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Published in final edited form as: Neuroscience. 2020 Jul 21;443:84–92. doi: 10.1016/j.neuroscience.2020.07.024

Bidirectional control of alcohol-drinking behaviors through locus coeruleus optoactivation

Alex L Deal 1, Caroline E Bass 2, Valentina P Grinevich 1, Osvaldo Delbono 3, Keith D Bonin 4, Jeff L Weiner 5, Evgeny A Budygin 1,*
PMCID: PMC8074022  NIHMSID: NIHMS1614725  PMID: 32707291

Abstract

The relationship between stress and alcohol-drinking behaviors has been intensively explored; however, neuronal substrates and neurotransmitter dynamics responsible for a causal link between these conditions are still unclear. Here, we optogenetically manipulated locus coeruleus (LC) norepinephrine (NE) activity by applying distinct stimulation protocols in order to explore how phasic and tonic NE release dynamics control alcohol-drinking behaviors. Our results clearly demonstrate contrasting behavioral consequences of LC-NE circuitry activation during low and high frequency stimulation. Specifically, applying tonic stimulation during a standard operant drinking session resulted in increased intake, while phasic stimulation decreased this measure. Furthermore, stimulation during extinction probe trials, when the lever press response was not reinforced, did not significantly alter alcohol-seeking behavior if a tonic pattern was applied. However, phasic stimulation substantially suppressed the number of lever presses, indicating decreased alcohol seeking under the same experimental condition. Given the well-established correlative link between stress and increased alcohol consumption, here we provide the first evidence that tonic LC-NE activity plays a causal role in stress-associated increases in drinking.

Keywords: optogenetics, alcohol seeking and drinking, stress, norepinephrine, locus coeruleus

INTRODUCTION

Alcohol use disorder (AUD) and stress-related syndromes are highly comorbid in humans (Jacobson et al., 2008; Debell et al., 2014; Shorter et al., 2015). Acute alcohol consumption can be anxiolytic, which may explain the frequently observed increases in intake under anxiety-associated conditions following stressful events. However, withdrawal from chronic alcohol can increase anxiety and other stress-related symptoms. Therefore, while the affective disorders promote excessive alcohol drinking, excessive drinking worsens their symptoms (Gilpin and Weiner, 2017).

In fact, activation of brain stress systems is hypothesized to be a key element of the negative emotional state produced by AUD that drives drug and alcohol seeking and intake through negative reinforcement mechanisms (Koob, 2009). It has been speculated for some time that norepinephrine (NE) release, which is enhanced by stress, might have a significant impact on alcohol-drinking behaviors. However, the mechanisms and exact neural circuitries responsible for these interactions are still unclear.

Recent studies have identified numerous neurochemical alterations in different brain circuits following stress and alcohol drinking (Deal et al., 2018; Weera and Gilpin, 2019; Orrù et al., 2016; Caffino et al., 2015; Holly et al., 2015). However, a major challenge has been to establish whether these changes play a causal role in the shaping of alcohol-drinking behaviors. Identifying the specific NE mechanisms responsible for the development, maintenance and escalation of alcohol drinking should help to harness the untapped potential of targeting the NE system in the treatment of AUD, especially in individuals with stress-associated etiologies.

Previously, we used optogenetic methods to make a number of advances demonstrating how specific manipulations of the temporal characteristics of dopamine release influence addictive drinking behaviors (Adamantidis et al., 2011; Bass et al., 2013; Mikhailova et al., 2016; Budygin et al., 2020). One important conclusion from these studies is that research should focus not only on the strength of neuronal signaling but also on the patterns of neurotransmitter release. For example, our latest work revealed that alcohol seeking could be inhibited by a tonic pattern of dopamine release in the nucleus accumbens while phasic stimulation of this circuit actually increased this behavior (Budygin et al., 2020). It should be noted that NE is released in the brain with patterns, which are quite similar to dopamine transmission. Thus, locus coeruleus (LC) neurons fire in two distinct modes: the first (tonic) is characterized by irregular but continuous baseline activity (one to six spikes per second), while during the second mode (phasic), cells fire short bursts at higher frequencies (Aston-Jones and Bloom, 1981; Clayton et al., 2004; Aston-Jones and Cohen, 2005). How these patterns within the LC-NE circuitry influence alcohol-drinking behaviors is unknown.

