulation of many behaviors including ethanol consumption (Chen et al., 2013, 2014; Navarro et al., 2016; Sprow et al., 2016; Wayner et al., 1971) and seeking (Marchant et al., 2009, 2014). Though brainstem NE nuclei innervate numerous regions (Robertson et al., 2016), the LH connects reciprocally with the LC (Jones and Moore, 1977; Papp and Palkovits, 2014) and contains rich populations of α-1, α-2, and β-adrenergic receptors (ARs) (Leibowitz et al., 1982). Thus, the LC may signal through the LH to regulate binge-like ethanol consumption. Experiments herein elucidated the role of the LC in binge-like ethanol intake. First, we show that peripheral administration of a NE reuptake inhibitor (NRI) significantly blunted binge-like ethanol consumption without altering overall motor behavior or anxiety-like behavior, consistent with our previous studies using BUP. Using excitatory Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), we demonstrated that general activation the LC blunted binge-like ethanol and sucrose consumption. Specific activation of TH-expressing LC neuron projecting to the LH similarly reduced binge-like ethanol consumption without altering sucrose drinking in ethanol-experienced or ethanol-naïve mice. Finally, we show that LH-directed adrenergic receptor (AR) manipulations reduced ethanol consumption that were compound-specific, and in some cases sex-specific and dependent on baseline levels of ethanol intake. The present results provide novel evidence that increased NE tone in a circuit arising from the LC and projecting to the LH reduces binge-like ethanol drinking, and may represent a novel strategy for treating binge drinking disorders prior to the development of ethanol dependence.
Section snippets
Animals
Male and female TH-ires-Cre mice (Savitt et al., 2005), bred in house and backcrossed on a C57BL6/J strain, were utilized for DREADD manipulations and tracing studies. Male and female C57BL6/J mice (stock # 000664, Jackson Laboratory), 6–8 weeks old upon arrival were utilized for pharmacological manipulations. All mice were individually housed at least 1 week prior to testing onset with ad libitum access to Prolab® RMH 3000 (Purina labDiet®; St. Louis, MO) and water except where noted. The
Experiment 1: effects of systemic administration of a peripherally bioavailable NRI on binge-like ethanol intake
Data from this experiment are presented in Fig. 1. A repeated-measures ANOVA (sex x treatment) failed to reveal a main effect of sex or sex by treatment interaction, so data from males and females were collapsed. Paired t-test for total ethanol intake revealed a significant reduction in intake following 10 mg/kg reboxetine pretreatment (Fig. 1A) [t-test: t (20) = 4.199, p < 0.001]. 10 mg/kg reboxetine similarly blunted BECs (Fig. 1B) [t-test: t (20) = 3.473, p = 0.002]. Fig. 1C shows a
Discussion
This series of studies sought to uncover the roles of the LC and its projection to the LH in modulation of voluntary binge-like ethanol consumption. First, we first showed that systemic administration of the NRI, reboxetine, blunted binge-like ethanol intake in both sexes without altering locomotor or anxiety-like behavior. These findings are consistent with our previous observation that the NE and dopamine re-uptake inhibitor, BUP, significantly blunts binge-like ethanol drinking mice (Navarro
Conclusions
Though well-established as playing a critical role in depression and anxiety disorders (Montoya et al., 2016), a growing body of literature is unpacking a role for central NE signaling in the modulation of drug and alcohol intake. Our results indicate that elevated NE signaling in the NE LC→LH circuit plays protective roles against binge-like ethanol intake. We recently reported that the dopamine/norepinephrine reuptake inhibitor, bupropion, blunts binge-like ethanol intake in mice (Navarro et
Declaration of competing interest
The authors declare no competing financial interests. Dr. Thiele owns shares of Glauser Life Sciences, a copy the aims to develop therapeutics for mental health disorders. The work that is presented in this paper is not directly related to the scientific aims of Glauser Life Sciences.
Acknowledgments
We thank Rhiannon Thomas, Timothy Gilliam, Sophie Bendrath, and Sonia Sabater for their expert assistance with the present projects. This research was funded by NIH grants AA013573, AA022048, & AA025809.
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