Septo-hypothalamic regulation of binge-like alcohol consumption by the nociceptin system

SUMMARY

High-intensity alcohol drinking during binge episodes contributes to the socioeconomic burden created by alcohol use disorders (AUDs), and nociceptin receptor (NOP) antagonists have emerged as a promising intervention. To better understand the contribution of the NOP system to binge drinking, we found that nociceptin-containing neurons of the lateral septum (LS^Pnoc) displayed increased excitability during withdrawal from binge-like alcohol drinking. LS^Pnoc activation promoted active avoidance and potentiated binge-like drinking behavior, whereas silencing of this population reduced alcohol drinking. LS^Pnoc form robust monosynaptic inputs locally within the LS and genetic deletion of NOP or microinjection of a NOP antagonist into the LS decreased alcohol intake. LS^Pnoc also project to the lateral hypothalamus and supramammillary nucleus of the hypothalamus, and genetic deletion of NOP from each site reduced alcohol drinking. Together, these findings implicate the septo-hypothalamic nociceptin system in excessive alcohol consumption and support NOP antagonist development for the treatment of AUD.

Graphical Abstract

In brief

Haun et al. demonstrate that LS^Pnoc are responsive to aversive stimuli and sensitive to a history of alcohol drinking. LS^Pnoc activation is both aversive and enhances alcohol intake, whereas silencing decreased alcohol drinking. NOP antagonist or receptor deletion decreased alcohol intake in septohypothalamic sites.

INTRODUCTION

Excessive alcohol consumption is highly prevalent in the United States and contributes to the ever-increasing socioeconomic burden produced by alcohol use disorders (AUDs).^1–3 Binge drinking is the most common pattern of excessive drinking behavior shared in individuals diagnosed with an AUD, as well as those who do not meet the Diagnostic and Statistical Manual of Mental Disorders criteria.^4–7 Binge drinking is defined as the rapid consumption of alcohol containing beverages resulting in blood alcohol concentrations (BACs) in excess of the legal limit of intoxication (0.08 g/dL), typically consisting of five or more standard drinks in men and four or more for women in a session.^8–10 This risky pattern of drinking is the leading cause of emergency room visits in the United States, is correlated with a plethora of negative health outcomes, and is a strong predictor of the development of an AUD.^5,6,11,12 Presently, there are three U.S. Food and Drug Administration-approved medications for the treatment of AUD; however, there is a high prevalence of relapse and resistance to pharmacological intervention,^13–16 creating a pressing need to expand our therapeutic toolkit.

Clinical and preclinical studies have identified the nociceptin/orphanin-FQ (nociceptin) peptide system as a candidate for AUD treatment development. In fact, a nociceptin receptor (NOP) antagonist was found to reduce heavy drinking days and increase days abstinent in treatment-seeking patients with an AUD.¹⁷ Preclinical studies support this finding in that selective NOP antagonists attenuate alcohol self-administration and stress-induced reinstatement of alcohol seeking behavior in alcohol-preferring rats^18–20 and reduce binge-like alcohol consumption in mice.²¹ The ability of NOP antagonists to decrease alcohol drinking across preclinical models and rodent species is highly encouraging, warranting in-depth neuroanatomical study to isolate potential sites of action. Interrogating endogenous nociceptin-NOP circuitry is critical to understanding how the system may be dysregulated by alcohol and better inform treatment strategies. The lateral septum (LS) is a site of interest given that it is enriched in mRNA coding for the nociceptin peptide (prepronociceptin [Pnoc]),^22,23 promotes drug-seeking behavior,^24–26 and drives binge-like alcohol drinking in male mice.²⁷ Furthermore, the LS is situated as an integral node in addiction circuitry, interfacing directly with regions involved in binge drinking behavior such as the ventral tegmental area (VTA), nucleus accumbens (NAc), and hypothalamus.^28–30 Thus, nociceptin-containing neurons in the LS (LS^Pnoc) could be a previously unidentified node in circuitry mediating excessive drinking behavior.

The first goal of the present experiments was to classify expression patterns of Pnoc and NOP (opioid receptor like-1 [Oprl1]) mRNA throughout the neuroanatomical subdivisions of the LS. We then sought to determine activity patterns of LS^Pnoc during alcohol drinking and general appetitive and aversive behaviors with fiber photometry. LS^Pnoc sensitivity to a history of binge-like alcohol consumption was assessed using electrophysiology, and the reinforcing properties of LS^Pnoc activation was explored using optogenetics.³¹ A chemogenetic approach was then used to determine a causal role for LS^Pnoc in binge drinking behavior.³² Last, we mapped functional LS^Pnoc projection sites and assessed the contribution of NOP in these downstream targets to binge-drinking behavior through genetic deletion and local pharmacology.

RESULTS

The LS is enriched in Pnoc and Oprl1 mRNA

The LS is a GABAergic basal forebrain structure comprised of dorsal subdivisions (dLS), intermediate subdivisions (iLS), and ventral subdivisions (vLS) along the dorsal-ventral (D-V) gradient, and spans roughly 1.5 mm along the anterior-posterior (A-P) gradient in mice.^33–35 Because nociceptin and NOP expression has not been rigorously canvased within the LS, we used RNA-scope to quantify Pnoc and Oprl1 mRNA in VGAT-expressing cells in male and female C57BL/6J mice (Figure 1). Quantification was conducted in DAPI⁺ cells within the three subdivisions of the LS (dLS, iLS, and vLS) across the A-P gradient spanning from 1.10 to 0.14 mm relative to bregma (Figure 1A). For analysis, male and female data were collapsed across sex given a lack of main effect or factor interaction for Pnoc and Oprl1 probes. Because the LS is primarily GABAergic, a VGAT probe was used to define the boundaries of the LS and data were expressed as percent VGAT⁺ cells.^33,36

Figure 1. The LS is enriched in Pnoc and Oprl1 mRNA.

(A) Atlas image of regions of interest for the dLS, iLS, and vLS for analysis of Pnoc and Oprl1 expression across the A-P gradient.

(B) Representative image of the LS visualizing probes for VGAT (GABA), Pnoc (nociceptin), and Oprl1 (NOP) mRNA in DAPI⁺ nuclei. Scale bar, 100 μM.

(C) Representative images of VGAT, Pnoc, and Oprl1 overlay with DAPI. Scale bar, 50 μM.

(D–F) Quantification of mRNA within the dLS, iLS, and vLS. Values are collapsed across sex given the lack of main effect and factor interaction.

(D) Pnoc and Oprl1 are expressed to a similar extent in VGAT⁺ cells throughout the A-P gradient of the dLS, with the exception of 0.86 mm where Oprl1 expression is less than Pnoc (*p < 0.05). Cells expressing both Pnoc and Oprl1 are expressed to a lesser extent than Pnoc ($p < 0.01) and Oprl1 (∧p < 0.001).

(E) Oprl1 expression is greater than Pnoc (*p < 0.001) and Pnoc and Oprl1 colocalization ($p < 0.001) across the A-P gradient in the iLS. Pnoc expression was also greater than Pnoc and Oprl1 colocalization ($p < 0.001).

(F) Oprl1 expression is greater than Pnoc (*p < 0.001) and Pnoc and Oprl1 colocalization ($p < 0.001) across the A-P gradient in the vLS. Pnoc expression was also greater than Pnoc and Oprl1 colocalization across the A-P gradient ($p < 0.001).

(G) Pnoc expression is most abundant in the dLS, being greater than iLS and vLS in all but the posterior-most LS (^**p < 0.01, ^***p < 0.005, ^****p < 0.001).

(H) Oprl1 mRNA is fairly evenly represented across the LS.

(I) Colocalized Pnoc and Oprl1 mRNA was most abundant in the dLS, exceeding the iLS and vLS (*p < 0.05, ^**p < 0.01, ^***p < 0.005, ^****p < 0.001).

Data are presented as mean ± SEM. Triangles, females; Circles, males.

The LS is enriched in VGAT⁺ cells that express Pnoc and/or Oprl1 (Figures 1D–1F). Pnoc and Oprl1 are fairly evenly expressed within the dLS, accounting for 50% and 44.7% of GABAergic cells, respectively (Figure 1D), with co-expression of both in 28.8% of this population. However, Pnoc is expressed to a lesser extent than Oprl1 in the iLS (Figure 1E) and vLS (Figure 1F) across the A-P gradient. Pnoc is most abundant in the dLS (50%) and expression sharply decreases in the anterior subdivisions (1.10 and 0.86 mm) of the iLS (20.6%) and vLS (18.6%) (Figure 1G). In contrast, Oprl1 was consistently expressed across D-V gradients of the LS, but differed along the A-P axis with expression being slightly less in the posterior subdivisions (Figure 1H). Together, these data suggest that Pnoc-expressing cells are most dense within the dLS, independent of A-P position, while Oprl1 is somewhat evenly expressed, and co-expression (Figure 1I) indicates the possibility of feedback regulation of nociceptin and/or GABA release locally within the LS, as this structure is reported to be highly interconnected.^34,37,38

LS^Pnoc responds to stimuli associated with negative valence

An abundance of Pnoc-expressing neurons were observed in the LS, and this region as a whole is reported to be involved in consummatory behavior as well as aversion-related behaviors.^29,30 A series of fiber photometry experiments were conducted to selectively assess LS^Pnoc responsivity to appetitive drinking behavior, feeding behavior, and exposure to aversive stimuli. Adeno-associated virus 8 (AAV8)-hSyn-FLEX-GCaMP7f was infused into the LS of Pnoc-cre mice and fibers were positioned above this site to record calcium transients (Figure 2A). LS^Pnoc activity was increased preceding bouts of water drinking in male but not female mice (Figure 2B). While no change in activity was detected during bouts of alcohol drinking (Figure 2C), LS^Pnoc activity decreased during active licking bouts for sucrose in males (Figure 2D). This is consistent with activity recorded from LS -NTS³⁹ and -CRFR2,⁴⁰ which display decreased activity during consumption of a palatable reward. Yet, there was no change in LS^Pnoc activity during investigation or consumption of a highly palatable HFD pellet (Figures 2E and 2F). We then assessed LS^Pnoc activity in response to air puff exposure and fear conditioning given that discrete cell types within the LS are tuned to aversive stimuli.^40–42 An acute air puff elicited a robust response from LS^Pnoc (Figure 2G) and while a neutral tone was without effect (Figure 2H), footshock drove robust activity (Figure 2I). Interestingly, exposure to a conditioned stimulus (tone) did not provoke activity in LS^Pnoc after four tone-shock pairings (Figure 2J). A sucrose splash test was then conducted to query LS^Pnoc activity during provoked grooming behavior. As with air puff and footshock exposure, acute sucrose spray increased LS^Pnoc activity (Figure 2K). However, activity decreased during active bouts of grooming behavior after splash exposure (Figure 2L). Anxiety-like behavior was assessed in an elevated plus maze (EPM) and LS^Pnoc activity was elevated during exploration of the open arm, which normalized upon return to the closed arm in males (Figures 2M and 2N). Last, acute restraint stress blunted LS^Pnoc activity during the physical restraint, but activity sharply increased immediately following release (Figures 2O and 2P). Together, these findings indicate that LS^Pnoc are highly responsive to aversive stimuli and stimuli associated with approach/avoidance behavior, whereas activity is modestly decreased during sucrose consumption.