Here, we used optogenetics to mimic tonic and phasic increases in NE transmission in rats during an operant alcohol self-administration paradigm in order to reveal the role of distinct NE patterns in alcohol-drinking behavior.

METHODS

Subjects

Adult male Long Evans rats (Envigo) were housed in acrylic cages on a 12/12 h light/dark cycle (lights off at 1800) in a temperature-controlled vivarium. Food and water were available ad libitum except during the experimental trials, which were conducted in the light phase. Rats were maintained in group-housing conditions (2 per cage) prior to surgery and then single-housed after viral infusion and implantation of the optical cannula. Animal procedures and protocols were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Wake Forest University School of Medicine Institutional Animal Care and Use Committee.

Virus Injection and Cannula Implantation

Rats were anesthetized using ketamine hydrochloride (80 mg/kg, i.p.) and xylazine hydrochloride (10 mg/kg, i.p.). Once placed in a stereotaxic frame, the scalp was shaved and wiped with iodine. The skull was uncovered by making an incision centrally along the scalp. Two small drill holes were fashioned for two skull screws to stabilize a cement cap. A final hole was drilled on the right side above the LC (from bregma: anterior 9.8 mm; lateral, 1.4 mm) for the virus injection and for implantation of the optic cannula (Doric Lenses, Canada). Next, a combination of DIO-ChR2-EYFP-AAV2/10 and TH-iCRE-AAV2/10 were coinjected (1.0 μl total) gradually into the LC over 13 min via a Hamilton syringe (ventral 6.8 mm). Dental cement stabilized by skull screws was used to cover the exposed skull following the insertion of the optic cannula above the LC. Subjects were returned to their home cages for recovery once the cement was dry.

Operant Conditioning for Alcohol Self-Administration

Training

Training sessions were performed daily in sound-attenuated operant chambers (Med Associates, East Fairfield, VT) as previously described (Samson et al., 1999; Budygin et al., 2020). These chambers contained a house light (used to indicate the beginning of each session), a retractable lever, and a sipper tube. Rats were trained to self-administer alcohol (ethanol) using an abbreviated substitution protocol based on previously established methods (Samson, 1986; Budygin et al., 2020). Briefly, rats were acclimated for 2 h to the operant chamber on the first day with a 10% sucrose solution on a fixed ratio (FR1) schedule (sipper tube available for 45 s per lever press). On consecutive days following this initial session, the session duration, FR schedule, and sipper tube availability were altered. Additionally, the sucrose concentration was lowered by 1% daily and replaced with increasing ethanol concentrations. Over 8 days, the FR schedule was modified from a FR1 to FR4. On day 9 of training, the reinforcement schedule was changed to a response requirement of eight lever presses (RR8) resulting in a 20 min reinforcer presentation. This response requirement was increased during the following consecutive sessions from RR10 to RR15, 20, 25, and 30 for at least 2 days per response requirement (customized to each rat’s response performance). Rats had 20 min to complete the RR30. With this approach, all rats achieved the 30-presses-persession criterion within a six-week period. Data of appetitive and consummatory behaviors were collected using Med Associates software.

Extinction

Extinction trials were conducted to quantify appetitive-like behavior separate from consumption. Rats were placed into the operant chamber for 30 min where the lever was present and able to be pressed without limit but alcohol was not provided.

Optical Stimulation

To test the effect of NE activation patterns on alcohol consummatory and seeking behaviors, we performed optical stimulations during operant self-administration and extinction trials at least 4 weeks after surgery. This delay allowed for adequate expression of ChR2 at the cell body to elicit catecholamine release via light stimuli (Bass et al., 2010, 2013; Mikhailova et al., 2016). Rats performed in sessions of operant self-administration with optical stimulation twice though staggered so that baseline and post-stimulation sessions were conducted on days before and after the stimulated sessions. Optical stimulations were conducted with a laser at a wavelength of 473 nm (Beijing Viasho Technology Co., Ltd, Beijing, China) with a 100 mW max power output. Control signals were sent by a programmable function generator (Hewlett-Packard model 8116A) to modulate the laser via the TTL input control port on the laser power supply. The light modulation parameters for tonic stimulation were 150 pulses at 5 Hz (30 s total light exposure) and for phasic stimulation were 50 pulses at 50 Hz (4 stim/min per session). Manually firing a pulse generator (Systron Donner Model 100C) initiated the optical pulse procedure that then activated a digital delay generator (SRS Model DG535). The digital delay generator ensured selection of a finite number of pulses from the continuous waveform produced by the function generator by ensuring the function generator was sufficiently gated. A series of 5 Hz and 50 Hz square pulses were produced by the function generator. The digital delay generator gated the total number of pulses in one data stream because the temporal length of a gate pulse received by the function generator determined the number of square pulses elicited for each trigger by the function generator. Each series of pulses contained individual pulses with a 4 ms temporal width. The laser power output was measured by a commercial power meter (Thorlabs Model S121C, Newton, New Jersey).

Fast-Scan Cyclic Voltammetry

To verify the efficacy of the light stimulation to elicit NE release in regions efferent to the LC, we performed fast-scan cyclic voltammetry (FSCV) to record NE release in the prefrontal cortex (PFC) and basolateral amygdala (BLA) in anesthetized rats. Briefly, rats were anesthetized via urethane (1.5 g/kg, i.p.) and placed into a stereotaxic frame. The scalp was shaved, cleaned, and removed to reveal the skull. One hole was made in the skull above either the PFC (from bregma: 3.0 mm anterior, 0.5 mm lateral) or BLA (from bregma: 2.9 mm posterior, 4.6 mm lateral) for placement of the recording electrode and a second hole was fashioned in the contralateral side for insertion of a Ag/AgCl reference electrode connected to a voltammetric amplifier (UNC Electronics Design Facility, Chapel Hill, NC). The recording electrode was a carbon fiber microelectrode (6 μm diameter; 100 μm exposed fiber length) connected to a voltage amplifier and secured to the stereotaxic frame in order to be lowered into the PFC (4.5 mm ventral) or BLA (8.4 mm ventral) for voltammetric recordings. An optical fiber (200 μm diameter) was lowered into the guide cannula used for light stimulation of the LC during the operant responding and extinction trials and connected to a laser (Viasho, China). The release of NE was elicited using the same phasic stimulation parameters as during the operant self-administration and extinction trials. Voltammetric recordings were taken at the carbon fiber electrode every 100 ms by applying a triangular waveform from −0.4 to +1.3 and back to −0.4 V (Mateo et al., 2004; Jones et al., 2006; Oleson et al., 2009; Fox et al., 2016). Identification of the NE signal was made by observing oxidation and reduction peaks at +0.6 V and −0.2 V, respectively (vs. Ag/AgCl reference), as well as pharmacologically via intraperitoneal injection of idazoxan (α2 adrenergic receptor antagonist; 5 mg/kg) and raclopride (D2 DA receptor antagonist; 2 mg/kg). Data were digitized (National Instruments, Austin, TX) and stored on a computer. The carbon fiber electrodes were calibrated after each experiment with a known concentration (1 μM) of NE in vitro.

Histology

Following the experiments, histology was conducted to confirm placement of virus. Briefly, subjects were anesthetized and then transcardially perfused with 10% normal buffered formalin. Brains were taken and soaked overnight in fixative at 4º C and then incubated in a 25% sucrose solution until the brains sank. The sections (50 μm thick) were obtained on an American Optical 860 sliding microtome. Free-floating sections of rat brains were processed for immunohistochemistry. Briefly, sections were washed in PBS for 5 min followed by 3 × 10 min rinses in PBS + 0.5% triton X-100. Primary antibody diluted in PBS + 0.3% triton X-100 was applied overnight at 4ºC while shaking. Primary antibodies used were mouse anti-tyrosine hydroxylase (ImmunoStar #22941) at a 1:4000 dilution and a rabbit anti-GFP (Invitrogen #A6455, also cross reacts with EYFP) at a 1:2000 dilution. The following day, sections underwent 3 × 10 min PBS rinses and then were incubated with secondary antibodies of Alexa 555 donkey anti-mouse (Invitrogen, #A31570, 1:4000) and Alexa 488 goat anti-rabbit (Invitrogen #A11034, 1:2000) at room temperature for 2 hours while shaking. A last set of 3 × 10 min PBS rinses were applied to the sections that were then mounted onto slides and coverslipped with Prolong Gold media. Slides were visualized via Zeiss Axio Observer Confocal microscope.