Figure 2. LS^Pnoc respond to stimuli associated with negative valence).

(A) Schematic and representative image of viral infusion, expression, and fiber placement within the LS.

(B) LS^Pnoc activity during bouts of water drinking was increased in male mice (p = 0.013) preceding licking onset (bout [F (1,10) = 5.90, p = 0.036]).

(C) LS^Pnoc activity was not changed when mice were given access to alcohol.).

(D) LS^Pnoc activity decreased in male mice (p = 0.049) during licking bouts for sucrose (bout [F (1,11) = 6.54, p = 0.027]).

(E) Investigation of an HFD pellet did not affect LS^Pnoc activity.

(F) LS^Pnoc activity was unchanged during active consumption of an HFD pellet.

(G) Air puff applied to the dorsal coat elevated LS^Pnoc activity in males (p = 0.002) and females (p = 0.003) (bout [F (1,11) = 39.47, p < 0.001]).

(H) Exposure to a neutral tone did not affect LS^Pnoc activity.

(I) Foot shock elicited robust LS^Pnoc activity in both male (p < 0.001) and female (p < 0.001) mice (bout [F (1,11) = 170.10, p < 0.001]).

(J) There was no change in LS^Pnoc activity during exposure to a conditioned stimulus (tone).

(K) Application of an atomized sucrose solution to the dorsal coat was associated with increased LS^Pnoc activity in male (p = 0.015) and female (p = 0.02) mice (bout [F (1,11) = 20.16, p < 0.001]).

(L) Grooming behavior after sucrose splash was accompanied by a reduction in LS^Pnoc activity in both males (p = 0.002) and females (p = 0.002); (bout [F (1,11) = 39.87, p < 0.001]).

(M) Mice were tested in the EPM and entry into the open arm trended toward increased LS^Pnoc activity in males (p = 0.052); (bout [F (1,10) = 7.38, p = 0.022]).

(N) Entry into the closed arm was associated with decreased LS^Pnoc activity in males (p = 0.034) when compared with the open arm (bout [F (1,10) = 9.19, p = 0.013]).

(O) Onset of physical restraint decreased LS^Pnoc activity in males and females (ps < 0.001); (bout [F (1,9) = 122.4, p < 0.001]).

(P) LS^Pnoc activity increased in males and females upon release from restraint (ps < 0.001); (bout [F (1,9) = 124.9, p < 0.001]). See also Figure S1.

Data are presented as mean ± SEM (*p < 0.05, ^**p < 0.01, ^***p < 0.005, ^****p < 0.001).

Binge drinking increases excitability of LS^Pnoc

We sought to assess LS^Pnoc excitability during withdrawal from binge drinking when signs of negative affect emerge using whole-cell patch-clamp electrophysiology.^43,44 AAV8-hSyn-DIO-mCherry was infused into the LS of Pnoc-cre mice to selectively target LS^Pnoc and tissue was prepared for recording 24 h after the final alcohol drinking session (Figures 3A and 3B).

Figure 3. Binge drinking increases excitability of LS^Pnoc.

(A) Depiction of study design.

(B) Average alcohol intake (g/kg) for males and females across 3 weeks of drinking. Females generally consumed more alcohol than males (sex [F (1, 8) = 10.16, p= 0.013]) and drinking was greater during the 4-h drinking session (time [F (3,24) = 30.19, p < 0.001]).

(C and D) Input resistance and capacitance of LS^Pnoc were not altered as a function of drinking history.

(E) A history of alcohol drinking depolarized LS^Pnoc RMP (history [F (1, 56) = 11.78, p= 0.001]), and this effect was most pronounced in males (p < 0.001).

(F) LS^Pnoc AP frequency (Hz) was greater in males (p < 0.001) consuming alcohol compared with water (history [F (1, 56) = 11.23, p = 0.001]; sex*history [F (1, 56) = 5.583, p = 0.025]).

(G) Rheobase was significantly decreased in LS^Pnoc as a function of alcohol drinking (history [F (1, 56) = 11.88, p = 0.001]), and this effect was most prominent in males (p = 0.022).

(H) Current step protocols indicated increased firing at 40–180 pA in males with a history of alcohol drinking (ps < 0.05) compared with water drinkers (current [F (1, 56) = 367.22, p < 0.001]; history [F (1, 56) = 10.89, p = 0.002]; current*history [F (1, 56) = 5.70, p = 0.02]).

(I) Alcohol drinking did not alter AP activity in female mice.

Data are presented as mean ± SEM (*p < 0.05, ^**p < 0.01, ^***p < 0.005, ^****p < 0.001).

Excitability was assessed through four metrics: resting membrane potential (RMP), spontaneous action potential (AP) frequency, rheobase, and APs generated through an increasing current-step protocol. Three weeks of binge-like alcohol consumption had no effect on input resistance or capacitance in LS^Pnoc, but promoted a more depolarized RMP and increased AP frequency 24 h after alcohol drinking compared with water-drinking control mice. The ability of alcohol drinking to influence LS^Pnoc RMP was driven in large part by male mice, as RMP was more depolarized in alcohol drinkers (Figure 3C). Similarly, LS^Pnoc AP frequency was greater in males with a history of alcohol drinking compared with water, with no difference in females (Figure 3D). Alcohol drinking decreased rheobase in male mice compared with water drinkers, and this effect neared significance in females (Figure 3F). Excitability assessed through a current step protocol revealed an increased number of AP events in male mice with a history of binge-like alcohol consumption with no difference in females (Figures 3H and 3I).

LS^Pnoc activation is aversive and increases alcohol drinking

A real-time place preference assay was used to determine if LS^Pnoc activation has aversive properties. AAV-hSyn-DIO-ChR2/mCherry was expressed in the LS of Pnoc-cre mice and fibers were positioned above this site (Figure 4A). Mice were introduced to a two-chamber apparatus with identical sides and time spent on either side of the chamber was recorded during a pre-test session (Figures 4B and 4C). After controlling for pre-test side bias, laser stimulation of LS^Pnoc in mCherry and ChR2-expressing groups was conducted the following day. ChR2-expressing mice spent less time on the laser paired side of the chamber, suggesting that LS^Pnoc activation promotes avoidance behavior (Figures 4C and 4D), an effect absent in the control group (mCherry).

Figure 4. LS^Pnoc activation is aversive and increases alcohol drinking.

(A) Depiction of viral infusion and representative image of mCherry expression and fiber placement in LS^Pnoc for optogenetic studies.

(B) Diagram of two-chamber testing apparatus with laser-paired side.

(C) While there was no difference in basal side preference during the pre-test phase, blue light laser stimulation decreased side preference in ChR2-expressing mice (p = 0.002) with no change in mCherry controls (day*AAV [F (1, 19) = 5.13, p = 0.035]). Data are collapsed across sex given the lack of main effect. Triangles, females; circles, males.

(D) Representative heatmap of time spent in either side of the two-chamber apparatus during laser stimulation.

(E) Depiction of viral infusion and representative image of mCherry expression in LS^Pnoc for chemogenetic studies.

(F) Mice were challenged with vehicle (Veh, saline) or CNO (3 mg/kg) prior to alcohol drinking. CNO treatment in hM4Di-expressing male (p = 0.021) and female (p = 0.039) mice decreased alcohol intake compared with vehicle. Male (p = 0.032) and female (p = 0.027) mice expressing hM3Dq in LS^Pnoc consumed more alcohol when challenged with CNO. There was no effect of CNO in mCherry controls, but females consumed more alcohol than males in all groups (sex [F (1, 49) = 50.94, p < 0.001]; AAV [F (2, 49) = 5.86, p = 0.005]; drug*AAV [F (2, 49) = 10.06, p < 0.001]).

(G) There was no effect of LS^Pnoc manipulation on sucrose intake, although females consumed more sucrose than males (sex [F (1, 48) = 25.16, p < 0.001]).

(H) There was no change in locomotor activity resulting from LS^Pnoc silencing or activation, but females displayed a greater distance traveled than males (sex [F (1, 49) = 5.66, p = 0.021]). See also Figure S2.

Data are presented as mean ± SEM (*p < 0.05, ^**p < 0.01, ^***p < 0.005, ^****p < 0.001).