Data Analysis

Data were analyzed using GraphPad Prism (GraphPad Software version 7.04, San Diego, CA, USA). Parametric and nonparametric analyses were performed where appropriate with the criterion for significance set at p<0.05.

RESULTS

Expressing ChR2 in NE neurons of the rat LC

To permit neuron subtype specific gene expression in wild type animals, we have developed a combinational AAV targeting system that drives, in combination, subtype specific Cre-recombinase expression with a strong but non-specific Cre-conditional transgene (Bass et al., 2013; Gompf et al., 2015). Using this system we previously demonstrated that the tyrosine hydroxylase (TH) promotor restricted expression of ChR2 to TH positive neurons of rat ventral tegmental area (VTA) (Gompf et al., 2015; Mikhailova et al., 2016; Budygin et al., 2020), which are dopamine cells, or hM3Dq transgenes in the LC (Gompf et al., 2015), that is a main source of NE cell bodies. Here, we confirmed expression of ChR2 in the LC of rats injected with the same construct (Fig.1A). The fact that optoactivation of the LC resulted in NE efflux in terminal fields (Fig. 1B) further proves the expression of this opsin on NE cell bodies (see below).

Figure 1.

Figure 1.

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Optogenetically targeting LC NE neurotransmission in rat brain. (A) Rats were anesthetized and injected in the LC with a combination of DIO-ChR2-EYFP-AAV2/10 and TH-iCRE-AA2/10 via a Hamilton syringe (left panel). Histochemical analysis confirmed that ChR2-EYFP was restricted to the targeted area (right panel). (B) Optoactivation of the LC resulted in a robust catecholamine release detected with FSCV from NE terminals. A two dimensional color plot topographically represents electrochemical changes measured in real time in the BLA of a single anesthetized rat before, during (denoted by black line) and after optical stimulation of the LC. The green spot denotes changes in current, which are due to NE oxidation (dotted line indicates the oxidation potential). (C) The measured signal was pharmacologically confirmed to be NE, and not DA, by a significant increase in release following injection of idazoxan (top; n=5) and no effect following raclopride (bottom; n=4) when compared to a preinjection baseline. *p<0.05

Robust axonal NE release can be triggered through the optostimulation of the LC

Our previous voltammetric study has shown that an electrical stimulation of the LC results in a catecholaminergic signal in the PFC that is predominantly noradrenergic (Deal et al., 2019). Here, we used the same electrochemical approach (FSCV) to explore whether the level of ChR2 expression reached in the LC is sufficient to generate optogenetic NE release in terminal fields. We found that optostimulation of NE cell bodies at the level of the LC resulted in robust catecholamine effluxes in the infralimbic PFC and BLA (Fig.1, 2). Additionally, the NE signal in the BLA was pharmacologically confirmed by administration of idazoxan or raclopride. A significant increase in the catecholamine release was measured 10 min following idazoxan administration (paired t test; t(4)=22.19; p<0.0001) but not raclopride (t(3)=1.53; p=0.224), indicating that NE, and not DA, is the detected catecholamine (Fig. 1C).

Figure 2.

Figure 2.

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Voltammetric recordings indicated that optostimulation of the LC triggered NE release in the PFC (left) and BLA (right). Hashed bars indicate optical stimulation (50 Hz, 100 pulses, 4 ms pulse). Inset: background-subtracted cyclic voltammograms demonstrating characteristic oxidation and reduction peak potentials that identify NE. n=4 rats per group.

Tonic and phasic NE release differentially modulate alcohol intake

To determine the effect of tonic LC optical stimulation on alcohol intake, we first established a stable baseline of operant alcohol intake for three consequent sessions. We then conducted a session in which rats received LC stimulation and then, the following day, the sessions were conducted in the absence of stimulation to determine if there were any persistent effects of optically released NE on alcohol intake. This experiment was then repeated in order to replicate initial findings. After confirming that the data were consistent with a normal distribution (Shapiro-Wilk normality test; p>0.05), a repeated measures two-way ANOVA (Session Condition (i.e., before, during, and after stimulation) X Trial) calculated a main effect of session condition (F(2,12)=11.25; p=0.0018) and no significant effect of trial (F(5,6)=2.20; p=0.183), suggesting the second trial of the experiment was not significantly different than the initial run, and no interaction (F(10,12)=1.60; p=0.218) (Fig. 3). A post-hoc Tukey’s multiple comparisons test revealed significantly increased alcohol intake during Stimulated sessions compared to intake during the Before stimulation session (p=0.0124) and intake during the After stimulation session (p=0.0018).

Figure 3.

Figure 3.

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Optical tonic stimulation of the locus coeruleus (LC) significantly increased alcohol intake. Rats were operantly trained to self-administer alcohol on an FR1 schedule. Tonic stimulation (5 Hz, 30s) significantly increased alcohol consumption compared to prestimulation baseline sessions. This augmentation was not persistent as intake levels returned to baseline levels in subsequent non-stimulated sessions. n=6; *p<0.05

Following the same procedure, the effect of phasic LC optical stimulation on alcohol consumption was measured by altering the stimulation pattern during a session after a stable baseline was confirmed (Fig. 4). The data were found to be consistent with a normal distribution (Shapiro-Wilk; p>0.05) and analyzed via a repeated measures two-way ANOVA which found a main effect of session (F(2,10)=5.49; p=0.0246) and no effect of trial (F(4,5)=0.950; p=0.506) and no significant interaction (F(8,10)=1.75; p=0.200). A post-hoc Tukey’s multiple comparisons test found a significant attenuation of intake during the Stimulated session compared to intake during the Before session (p=0.0214).

Figure 4.

Figure 4.

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Optical phasic stimulation of the locus coeruleus (LC) significantly reduced alcohol intake. Rats were operantly trained to self-administer alcohol on an FR1 schedule. Phasic stimulation (50 Hz, 1s stimulation, 4x/min) resulted in a significant decrease in alcohol consumed compared to prestimulation baseline sessions. This reduction was attenuated in subsequent non-stimulated sessions though not back to baseline levels. n=6; *p<0.05

Phasic but not tonic NE release affects alcohol-seeking behavior

In order to distinguish between alcohol seeking and consummatory behaviors, the rats underwent extinction trials (Fig. 5). The alcohol-related cues were present during these trials, though lever pressing did not result in alcohol availability. Quantification of lever presses during these extinction sessions provides a validated measure of seeking behavior (Samson and Czachowski, 2003). A test for normality found that the data were significantly different from a normal distribution (Shapiro-Wilk; p<0.05). Therefore, these data were analyzed using the nonparametric Friedman test which found a significant main effect (Χ2=12.67; p<0.001). A post-hoc Dunn’s multiple comparisons test found a significant attenuation of lever pressing during sessions with phasic stimulation compared to the Before stimulation session (p<0.01). There was no significant difference in lever presses during tonic stimulation compared to the sessions before or stimulation (p>0.05). Furthermore, neither pattern of stimulation significantly changed the latency to the first lever press (Shapiro-Wilk normality test: p>0.05; RM One-way ANOVA: F(1.145,5.735)=1.21, p=0.326; Fig. 6). However, there was some trend to increased latency during phasic stimulation that is consistent with lessened motivated behavior, which was clearly indicated by decreased lever presses.

Figure 5.

Figure 5.

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Phasic stimulation of the locus coeruleus (LC) significantly reduced the number of lever presses during extinction trials. Rats were optically stimulated with two different stimulation patterns, tonic or phasic, in the LC during extinction trials performed one week apart. Phasic stimulation, and not tonic, showed a significant impairment of lever pressing during stimulated sessions. This decrease was not persistent as rats returned to lever pressing levels similar to baseline extinction sessions in a subsequent non-stimulated session. Dashed line shows poststimulation lever pressing returning to prestimulation levels. n=5; *p<0.05

Figure 6.

Figure 6.