A chemogenetic experiment was then conducted to determine the functional contribution of LS^Pnoc to alcohol drinking. AAV8-hSyn-DIO-hM4Di/hM3Dq/mCherry was infused into the LS of male and female Pnoc-cre mice (Figure 4E). Mice were challenged with vehicle and clozapine N-oxide (CNO) in a within-subjects counterbalanced design after habituation to alcohol drinking, sucrose drinking, and locomotor activity. Generally, females consumed more alcohol than males and CNO challenge was without effect in the control groups (mCherry). However, silencing of LS^Pnoc mice reduced alcohol intake in both males and females compared with vehicle (Figure 4F). Conversely, activation of LS^Pnoc increased alcohol drinking in males and females. LS^Pnoc silencing/activation was then assessed in the context of sucrose drinking in an identical design (Figure 4G). Sucrose was chosen as a contrast to alcohol given its reinforcing and caloric properties, and the concentration was selected as mice consume similar volumes of each solution within a 2-h session. Females consumed more sucrose than males, but there was no difference between vehicle and CNO treatment among AAV groups. Last, mice were assessed in an open field test to determine if the changes in alcohol intake could be attributed to a non-specific perturbation of locomotor activity. While females displayed greater overall locomotor activity, there was no effect LS^Pnoc manipulation on distance traveled (Figure 4H) or time spent in the center of the open field (Figure S2C). Together, these findings indicate that LS^Pnoc activation has aversive properties and plays a role in alcohol consumption.

LS^Pnoc forms GABAergic synapses in LS, LH, and SuM

We then evaluated the projection patterns of LS^Pnoc neurons in two complementary circuit mapping experiments. An AAV harboring synaptophysin-mCherry was expressed in LS^Pnoc (Figure 5A) and terminal expression was observed within the LS (Figure 5B), lateral hypothalamus (LH; Figure 5C), and supramammillary nucleus of the hypothalamus (SuM; Figure 5D). To confirm functionality in these projection sites, ChR2-assisted circuit mapping was conducted by expressing ChR2 in LS^Pnoc and optically evoked synaptic input was recorded using whole-cell patch-clamp electrophysiology (Figure 5E). Blue light-evoked post-synaptic current was observed in non-Pnoc-expressing neurons within the LS, and this effect was absent in the presence of picrotoxin, suggesting that LS^Pnoc form local functional GABAergic synapses (Figure 5F). An additional series of experiments was conducted in ChR2-exrpressing mice to determine the number, amplitude, and strength of local LS^Pnoc GABAergic synapses using a paired pulse protocol. The overwhelming majority (96%) of cells within the LS received mono-synaptic input from LS^Pnoc (Figure 5G). The observed optically evoked post-synaptic current (oIPSC) amplitude, as measured from the first oIPSC event, was not different as a function of sex (Figure 5H). A paired pulse ratio was then calculated and paired pulse inhibition was observed, but was not dependent upon sex (Figure 5I). This suggests similar levels of synaptic strength of local LS^Pnoc inputs.

Figure 5. LS^Pnoc form GABAergic synapses in the LS, LH, and SuM.

(A) Depiction of viral infusion into the LS for circuit mapping.

(B–D) Representative images of LS^Pnoc terminal expression (mCherry) in the (B) LS, (C) LH, and (D) SuM.

(E) Diagram depicting ChR2 expression in LS^Pnoc and electrophysiology in the LS.

(F) The oIPSC resulting from LS^Pnoc stimulation produced a robust current and this effect was absent in the presence of the GABA-A and -B receptor antagonist, picrotoxin (500 μM).

(G) An oIPSC was observed after blue light stimulation in the majority of cells in the LS (responsive; 96%).

(H and I) LS oIPSC amplitude and paired pulse ratio (PPR) were collapsed across sex given the lack of difference between sexes.

(J) Diagram depicting electrophysiology in LS^Pnoc terminal fields in the LH.

(K) Example trace of oIPSC in the LH resulting from LS^Pnoc stimulation and blockade by picrotoxin.

(L) An oIPSC was observed after blue light stimulation in 32% of cells in the LH (responsive).

(M and N) The amplitude of oIPSCs and PPR observed in the LH were collapsed across sex, although this effect neared significance for PPR (sex:t (8) =2.213, p = 0.058).

(O) Diagram depicting electrophysiology in LS^Pnoc terminal fields in the SuM.

(P) Example trace of oIPSC in the SuM resulting from LS^Pnoc stimulation and blockade by picrotoxin.

(Q) An oIPSC was observed after blue light stimulation in 33% of cells in the SuM (responsive).

(R and S) The amplitude of oIPSCs and PPR in the SuM were collapsed across sex.

Data are presented as mean ± SEM (*p < 0.05, ^**p < 0.01, ^***p < 0.005, ^****p < 0.001). Triangles, females; Circles, Males.

Synaptophysin-mCherry immunofluorescence was also observed in the LH (Figure 5C). ChR2-assisted circuit mapping revealed oIPSC within the LH that was blocked by picrotoxin, confirming a functional GABAergic synapse (Figure 5K). Stimulation of LS^Pnoc terminals in the LH drove oIPSCs is 32% of cells and promoted paired-pulse inhibition (Figures 5L–5N). Similarly, synaptophysin-mCherry immunofluorescence arising from LS^Pnoc was observed in the SuM (Figure 5D). LS^Pnoc terminal activation in the SuM evoked a picrotoxin-sensitive current, suggesting monosynaptic GABAergic connectivity (Figure 5P) and oIPSCs were observed in 33% of cells (Figures 5Q–5S). Together, these circuit mapping studies revealed that LS^Pnoc provide monosynaptic GABAergic input to a high number of cells within the LS, suggesting a robust mechanism for local inhibitory control and, to a lesser extent, downstream within the LH and SuM.

NOP deletion from LS, LH, or SuM attenuates alcohol intake

Because functional synapses were observed in LS^Pnoc terminal fields, genetic deletion studies were conducted to determine the role of NOP in the LS, LH, and SuM in relation to binge-drinking behavior. AAV8-hSyn-GFP/cre was infused into the LS of male and female NOP^fl/fl mice (Figure 6A), which was largely confined to the dLS and iLS, and decreased Oprl1 mRNA (Figures S3A–S3D). Alcohol drinking was assessed in the 2-h modified drinking in the dark (DID) model for 3 weeks. Females consumed more alcohol than males, and genetic deletion of NOP from the LS generally reduced alcohol drinking, an effect most prominent in males (Figure 6B). Average alcohol drinking across weeks is shown in Figure S3E, and BACs, alcohol intake, and BAC/intake correlation from the final day of drinking are shown in Figures S3F and S3G. Sucrose drinking was not affected by NOP deletion (Figure 6C), but water intake was reduced, and this effect was primarily driven by females (Figure 6D). LS NOP deletion had no effect on locomotor activity or body weights (Figures S3H and S3I).

Figure 6. NOP deletion from the LS, LH, or SuM attenuates alcohol intake.

(A) Depiction of viral infusion in the LS and representative image of expression.

(B) Genetic deletion of NOP from the LS decreased alcohol intake and this effect was primarily driven by males (p = 0.016) when compared GFP-expressing controls (sex [F (1, 32) = 38.72, p < 0.001]; AAV [F (1, 32) = 5.61, p = 0.024]).

(C) Female mice consumed more sucrose than males (sex [F (1, 32) = 18.26, p < 0.001]), but NOP deletion from the LS did not affect intake.

(D) Deletion of NOP from the LS decreased water intake over a 24-h period (AAV [F (1, 32) = 6.49, p = 0.016]) and this effect was primarily driven by females (p = 0.033). Females also consumed more water over the drinking session (sex [F (1, 32) = 39.02, p < 0.001]).

(E) Depiction of viral infusion into the LH and representative image of expression.

(F) Genetic deletion of NOP from the LH in Cre expressing mice generally blunted alcohol intake (AAV [F (1, 30) = 13.50, p < 0.001]). Females consumed more alcohol than males (sex [F (1, 30) = 48.56, p < 0.001]) and the reduction in alcohol drinking was also primarily driven by female mice (p = 0.002).

(G) Sucrose intake was not affected by NOP deletion from the LH; however, females consumed more sucrose in 2 h (sex [F (1, 30) = 27.86, p < 0.001]).

(H) Water intake was not affected by NOP deletion from the LH, but females consumed more water over a 24-h period (sex [F (1, 30) = 36.21, p < 0.001]).

(I) Depiction of viral infusion into the SuM and representative image of expression.

(J) Genetic deletion of NOP from the SuM suppressed alcohol intake (AAV [F (1, 23) = 7.48, p = 0.012]). Females consumed more alcohol than males (sex [F (1, 23) = 39.91, p < 0.001]) and the ability of NOP to affect alcohol drinking was primarily driven by females (p = 0.047).

(K) Sucrose intake was not affected by NOP deletion from the SuM and females consumed more sucrose (sex [F (1, 23) = 19.53, p < 0.001]).

(L) Water intake over a 24-h period was greater in female mice (sex [F (1, 23) = 15.38, p < 0.001] and ANOVA revealed a factor interaction (sex*AAV [F (1, 23) = 5.86, p = 0.024]). However, a post hoc analysis indicated no difference in water intake as a function of SuM NOP deletion. See also Figures S3–S5.

Data are presented as mean ± SEM (*p < 0.05, ^**p < 0.01, ^***p < 0.005, ^****p < 0.001).

Genetic deletion of NOP from the LH was investigated in a separate study (Figure 6E). Viral expression was observed throughout the LH, with some expression in neighboring ventromedial hypothalamus, and cre expression was effective in reducing Oprl1 mRNA (Figures S4A–S4D). NOP deletion from the LH led to reduced alcohol intake, an effect that was more pronounced in females (Figure 6F). Alcohol intake by week, final day of drinking, BAC, and intake/BAC correlation can be found in Figures S4E–S4G. Sucrose and water intake were not affected by LH NOP deletion, but locomotor activity was attenuated in females (Figure S4I), perhaps influencing alcohol-drinking behavior.