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Latency to first lever press was not altered by tonic or phasic LC optostimulation. The time to the first lever press during the extinction trials was analyzed for each condition. There was no significant difference in latency to first press between no stimulation and stimulation trials. n=6

Discussion

Our findings demonstrate that viral-mediated gene delivery can be successfully used to express ChR2 in NE cell bodies in Long-Evans rats. Expression levels were high enough to permit real time NE release in terminal fields in the PFC and BLA through LC optostimulation. Combining this approach with an operant alcohol-drinking paradigm, we revealed that optostimulating the LC with a tonic pattern significantly increased alcohol intake, while phasic activation resulted in a decrease of this measure. Furthermore, stimulation during extinction probe trials, when the lever press response was not reinforced, did not significantly alter alcohol-seeking behavior if a tonic pattern was applied. However, phasic stimulation substantially suppressed the number of lever presses, indicating decreased alcohol seeking under the same experimental condition.

The relationship between stress-related and alcohol-drinking behaviors has been intensively explored, providing experimental and clinical evidence that a stressful experience is correlated with elevated alcohol craving and intake (Kudryavtseva et al., 2006; Advani et al., 2007; McCool and Chappell, 2009; Caldwell and Riccio, 2010; Sinha, 2013; Boden et al., 2014; Norman et al., 2015; Albrechet-Souza et al., 2017; Gilpin and Weiner, 2017; Karlsson et al., 2017; Laws et al., 2017; Newman et al., 2018). However, neuronal substrates and neurotransmitter dynamics responsible for a causal link between these conditions are still unclear. Notably, stress exposure interacting with alcohol drinking has been shown to alter multiple brain systems, including the hypothalamic-pituitary-adrenal axis, corticotropin releasing factor, dynorphin, neuropeptide Y, dopamine and NE (Deal et al., 2018; Weera and Gilpin, 2019). Though numerous prospective circuit-based targets exist, it seems that the NE LC system has the potential to directly drive stress-related behaviors, including anxiety and fear (McCall et al., 2015; 2017), and therefore represents a promising candidate for modulating the interaction between acute stress and alcohol drinking.

Here, we optogenetically manipulated LC-NE activity by applying distinct stimulation protocols in order to explore how phasic and tonic NE release dynamics affect alcohol-drinking behaviors. Our results clearly demonstrate contrasting behavioral consequences of LC-NE circuitry activation during low and high frequency stimulation. Specifically, applying tonic stimulation during a standard operant drinking session resulted in increased intake, while phasic stimulation decreased this measure. It should be noted that previous studies have provided evidence on spontaneous and pattern-specific (tonic versus phasic) activity of the LC during stress-associated behaviors (Valentino and Van Bockstaele, 2008; Curtis et al., 2012; McCall et al., 2015; Borodovitsyna et al., 2018). Indeed, recent work demonstrated that tonic, but not phasic, activation of noradrenergic LC projections can result in increases in anxiety-like behavior (McCall et al., 2017). Together the data clearly indicate that the optogenetic shifting of LC-NE transmission into tonic mode, and therefore inducing stress-like behavior, can efficiently promote alcohol intake in drinking subjects. This is in sharp contrast with the finding that an increase in tonic dopamine release within the VTA-nucleus accumbens circuitry suppresses both alcohol and sucrose intake (Bass et al., 2013; Mikhailova et al., 2016). Therefore, these two pathways play opposing roles in the regulation of consummatory behavior under conditions where the release of neurotransmitters is increasing with the same patterns.

Whereas the effect of tonic stimulation of the LC-NE on alcohol consumption can be easily interpreted, the consequence of the phasic pattern is more puzzling. According to a previous finding, phasic LC stimulation did not result in anxiety-related behaviors (McCall et al., 2015). Therefore, an increase in alcohol intake under phasic activation would be unexpected in our experiment. However, why did phasic patterns of NE transmission suppress alcohol drinking under the current experimental design? Prior studies have revealed that phasic activity of the LC-NE is associated with the encoding of salient stimuli (not necessarily stress-related) and behaviors elicited by such stimuli (Vankov et al., 1995; Sara and Bouret, 2012). Indeed a recent study demonstrated that phasic, but not tonic LC, stimulation elicits event-related potentials in the PFC and can enhance PFC encoding of salient stimuli in male Long-Evans rats (Vazey et al., 2018). Perhaps in the current experiment, the phasic activity induced by the optical stimulation in our studies elicited cortical activity that interfered with or competed with attentional processes associated with alcohol-seeking behavior in the operant chamber environment, and consequently attenuated the intake.