Last, the effect of NOP deletion from the SuM on alcohol drinking was examined (Figure 5M). Expression of cre in the SuM reduced Oprl1 mRNA and viral expression was centered on the SuM, with some spread into the anteromedial VTA (Figures S5A–S5D). Genetic deletion of NOP from the SuM reduced alcohol intake, an effect that was driven in large part by females (Figure 6J). Drinking data by week, BACs on the final day of drinking, and intake/BAC correlation can be found in Figures S5E–S5H. SuM NOP deletion had no effect on sucrose (Figure 6K) or water (Figure 6L) intake, or on locomotor activity (Figure S5I). However, SuM NOP deletion attenuated body weight in males (Figure S5J).

NOP antagonist reduces alcohol intake

NOP antagonists have been shown to decrease alcohol intake in rats and mice,^19,21 yet few studies have explored sex as a biological variable (SABV) in relation to NOP antagonist efficacy in the context of binge drinking. First, we tested the effect of systemic administration of the selective NOP antagonist, SB-612111, on alcohol drinking in C57BL/6J mice using the 2-h modified DID model (Figure 7A). This dose of SB-612111 was selected as it does not affect sucrose or water intake, but reduced binge-like alcohol intake in male mice.^21,45 Here, SB-612111 reduced binge-like alcohol consumption and this effect was primarily driven by males (Figure 7B). In a separate experiment, bilateral guide cannula were positioned above the LS and SB-612111 was infused prior to alcohol drinking (Figure 7C). Blockade of NOP in the LS blunted alcohol drinking and, like systemic SB-612111, this effect was primarily driven by males (Figure 7D). We then leveraged electrophysiology to assess the effect of SB-612111 and nociceptin on the membrane potential of cells in the LS (Figure 7E). Bath application of SB-612111 did not affect membrane potential (Figure 7F), but nociceptin drove a hyperpolarized shift in both males and females (Figures 7G and 7H). The ability of nociceptin to hyperpolarize LS cells was blocked in the presence of SB-612111 in both sexes, indicating efficacy of SB-612111 as a NOP antagonist. Together, these findings suggest that NOP within the LS contribute to alcohol drinking and are equally responsive to nociceptin and NOP antagonist in males and females.

Figure 7. NOP antagonist reduces alcohol intake.

(A) Depiction of systemic NOP antagonist (SB-612111; 10 mg/kg) treatment prior to alcohol drinking.

(B) Systemic NOP antagonist treatment decreased alcohol intake (drug [F (1, 30) = 5.55, p = 0.025]). While females generally consumed more alcohol than males (sex [F (1, 30) = 6.69, p = 0.015]), a post hoc analysis indicated that the ability of a NOP antagonist to reduce alcohol drinking was primarily driven by males (p = 0.02).

(C) Depiction of site-specific NOP antagonist (SB-612111; 100 μM) microinjection into the LS prior to alcohol drinking.

(D) Intra-LS injection of SB-612111 decreased alcohol intake (drug [F (1, 21) = 11.22, p = 0.003]) and post hoc indicated a reduction of alcohol drinking in males (p = 0.015).

(E) Depiction of patch-clamp electrophysiology assessing effect of nociceptin and SB-612111 on membrane potential in the LS.

(F) Bath application of SB-612111 did not affect membrane potential in male and female mice. However, membrane potential was more hyperpolarized in females compared with males (sex [F (1, 15) = 9.51, p = 0.008]).

(G) Representative traces depicting hyperpolarizing effect of nociceptin in the LS and blockade with SB-612111.

(H) Nociceptin application drove a hyperpolarizing shift in membrane potential in LS cells (agonist [F (1, 15) = 17.13, p < 0.001]) in males (p = 0.012) and females (p = 0.030). Pre-application with SB-612111 blocked the effect of nociceptin. See also Figure S6.

Data are presented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001).

DISCUSSION

NOP as a target for AUD pharmacotherapy

The ability of NOP antagonists to reduce alcohol drinking in rodent models is well documented, where treatment has reduced binge-like alcohol consumption in male mice²¹ and blunts drinking and reinstatement of alcohol-seeking behavior in rats.^18–20 Here, we replicate the findings of Brunori and colleagues²¹ and extend this work to include SABV, demonstrating that reduced alcohol intake post SB-612111 treatment is most apparent in males. In contrast, the NOP antagonist LY2817412 attenuated alcohol consumption in male and female alcohol-preferring rats,¹⁹ and global NOP deletion blocks alcohol-induced conditioned place preference in female mice.⁴⁶ Additional studies indicating NOP antagonist efficacy in females used continuous access drinking or self-administration models in alcohol-preferring rat strains^19,20; thus, it is possible that differences in species, strain, and model account for these variable findings that make interpreting sex differences in NOP antagonist effects difficult.

The NOP receptor presents an interesting challenge in developing therapeutic interventions for AUD in that both NOP agonists and antagonists reduce alcohol intake.^47–49 One explanation for this phenomenon is that agonists can rapidly desensitize and internalize NOP,^50–52 resulting in a presumed antagonist-like effect on alcohol drinking especially under conditions of chronic dosing. For example, acute intracerebroventricular administration of nociceptin-potentiated alcohol intake in alcohol-preferring rats whereas a suppressive effect on drinking emerged after multiple doses.⁵³ Furthermore, the highly selective and brain penetrant NOP agonist MT-7716 reduced alcohol drinking after 3–4 days of treatment with twice daily oral administration.⁵⁴ While desensitization effects have not been fully ruled out, it is likely that more complicated mechanisms are at play. To underscore this point, acute NOP agonist treatment can increase,⁵³ decrease,⁵⁵ or have no effect on alcohol drinking or self-adminstration.²¹ Second, NOP agonists and antagonists can have similar physiological effects on cellular activity depending on the site of action. This was recently identified in the VTA, where NOP agonist or antagonist bath application suppressed firing of dopaminergic neurons through direct and indirect inhibition, respectively.⁵⁶ This suggests that the ability of a NOP antagonist to reduce drinking may be dependent on site of expression, potentially in a cell-type-specific manner.

A few discrete sites of NOP antagonist action in male rats have been explored in vivo, indicating that NOP in the VTA and CeA, but not NAc, regulate excessive alcohol drinking.^19,20 Here we report that NOP in the LS, LH, and SuM also regulate excessive drinking and argue for the exploration of antagonist effects outside of canonical mesolimbic and extended amygdala addiction circuity, and this is consistent with global genetic NOP knockout that suppresses binge drinking²¹ and alcohol self-administration.⁵⁷ Genetic deletion of NOP from the LS reduced alcohol and water intake without affecting sucrose drinking. Similarly, NOP deletion from the LH attenuated alcohol drinking without affecting sucrose or water intake, but was accompanied by a nonspecific attenuation of locomotor activity, making interpretation difficult. A more clear effect emerged at the level of the SuM, where NOP deletion decreased alcohol intake with no effect on sucrose intake, water intake, or locomotor activity. Because chemogenetic activation of the SuM is anxiolytic,⁵⁸ we speculate that NOP deletion may disinhibit the SuM and thereby decrease the motivation to consume alcohol, given that decreased anxiety is associated with decreased alcohol consumption.⁵⁸

Role for the LS in binge drinking

The LS is a frequently overlooked node in the theoretical framework proposed for AUD, contributing to binge drinking and displaying increased activity during withdrawal. We have recently shown that activation of the dorsal septum (comprising the LS and dorsal tip of the medial septum) is sufficient to increase alcohol and sucrose intake in males, and increase locomotor activity independent of sex.²⁷ Here we report that more discrete isolation of LS^Pnoc revealed a selective contribution to alcohol drinking, and the ability of this population to affect alcohol intake was not influenced by SABV. The LS is heterogeneous in terms of peptide expression and it is likely that targeting nociceptin-expressing neurons allowed for the uncovering of neuromodulation specific to alcohol. Regions governing binge-drinking behavior are often associated with reward, and initial observations by Olds and Milner⁵⁹ indicated that activation of the septal area is innately rewarding. Subsequent work has outlined a role for the LS in the rewarding properties of cocaine and in regulating reinstatement of drug-seeking behavior.^24–26 Further, ablation of the LS attenuates the ability of alcohol to increase dopaminergic tone in the NAc and supports a role of the LS in the rewarding properties of alcohol.⁶⁰

Projections from the LS directly modulate reward circuitry by innervating the VTA,^61,62 yet activation of LS projections to the VTA have yield mixed results. For example, rodents will not self-stimulate for non-specific LS-VTA activation nor does activation of this pathway drive place preference,⁶³ but mice will actively nose-poke for selective activation of the LS-Esr1-VTA circuit.⁶⁴ Here, synaptophysin tracing experiments revealed a lack of LS^Pnoc innervation of the VTA, which agrees with retrograde tracing of VTA afferents in Pnoc-cre mice conducted by Parker and colleagues.⁶⁵ It is likely that any modulation of the VTA by LS^Pnoc is through indirect means, such as local microcircuits impinging upon select populations of non-Pnoc outputs to the VTA.^34,38 This is consistent with our findings indicating that nearly all non-Pnoc-expressing cells targeted at the level of the LS receive robust monosynaptic GABAergic input from LS^Pnoc.

Approximately 30% of cells observed in the present studies within the LH and SuM receive monosynaptic input from LS^Pnoc. At the level of the LH, specific subclasses of neurons innervate the VTA and regulate binge drinking and consummatory behaviors and promote aversive behaviors.^66,67 GABAergic neurons within the LH that project to the VTA directly promote consummatory behaviors,^68–70 while activation of glutamatergic neurons reduces feeding and reward-associated behaviors.^71,72 Given that LS^Pnoc form GABAergic synapses in the LH, it is likely that activation of this circuit results in a suppression of LH-VGLUT neuronal activity, allowing for the promotion of alcohol drinking behavior. In contrast, the SuM is involved in arousal,⁷³ motivation,⁷⁴ and anxiety-like behavior.⁵⁸ Chemogenetic activation of the SuM is anxiolytic,⁵⁸ and silencing of LS^Pnoc may result in SuM disinhibition, thereby reducing alcohol-associated anxiety during withdrawal and subsequent motivation to consume alcohol. Consummatory behavior decreases SuM activity and inhibition disrupts reward seeking that is likely involved in binge drinking.⁷⁵

The LS has been implicated in stress and anxiety states and recent studies leveraging in vivo Ca imaging has revealed heightened activity in genetically defined LS populations during exposure to stimuli associated with negative valence. For example, NTS-expressing,^42,76 SST- expressing,⁴¹ and CRFR2-expressing⁴⁰ neurons in the LS are responsive to footshock, and VGAT-expressing neurons are highly responsive to pain.⁷⁷ LS^Pnoc activity follows this pattern, displaying increased activity during footshock, air puff stimulus, and sucrose spray, as well as exploration of the open arm in an EPM. However, LS^Pnoc activity is reduced during active grooming behavior when mice experience discomfort from a soiled coat. Silencing of hyperactive LS activity provoked by inflammatory pain has analgesic and anxiolytic properties,⁷⁷ suggesting that quiescence of LS activity may provide relief from aversive stimuli. Indeed, high-level alcohol consumption acutely suppresses activity of the dLS,⁷⁸ whereas acute withdrawal is associated with rebound excitation.⁷⁹ However, LS^Pnoc Ca activity was not altered during active alcohol consumption and we speculate this was due to acute alcohol presentation, which was experimentally controlled as to not confound subsequent photometry experiments in the same mice. It is possible that LS^Pnoc recruitment may be observed under experimental conditions engendering more robust levels of alcohol drinking resulting in binge levels of intoxication.

Toward a deeper understanding of nociceptin circuitry

Few studies have directly manipulated endogenous nociceptin-expressing populations in the context of motivated behaviors such as alcohol drinking. Modulation of Pnoc-expressing neurons thus far has been somewhat limited to midbrain,^65,80 hypothalamic,^80–82 and extended amygdala structures.^23,83 For example, Pnoc-expressing neurons of the perinigral VTA function as a break on progressive ratio responding for a natural reward and acute activation of VTA-Pnoc suppresses binge-like feeding behavior.^65,80 Conversely, activation of Pnoc-expressing neurons within the central amygdala is rewarding and silencing of this population reduces binge-like feeding behavior,²³ similar to arcuate-Pnoc that promotes binge-like feeding behavior upon activation.^80–82 Thus, endogenous nociceptin circuitry does not uniformly regulate appetitive behavior and may have opposing actions depending on the site of expression and projection target. Pnoc-expressing neurons of the BNST are located ventral to LS^Pnoc and display a heterogeneous response to salient stimuli.⁸³ The authors conclude that this population is recruited with high temporal specificity to salient stimuli that gates an acute anxiety response. It is possible that LS^Pnoc functions similarly, given the acute responsivity to aversive stimuli and discrete projection sites to the LS, LH, and SuM, all regions implicated in arousal.^40,73,84

Sex-specific effects were observed in relation to LS^Pnoc excitability, where increased spontaneous action potential firing was not observed in females with a history of binge-like alcohol consumption. It is important to note that 3 weeks of binge drinking in the DID model is a relatively brief window of voluntary alcohol drinking, and more robust models of alcohol drinking and/or exposure, such as the chronic intermittent ethanol (CIE) exposure and drinking model, may drive adaptations in LS^Pnoc in females that do not emerge in the DID model. In fact, the CIE model may serve as a more optimal platform for future studies in that basal sex differences (and individual variability) in voluntary alcohol consumption can be experimentally controlled. Further, we did not assess LS^Pnoc neurotransmitter release dynamics after alcohol drinking, leaving effects at the synaptic level either within the LS itself or in downstream projection sites open to further study.

In conclusion, the present studies identify LS^Pnoc as a GABAergic population that is enriched in the dLS. These neurons are primarily responsive to acute aversive stimuli presentation and activation has aversive qualities. Furthermore, these neurons display increased excitability during withdrawal from binge-like alcohol consumption and play a causal role in alcohol drinking in that chemogenetic activation increased alcohol drinking, whereas inactivation decreased alcohol intake. The effect of LS^Pnoc manipulation to influence alcohol drinking was not due to a general shift in consumption of a palatable fluid as sucrose drinking was unaffected, nor was this due to nonspecific locomotor effects. LS^Pnoc form strong local GABAergic synapses within the LS, as well as the LH and SuM. Genetic deletion of NOP of from the LS, LH, or SuM reduced alcohol drinking and site-specific direction of a NOP antagonist to the LS had the same effect. Together, these findings point to septo-hypothalamic nociceptin signaling as a druggable target system regulating excessive alcohol consumption.

Limitations of the study

While our study implicates LS^Pnoc and NOP in modulating alcohol consumption and demonstrates that LS^Pnoc form GABAergic synapses in NOP-rich regions, it remains uncertain whether nociceptin itself is released from LS^Pnoc to directly act on NOP in these sites. Thus, it is unclear if nociceptin, GABA, or another peptide derived from LS^Pnoc directly affects alcohol drinking. The development of tools for selective Pnoc genetic deletion and/or genetically encoded nociceptin sensors offer promising techniques for manipulation and detection that may shed light on these shortcomings in the future. Furthermore, discrete LS^Pnoc projections (e.g., LS^Pnoc-LS, LS^Pnoc-LH, and LS^Pnoc-SuM) were not directly isolated and manipulated, leaving potential for future studies that manipulate LS^Pnoc projections in the context of alcohol drinking while isolating the contribution of nociceptin. Last, the modest effects observed with chemogenetic manipulation, NOP receptor deletion, and antagonist treatments on alcohol intake warrant cautious interpretation. Nonetheless, these findings are encouraging as they are consistent with preclinical studies and clinical trials, which similarly reported modest but reliable reductions in alcohol consumption with NOP antagonists.^17–21 This alignment across experimental models reinforces the potential therapeutic value of targeting the nociceptin/NOP system for AUD treatment.

RESOURCE AVAILABILITY

Lead contact

Thomas Kash. tkash@email.unc.edu.

Materials availability

No new materials were generated.

Data and code availability

• All data reported in this paper will be shared by the lead contact upon request.

• This paper does not report original code.

• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

STAR★METHODS

EXPERIMENTAL MODEL AND STUDY PARTICIPANTS DETAILS

Male and female Pnoc-IRES-cre (Stock# 034278, Jackson Laboratories),⁶⁵ NOP^fl/fl (Stock# 036308, Jackson Laboratories),⁶⁵ and C57BL/6J (Stock# 000664, Jackson Laboratories) mice were housed in a temperature (70–75F) and humidity (40–60%) controlled AAALAC approved facility for all studies. Pnoc-IRES-cre and NOP^fl/fl mice of a C57BL/6J background were used from an established breeding colony, generated from breeders acquired from Jackson Laboratories. Tail snips from all Pnoc-IRES-cre and NOP^fl/fl mice were collected and samples genotyped by Transnetyx prior to use. Male and female C57BL/6J mice were also acquired from Jackson Laboratories and, for all experiments, mice were given at least 1 week of acclimation to the colony room prior to testing. All mice were singly housed at 8–12 weeks of age and maintained on a reverse 12-h light/dark cycle within a ventilated rack system. Mice were housed in standard cobb bedding in polycarbonate cages (GM500, Techniplast) and provided with food (Prolab Isopro, RMH3000) and water ad libitum. For all studies, experimentally naive mice were randomly assigned to groups to prior to testing. Sex as a biological variable was defined in terms of chromosomal and gonadal sex as male or female for all studies. All studies and experiments were conducted in accordance with NIH guidelines and under a UNC School of Medicine-approved Institutional Animal Care and Use Committee protocol.

METHOD DETAILS

RNAscope

Fresh frozen tissue (N = 8, 4 male and 4 female) was collected from C57BL/6J mice for identification of cell type within the LS (Figure 1). Mice were deeply anesthetized with tribromoethanol (Avertin, 1 mL, ip.) and the brain was removed then immediately frozen on dry ice, and stored at −80c until sectioning. Serial slices from each region (14 μM) were collected on a cryostat (Leica CM3050 S) and mounted on Superfrost Plus slides. Slides were then processed using Advanced Cell Diagnostics RNAscope Fluorescence Multiplex Assay Kit according to the provided protocol. Briefly, mRNA was visualized with TSA Vivid Dyes (AKOYA Biosciences) targeting the vesicular GABA transporter (VGAT; Slc32a1 Probe# 319191), prepronocicpetin (Pnoc; Probe# 437881), and the nociceptin receptor (Oprl1; Probe# 514301). Slides were coverslipped with Prolong Diamond mounting medium and imaged on a VS200 Slide scanner (Olympus Life Sciences). Intensity and duration of exposure for each channel was optimized in consultation with the Hooker Imaging Core (UNC Chapel Hill) and all images were collected under identical settings. Image analysis was conducted using QuPath software to determine VGAT, Pnoc, and Oprl1 expression in DAPI-expressing cells.⁸⁵ Circular ROIs of identical size were positioned over the left and right dLS, iLS, and vLS for each section along the A-P gradient. A background threshold was applied to all images equally and DAPI+ cells were identified for each ROI. Puncta counts were generated within DAPI+ cells for each probe (VGAT, Pnoc, Oprl1) and data were exported to excel. Data were then expressed as a percentage of VGAT+ cells for each probe and the final data represent the average of the left and right ROI for each subdivision.