To explore how different patterns of LC-NE transmission affect an appetitive (seeking) measure of alcohol-drinking behavior, nonreinforced extinction probe trials were conducted where rats were allowed to press the lever for the entire twenty-minute session, regardless of the number of lever presses completed. Alcohol-seeking behavior was not significantly altered by tonic patterns of stimulation under this experimental condition. Similarly, adolescent social isolation, which leads to increases in anxiety-like behaviors and alcohol intake did not affect extinction probe trial responding using the same operant procedure employed in our study (McCool and Chappell, 2009). It should be noted that appetitive (seeking) and consummatory alcohol drinking measures do not correlate with each other in this operant procedure (Samson and Czachowski, 2003; Budygin et al., 2020) and microinjection studies have revealed that these measures can be pharmacologically dissociated (Butler et al., 2014; McCool et al., 2014). This is probably because these behaviors are encoded by divergent, although interconnected, neurobiological processes (Sharpe and Samson, 2001; Slawecki and Roth, 2003). For example, shifting dopamine transmission into a phasic mode did not change alcohol intake in a two-bottle choice test (Bass et al., 2013), while alcohol-seeking behavior was enhanced during an extinction trial (Budygin et al., 2020). On the other hand, tonic increases in dopamine release resulted in the suppression of both consummatory and appetitive components (Bass et al., 2013; Budygin et al., 2020). The present findings indicate that replicating a stress-like condition through optogenetic activation of the LC-NE can selectively affect alcohol intake without changes in motivational behavior. Nevertheless, alcohol seeking was altered in the same manner as the intake when phasic patterns of NE release were triggered. This observation would seem to suggest that both components of alcohol-drinking behavior are equally sensitive to the distractive action of high frequency LC stimulation. Notably, the same stimulation of the VTA-nucleus accumbens DA circuitry facilitated alcohol seeking (Budygin et al., 2020). Consequently, phasic stimulation of LC-NE and VTA-dopamine circuits results in opposite effects on appetitive alcohol drinking-related behaviors, as observed with alcohol intake under the effect of tonic stimulation.

Given that tonic LC stimulation can elicit increases in anxiety-like behavior (McCall et al., 2017) and the well-established association between stress and increased alcohol consumption (Weera and Gilpin, 2019), here we provide the first evidence that tonic LC-NE activity plays a causal role in stress-associated increases in alcohol intake. However, the LC, a primary source of NE, sends two essential projections to the BLA and PFC, which can be differentially involved in stress response and alcohol self-administration. Therefore, future studies should dissect the role of these circuitries providing specific insight into the BLA and PFC contributions during stress-associated alcohol-seeking and drinking behaviors.

In summary, by integrating in vivo optogenetics and an operant drinking regimen that procedurally dissociated appetitive and consummatory alcohol drinking behaviors, we discovered pattern-specific influences LC-NE circuitry on alcohol-drinking behaviors. Our findings demonstrate a causal link between tonic patterns of LC-NE functioning, which elicits a stress-related condition, and increased alcohol intake. Furthermore, our data suggest that driving LC NE neurotransmission into a phasic mode reduces both alcohol consumption and seeking behavior, possibly by engaging PFC attentional processing. These results may guide future studies looking to pharmacologically or nonpharmacologically (transcranial magnetic stimulation, deep brain stimulation) manipulate NE release dynamics in order to treat AUD, especially in the common condition where there is also a comorbid anxiety/stressor disorder.

HIGHLIGHTS.

Alcohol-drinking behaviors can be controlled through LC optostimulation

Tonic stimulation during a drinking session results in increased alcohol intake

Phasic stimulation during a drinking session results in decreased alcohol intake

Tonic stimulation during an extinction trial does not alter alcohol-seeking behavior

Phasic stimulation during an extinction trial suppresses alcohol-seeking behavior

Acknowledgements

We thank A. Chappell for excellent guidance with behavioral experiments and Dr. Benjamin Rowland for his assistance with statistical analysis. Funding: This work was supported by the National Institutes of Health [grant numbers AA022449 (EAB), AA17531 (JLW), P50 AA026117 (EAB and JLW), DA024763 (CEB), AG057013 (OD), T32AA007565 (ALD)] and the Tab Williams Family Endowment Fund (EAB).

Footnotes

Declarations of Interest: none

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