Surgical procedures

Mice underwent intracranial stereotactic surgery at 8–12 weeks of age using procedures previously described.²⁷ Mice were anesthetized with isoflurane (2%) in oxygen (1–2 L/min), placed in the stereotaxic instrument (Angle 2, Leica), and hair clippers were used to prepare the scalp. After sterilization of the scalp, a vertical incision was made and bregma was identified. A burr hole was made above the injection site and a Hamilton Neuros syringe (1 μL) was used to infuse adeno-associated virus (AAV) into either the LS, LH, or SuM at a flow rate of 0.05 μL/min, followed by a 5 min diffusion period, and 5 min retraction. For fiber photometry (Figure 2), electrophysiology (Figure 3), chemogenetics (Figure 4), optogenetics (Figure 4), and circuit mapping experiments (Figure 5), mice received bilateral infusion (0.3 μL/side) of AAV constructs into the LS (A-P: +0.8, M-L: +/− 0.3, D-V: −3.8), including: AAV8-hSyn-FLEX-jGCaMP7f (Addgene: 104492); AAV8-hSyn-DIO-mCherry (Addgene: 50459), AAV8-hSyn-DIO-hM4Di-mCherry (Addgene: 44362), AAV8-hSyn-DIO-hM3Dq-mCherry (Addgene: 44361), AAV8-EF1a-hSyn-DIO-synaptophysin-mCherry (MIT Vector core), or AAV8-EF1a-DIO-ChR2-mCherry (Addgene: 20297). Fibers were positioned above the LS (A-P: 0.6; M-L: +/− 0.5; D-V: −2.7) for photometry and optogenetic experiments and secured to the skull using Metabond dental cement per the manufacturer provided protocol.⁴⁴ For NOP deletion experiments (Figure 6), mice received bilateral infusion (0.3 μL/side) of AAV8-hSyn-cre-GFP (UNC Vector Core) or AAV8-hSyn-GFP (UNC Vector Core) into the LS (A-P: +0.8, M-L: +/− 0.3, D-V: −3.8), LH (A-P: −1.0, M-L: +/−1.2, D-V: −5.55), or a unilateral infusion into the SuM (5° angle; A-P: −2.8, M-L: +/−0.3, D-V: −5.1). For microinjection studies (Figure 7), bilateral guide cannula (RWD; 0.6mm center to center made to match microinjectors with a 0.5mm projection) were positioned above the LS (A-P: 0.7; M-L: +/− 0.3; D-V: −2.5) and secured to the skull using Metabond dental cement. Mice were monitored daily post-surgery and received ketoprofen injections until pre-operative body weigh was restored. All mice were given at least 1 week of recovery after surgery prior to habitation for behavioral testing, and all experiments were conducted no sooner than 4 weeks after surgery to ensure sufficient viral expression.

Drinking procedures

Alcohol was prepared weekly and diluted in tap water from a 95% stock to a final concentration of 20% (v/v). Alcohol drinking consisted of two procedures that model binge-like alcohol consumption. Electrophysiology (Figure 3) used the well-established ‘‘drinking in the dark’’ (DID) procedure,^86,87 consisting of 2 h limited-access to either alcohol or water, 3 h into the dark cycle for 3 consecutive days. The 4^th day extended the drinking session to a 4 h period, and mice remained undisturbed in their cage for 3 days before starting the cycle again. This was repeated for 3 consecutive weeks, tissue was collected, and electrophysiology performed, 24 h after the final 4 h drinking session. Chemogenetic (Figure 4), genetic deletion (Figure 6), systemic NOP antagonist (Figure 7), and microinjection studies (Figure 7) used a modified DID procedure consisting of 2 h limited access to alcohol, 3 h into the dark cycle, for 5 consecutive days.²⁷ This modification was made to accommodate within-subjects testing during the 3^rd cycle of DID drinking, and produced mean BACs >80 mg/dL (Figures S3G, S4G and S5G). Testing of water drinking occurred over a 24 h period and sucrose (1%; w/v) drinking was tested over 2 weeks (Mon-Fri) in the 2 h limited-access DID model described above. All fluid bottles were weighed immediately after the drinking session and adjusted for spillage. Data are represented as g of alcohol consumed per kg of body weight (g/kg) for all alcohol drinking studies. Water and sucrose intake are presented as mL of solution consumed per kg of body weight (mL/kg). All animals were weighed weekly (at minimum) for the duration of the experiments.

Fiber photometry hardware and signal processing

Neurophotometrics Inc equipment was used to record GCaMP7f-Ca transients from LS^Pnoc during various behaviors (Figure 2).^44,88 Briefly, hardware consisted of an LED driver box producing 470 and 415 nM light interleaved at 40 fps that was reflected through a dichroic mirror after bandpass filtering and collimation. LED light was focused through a 20× objective into a multibranch cord and output was measured and adjusted to ensure ~50 μW output from the fiber tip for both 470 and 415 wavelengths. The emitted fluorescent signal was bandpass filtered and captured using a CCD camera at 40 fps. Bonsai software was used to trigger a USB camera to capture behavior aligned to the photometry signal and generate manual timestamps. Published MATLAB code was used to deinterleave the 470 and 415 nm signals and subtract background florescence.⁴⁴ Here, the signal was fit to a bioexponential curve to correct for baseline drift and ∆F/F (%) was then generated (100*(signal-fitted signal)/(fitted signal)) for 470 and 415 signals. A z-scored signal was then calculated based on the entire recording session for the 470 output and fit using non-negative robust linear regression. The 415 output was then fit to the z-scored 470 output and subtracted for motion correction. The z-scored output for GCaMP7f was then aligned to timestamps generated for each behavior. The Z score for each behavioral test was plotted 5 s before and after bout or stimulus onset (with the exception of footshock given the prolonged decay time and restraint given the 1 min test duration), and an average Z score was used for repeated behaviors or stimuli during a session (Ex: bouts of drinking). For analysis, the average Z score during the pre-bout interval was compared to the average post-bout Z score for all behavior/stimuli.

Fiber photometry behavioral testing

A total of N = 17 mice were used for all fiber photometry experiments (Figure 2). N = 4 were excluded for a lack of viral expression or missed fiber placements resulting in a final N = 13 (7 males and 6 females). After surgery and recovery, mice were thoroughly habituated to patch cord tethering in Noldus Phenotyper boxes. Mice were connected to a dummy patch cord and allowed to explore the box for 30 min/day for at least 2 weeks prior to testing with food and water provided. For testing, mice were moved from the colony and sat undisturbed for no less than 1 h prior to testing. A USB camera was used to record all behaviors and positioned for clear, unobstructed observation of behavior, and timestamps were manually recorded.

Drinking experiments were conducted during the dark cycle, under red light, in a modified home cage containing fresh cobb bedding with an open bonnet top. The home cage water bottle was removed prior to testing resulting in an acute 1–2 h period of water deprivation for drinking experiments to encourage fluid consumption during testing. After acclimation to the testing room, mice were tethered to the fiber cable and placed into the modified home cage. Mice acclimated to the home cage for 2 min then a single bottle containing alcohol (20% v/v), water, or sucrose (1%) was introduced. Mice were observed for up to 30 min and were returned to the home cage. Bouts of drinking lasting at least 5 s were analyzed and an average Z score was generated. The average was calculated for 3 bouts (when 3 bouts were observed) and included the 5 s prior to bout onset and 5 s during active drinking. Similarly, consumption of a palatable food reward, high fat diet (HFD; Research Diets Cat# D12492), was tested in the same modified home cage, where a single HFD pellet was introduced into the center of the cage after a 2 min acclimation period. Investigation of the pellet and bouts of eating were analyzed that lasted more than 5 s. An average Z score was generated for the 5 s before onset and during HFD consumption for the first 3 bouts meeting this criteria.

Air puff testing was conducted in an open field arena measuring 50 × 50 cm. The arena was illuminated by red light in an otherwise dark room with a camera positioned directly overhead. After tethering, mice were placed in the center of the open field and allowed to acclimate for 2 min. An air puff was then applied 3 times to the dorsal coat for roughly 500 ms, and each exposure was separated by a 1 min interval. An average Z score of the 3 air puff stimuli was used for the final analysis, consisting of the 5 s prior to air puff onset and 5 s immediately following.

Tone presentation and footshock testing were conducted in a Med Associates modular mouse chamber equipped with a quick change grid floor and waste pan. Mice were tethered and placed into the chamber for 2 min prior to testing. Recording began with a 3 min free exploration period followed by a novel tone (30 s) presentation. The tone presentation co-terminated with a foot-shock (0.6 mA for 2 s) and was repeated 5× with a 90 s inter-shock interval. A Z score was generated for the initial tone presentation and final tone presentation, and an average Z score was generated for the 5× shock stimuli. The Z score was calculated for the 5 s before and after tone onset. For footshock, the Z score was calculated for 2 s prior to tone onset and 8 s after to capture the long decay kinetics of the signal.

Splash testing was conducted in the modified home cage based on procedures from Quadir and colleagues.⁸⁹ Mice were tethered and placed in the modified home cage under red light after 2 min acclimation. Mice were then exposed to a spray of sucrose (10% w/v; in DI water), delivered from an atomizer (100 μL) to the dorsal coat. Mice were observed for 10 min and instances of grooming were recorded. A Z score was generated for the spray exposure and an average Z score was generated for 3 (when observed) instances of grooming lasting at least 5 s. The Z score was calculated for the 5 s proceeding spray onset and the 5 s immediately after. Grooming z-scores were calculated for the 5 s proceeding grooming onset and the first 5 s of the grooming behavior itself.

Elevated plus maze (EPM) testing was conducted under ambient room light. Mice were brought to the testing room for a 2 h acclimation period and remained in the dark until testing. Mice were tethered and placed in the center of the EPM (75 cm). Center crossings, open arm entry, and open arm exit times were recorded for a 10 min period. Events were analyzed when time spent in the open arm was greater than 5 s and average Z score was calculated for 3 instances of open arm entry and exit. Z-scores were calculated for the 5 s period proceeding arm entry and exit, and the 5 s period that followed in either zone.

An acute restraint test was conducted in the home cage, in the dark, under red light. Mice were tethered and allowed to explore the home cage for 5 min prior to testing. Mice were then restrained by swiftly securing the tail scruffing the subject for a 1 min period. Mice were then softly released back into the home cage where they explored freely for 1 min. A Z score was generated reflecting the 20 s period prior and during restraint and the 20 s period prior to and after release.

Electrophysiology

Tissue for all electrophysiology experiments (Figures 3, 5, and 7) was rapidly extracted from deeply anesthetized mice (isolflurane induction chamber, 2%), coronal sections were collected at 250 μM on a vibratome (Leica VT100 S) in ice-cold oxygenated sucrose (194 mM, NaCl: 20 mM, KCl: 4.4 mM, CaCl₂: 2 mM, MgCl₂: 1 mM, NaH₂PO₄: 1.2 mM, glucose: 10 mM, and NaHCO₃: 26 mM), and transferred into oxygenated aCSF (NaCl: 124 mM, KCl: 4.4 mM, NaH₂PO₄: 1 mM, MgSO₄: 1.2 mM, D-glucose: 10 mM, CaCl₂: 2 mM, and NaHCO₃: 26 mM) in a heated water bath (32°C–35°C). After a 30-min equilibration period, tissue was transferred to a submerged recording chamber (Warner Instruments) under constant perfusion of heated aCSF (30°C) at a flow rate of 2 mL/min. Cells were visualized with an Olympus BX51WI microscope and mCherry-expressing LS^Pnoc cells were identified with a CoolLED pE-100 (Andover, UK) optical stimulator. Thin walled borosilicate glass pipettes were pulled (P-97, Sutter Instruments) to 3–4 MΩ and filled with filtered, ice-cold, K-gluconate (135 mM, NaCl: 5 mM, MgCl2: 2 mM, HEPES: 10 mM, EGTA: 0.6 mM, Na2ATP: 4 mM, Na2GTP: 0.4 mM, pH 7.3, 289–292 mOsm) or CsCl (cesium methanesulfonate: 134 mM, KCl: 10 mM, MgCl2: 1 mM, EGTA: 0.2 mM, MgATP: 4 mM, Na2GTP: 0.3 mM, phosphocreatine: 20 mM, pH 7.3, 285–290 mOsm; with 1 mg/mL QX-314). Data acquisition, digitization (10 kHz), and filtering (3 kHz) occurred through an Axon Multiclamp 700B amplifier (Molecular Devices). Cells were discarded in the event that a change in Ra exceeded 20% of the initial value.

For excitability experiments (Figure 3), coronal tissue sections (250 μM) were collected 24 h after the final drinking 4 h DID drinking session (N = 23). Recordings were collected from 3 cells for each mouse from N = 5/sex/drinking history. Three mice were excluded for lack of viral expression resulting in a final N = 20. Fluorescent cells within the LS of Pnoc-cre mice expressing AAV8-hSyn-DIO-mCherry were targeted (CoolLED pE-100, Olympus; 550 nm) and recordings began in voltage-clamp mode after a 5 min equilibration period, with membrane potential held at −70 mV with K-gluconate internal. Capacitance (Cm), input resistance (Rm), and access resistance (Ra) were recorded and excitability experiments were conducted in current-clamp mode. After a 2 min equilibration period with no holding current, resting membrane potential (RMP) and spontaneous firing activity were recorded from a 2 min gap-free recording (1 min epoch analyzed from 30s to 90s). Current was then applied to hold the membrane potential at −70 mV (cells were excluded if the current necessary to hold at −70 mV exceeded 100 pA), and rheobase and voltage-current (V-I) plot protocols were run. The rheobase protocol consisted of ramping 100 pA steps and the V-I plot protocol consisted of 20 pA current steps (200 ms) starting at −100 pA and ending at 280 pA. Lastly, cells were returned to voltage-clamp mode and Cm, Rm, and Ra were recorded. Analysis was the conducted in pClamp (Molecular Devices) and EasyElectrophysiology.

For ChR2-assisted circuit mapping experiments (Figure 5), coronal tissue slices containing the LS, LH, or SuM were collected at 250 μM from N = 15 mice (8 male and 7 female, N = 1–3 cells/region/mouse) expressing AAV8-hSyn-DIO-ChR2-mCherry in LS^Pnoc. Four mice were excluded for lack of detectable fluorescent cells within the LS. Recordings were conducted in voltage-clamp mode with CsCl internal with QX-314 in cells lacking mCherry expression and 490 nm pulses were delivered by a CoolLED pE-100 (Olympus) light source. After a 5 min equilibration period, blue light pulses (100 ms) were introduced at 1, 5, and 20 hz in 5 sweeps and optically-evoked post synaptic currents (oIPSC) were recorded. Oxygenated aCSF containing picrotoxin (500 μM; Abcam) was then applied to the bath under continuous perfusion for 30 min. Sweeps of blue light stimulation were applied at 1 hz once every 5 min for the duration. Representative traces were selected under aCSF perfusion, and after 20 min of picrotoxin application. Additional experiments were conducted in Pnoc-cre mice expressing AAV8-hSyn-DIO-ChR2-mCherry in LS^Pnoc under the same slice preparation. Here, tissue was equilibrated in oxygenated aCSF containing CNQX (20 μM; Abcam) and AP-5 (50 μM; Abcam) to isolate oIPSCs. Recordings began after a 2 min equilibration period in voltage-clamp mode, where a paired pulse stimulus was then delivered. The paired pulse protocol consisted of a 50 ms baseline followed by two 1 ms pulses separated by 50 ms. The oIPSC amplitude was calculated by subtracting the oIPSC peak from a baseline measurement taken at 30 ms. The paired pulse ratio was then calculated as event#2/event#1. Analysis was the conducted in pClamp (Molecular Devices).

NOP antagonist studies (Figure 7) were conducted in similar fashion in current-clamp mode. Tissue was collected (250 μM coronal sections) from the LS of experimentally naive C57BL/6J mice (N = 9; 5 males and 4 females; N = 1–2 cells/mouse). Recordings were collected after at least 30 min of constant perfusion of heated and oxygenated solutions. After a 2 min equilibration period in current-clamp mode with no holding current, a baseline period was recorded for 4 min period and RMP was calculated for the average RMP of the 3–4 min period. Drug was bath applied for an 11 min period and membrane potential was calculated after 8 min of constant perfusion. RMP was calculated for the average RMP of the 12–13 min period. Drug perfusion experiments were conducted in two configurations: 1) aCSF followed by nociceptin (300 nM; Tocris) or 2) SB-612111 (1 μM; Tocris) pre-treatment followed by a cocktail of SB-612111 (1 μM) and nociceptin (300 nM). SB-612111 was diluted in DMSO and stocks (10 mM) were stored at −20 c for no more than 1 week prior to use. SB-612111 was diluted in aCSF to a final concentration of 1 μM in 0.1% DMSO. Nociceptin was dissolved in distilled water and prepared daily from a 1 mM stock solution and diluted to a final concentration of 300 nM in aCSF containing 0.1% DMSO. All bath solutions contained 0.1% DMSO to ensure consistency across conditions. Analysis was the conducted in pClamp (Molecular Devices).

Optogenetics and real time place testing

Pnoc-cre mice (N = 24, 12/sex) received bilateral infusion of AAV8-EF1a-DIO-ChR2-mCherry into the LS and recovered for 4 weeks prior to testing (Figure 4). A total of 3 mice were excluded for lack of viral expression or missed fiber placement, resulting in a final N = 21 (12 males and 9 females). Mice were habituated to daily handling and tethering to a dummy patch cord for no less than 1 week prior to testing. Tethering consisted of 10 min free exploration whilst tethered in a modified home cage. Testing was conducted in a custom 2-chamber box (25 cm × 50 cm) with identical sides with a cobb bedding floor. The box was cleaned with DI water and fresh bedding was introduced between subjects. Overhead video was collected and mice were tracked in real time using Ethovision software. TTL output was directed to an Arduino to generate a 20 hz frequency (2 ms pulse width) from a 473 nm laser (Shanghai Laser, ADR-800A).^23,90 Patch cords were routed through a fiber optic rotary joint (Doric; FRJ 1×1 FC-FC) and laser output (~5 mW) was checked from fiber tips prior to testing. Experimental testing consisted of a Pre-Test and Test phase. Pre-Testing was a 15 min free exploration session and time spent on either side was generated using Ethovision and was used to counterbalance laser pairing. Laser-pairing occurred the following day during a 30 min Test phase and time spent on the laser-paired side (% total time) was the main dependent variable.

Chemogenetic behavioral experiments

Pnoc-cre mice (N = 65, 33 males and 32 females) received bilateral infusion of AAV8-hSyn-DIO-hM4Di/hM3Dq/mCherry into the LS (Figure 4). A total of N = 10 were excluded for lack of viral expression resulting in final N = 55 (29 males and 26 females). After recovery from surgery, mice were habituated for 1 week prior to behavioral testing with daily intraperitoneal (ip.) injections of saline (0.9%; 20 mL/kg). Mice received daily saline injections 30 min prior to testing through each phase of the experiment (in this order: alcohol drinking, locomotor activity, and sucrose drinking).

Mice consumed alcohol for 2 weeks in the 2 h DID model prior to testing. Here, mice received a saline injection daily 30 min prior to alcohol drinking, where a single bottle of alcohol (20% v/v) replaced the home cage water bottle for a 2 h period. All testing occurred in the dark under red light in the colony room and bottles were introduced 3 h into the dark cycle. Alcohol bottles were promptly removed at the end of the drinking session, bottle weights recorded, and home cage bottle returned. In the third week, mice were tested with Clozapine-N-Oxide (CNO; 3 mg/kg; HelloBio) and saline in a within-subjects, counterbalanced design. CNO was prepared fresh daily by dissolution in saline, where saline alone served as vehicle. Testing was conducted on days 2–4 of week 3 of alcohol drinking. Mice were tested on day 5 if a subject did not receive the injection due to excessive handling or a technical difficulty occurred that precluded injection. Mice remained in the colony room undisturbed for at least 2 days prior to the initiation of open field locomotor testing.

Open field testing occurred in a 60 cm wide × 60 cm long × 40 cm deep SuperFlex open field arena (Omnitech Electronics, Accuscan) with a centralized house light (~500 lx).²⁷ Mice were brought to the testing room for 2 days where they remained undisturbed to habituate to movement from the colony room. Mice were then brought to the testing room at least 1 h prior to testing for subsequent days. For habituation, mice received an injection of saline (ip.) 30 min prior to testing and were introduced to the open field arena for 30 min. The habituation session was used to control for novelty prior to within-subjects testing with CNO, as the primary objective was to determine the effect of LS^Pnoc manipulation on locomotor activity. After habituation, mice were administered CNO or saline over the next 2 days in a counterbalanced, within-subjects design, 30-min prior to testing. This strategy was chosen allow for within-subjects testing and account for novelty and order effects that occur with repeated testing in an open field. Behavior was tracked through beam breaks and analyzed with Fusion v6.5 software. The center of the open field was defined as a 10 cm square in the center of the assay. Data were collected in 1 min bins for the 30-min testing session and distance traveled (cm) and time spent in the center of the arena (s) were analyzed. Mice were returned to the colony room where they remained undisturbed for at least 2 days prior to the initiation of sucrose drinking experiments.

Sucrose drinking was conducted in a in a similar fashion to the alcohol drinking experiment. Sucrose bottles were prepared weekly and kept refrigerated between drinking sessions. Bottles were allowed to return to room temperature prior to testing. Saline injections were administered daily 30 min prior to sucrose drinking. Sucrose bottles were placed on the home cage 3 h into the dark cycle, replacing the home cage water bottle. After 2 h, the sucrose bottles were removed, weighed, and water bottle was replaced on the home cage. Bottles were promptly covered and refrigerated. Mice were given 1 week to consume sucrose with daily saline injections. CNO challenge occurred in the second week in a within-subjects, counterbalanced design, under identical conditions to the alcohol drinking experiment.

NOP genetic deletion procedures

NOP^fl/fl mice received infusion of AAV8-hSyn-cre-GFP or AAV8-hSyn-GFP in the LS, LH, or SuM at least 4 weeks prior to the initiation of testing (Figure 6). Testing was conducted in the following order: alcohol drinking, water drinking, locomotor activity, then sucrose drinking. All experiments were conducted during the dark cycle under red light. N = 38 mice were used for LS study (20 males and 18 females), N = 34 mice were used for the LH study (18 males and 16 females), and N = 33 mice were used for the SuM study (18 males and 15 females). Eight mice were excluded for lack of viral expression.

Alcohol drinking experiments used the modified drinking in the dark model. Alcohol bottles were made fresh weekly and a single bottle containing 20% alcohol (v/v) replaced the home cage water bottle for 2 h, 3 h into the dark cycle. Bottles were weighed immediately after the drinking session and the water bottle was replaced. Drinking occurred for 5 consecutive days for 3 weeks. On the last drinking session, tail blood samples were collected immediately after alcohol bottles were removed. Blood samples were processed using an Analox Instruments AM1 Analox. Mice were returned to their home cage and were left undisturbed for at least 2 days.

Water was assessed the following week across 2 consecutive days. A single water bottle was placed on the home cage 3 h into the dark cycle and water intake was recorded every 24 h.

Locomotor activity was assessed using a 60 cm wide × 60 cm long × 40 cm deep SuperFlex open field arena (Omnitech Electronics, Accuscan) with a centralized house light (~500 lx). Mice were brought to the testing room for 2 consecutive days where they remained undisturbed and in the dark for at least 4 h before returning to the colony room. Food and water was provided for the duration. On the third day, mice were brought to the testing room and introduced to the open field arena no more than 3 h later. Mice were placed in the center of the arena and allowed to explore freely for 30 min and were returned to the home cage. The arena was cleaned with DI water between tests. Beam breaks were recorded with Fusion v6.5 software, data were collected in 1 min bins, and total distance traveled (cm) was analyzed. Mice remained undisturbed in the colony room for 2 days prior to sucrose drinking.

Sucrose drinking was measured across 2 weeks using the modified drinking in the dark procedure. A single bottle of sucrose (1%, g/v) replaced the home cage water bottle for 2 h, being introduced 3 h into the dark cycle, for 5 consecutive days. Sucrose bottles were prepared weekly and bottle weights were recorded daily.

NOP^fl/fl mice were used to confirm genetic deletion of Oprl1 mRNA from the LS, LH, and SuM (Figures S3–S5). Genetic deletion confirmation experiments for the LS used N = 5 (3 male and 2 female), LH used N = 4 (3 male and 1 female), and SuM used N = 6 (2 male and 4 female) experimentally naive mice. Four weeks after surgery, mice were deeply anesthetized with tribromoethanol (Avertin, 1 mL, ip.), the brain was extracted and frozen on dry ice, and tissue stored at −80c until sectioning. Serial sections (14 μM) were collected from each region on a cryostat (Leica CM3050 S) and mounted on Superfrost Plus slides. Slides were then processed using Advanced Cell Diagnostics RNAscope Fluorescence Multiplex Assay Kit according to the provided protocol. mRNA was visualized with TSA Vivid Dyes (AKOYA Biosciences) targeting the nociceptin receptor (Oprl1; Probe# 514301). Slides were coverslipped with Prolong Diamond mounting medium and imaged on a VS200 Slide scanner (Olympus Life Sciences). All images were collected using identical settings. Viral mediated NOP deletion was confirmed using QuPath by measuring mean Oprl1 fluorescent intensity within each region of interest. A target ROI of identical size was positioned over the LS, LH, or SuM, respectively, and mean fluorescent intensity for the Oprl1 channel was generated.

NOP antagonist behavioral procedures

C57BL/6J mice were used for all NOP antagonist behavioral experiments (Figure 7). N = 33 (17 male and 16 female) were used for the systemic NOP antagonist study and N = 23 (12 male and 11 female) were used for the microinjection study. For the systemic NOP antagonist study, mice were habituated to daily ip. injections of saline for 1 week prior to alcohol drinking in an identical fashion to chemogenetic experiments. Saline injections were then administered 30 min prior to alcohol drinking for 1 week. A single bottle of alcohol (20% v/v) replaced the home cage water bottle for 2 h, being introduced 3 h into the dark cycle, for 5 consecutive days. Alcohol bottles were removed, weighed, and the water bottle replaced. After baseline alcohol drinking was established, testing occurred the following week in a within-subjects counterbalanced design. SB-612111 (10 mg/kg, Tocris) was prepared consisting of 2.5% DMSO in saline. Vehicle was prepared as 2.5% DMSO in saline and both solutions were made immediately prior to testing. Mice were treated with vehicle and SB-612111 30 min prior to alcohol drinking in a within-subjects counterbalanced design.

For the microinjection experiment, mice received bilateral guide cannula above the LS and were allowed to recover for at least 1 week prior to habituation. All mice were habituated to the handling procedure daily for 2 weeks in the colony room where all testing occurred under red light. Habituation consisted of the microinjector dust cap being removed, dummy guide retracted, and then both were replaced. Mice were then introduced to the modified drinking in the dark procedure with microinjection habituation occurring daily 30 min prior to drinking. Mice consumed alcohol (20% v/v) for 2 weeks where bottles were presented 3 h into the dark cycle in place of the home cage water bottle for a 2 h period. Bottle weights were recorded daily and the alcohol solution was made fresh weekly. Mice were then tested with vehicle and SB-612111 in a within-subjects fashion in weeks 3 and 4. SB-612111 (Tocris) was dissolved in DMSO and a stock solution (10 mM) was stored at −20 c for no more than 1 week prior to testing. The stock solution was diluted in sterile 1xPBS to a final concentration of 100 μM (1% DMSO). Vehicle (1% DMSO in 1xPBS) and SB-612111 were prepared fresh daily and infused into the dLS 30 min prior to alcohol drinking. Microinjections were conducted using a PHD ULTRA (Harvard Apparatus) pump equipped with two 5 μL syringes (7000 series, Hamilton). Syringes were connected to microinjectors with FEP tubing and loaded with vehicle or SB-612111 immediately preceding testing. Vehicle and SB-612111 were delivered at a flow rate of 0.25 μL/min for 2 min and injectors were left in place for a 2 min diffusion period.^91,92 Injectors were retracted and dummy guide cannula replaced upon completion and drug delivery from injector tips was confirmed before and after each injection. After completion of the microinjection procedure, mice remained undisturbed in the home cage for 30 min. Alcohol bottles were then introduced for 2 h, bottle weights recorded, and water bottles returned. Mice were tested the following day in the event that a microinjection line was chewed or an injector was bent that precluded testing that day.

Immunohistochemistry

Tissue from fiber photometry (Figure 2) optogenetic and chemogenetic behavior (Figure 4), LS^Pnoc projection mapping (Figure 5), NOP genetic deletion (Figure 6), and NOP antagonist microinjection (Figure 7) was collected for immunohistochemistry after testing concluded.²⁷ Mice were deeply anesthetized and perfused with cold PBS (15 mL) followed by paraformaldehyde (PFA, 4%; 15 mL). Tissue was extracted, post-fixed in PFA overnight and moved to 1xPBS for at least 24 h prior to sectioning. Tissue was sectioned on a vibratome (VT 1200s, Leica Biosystems) at 40 μM and sections stored in PBS with azide (0.02%). Slices for photometry, optogenetics, genetic deletion, and microinjection studies were mounted directly on SuperFrost Plus slides, coverslipped with Prolong Diamond with DAPI, and imaged on a Keyence microscope. Immunohistochemisty was used to amplify the mCherry tag for chemogenetic and synaptophysin tracing studies. Briefly, tissue was permeabilized in Triton X-100 (0.5%, 30 min) in PBS and blocked with normal donkey serum (NDS; 10%; 1 h). Tissue was then incubated overnight at room temperature on a rocker in the primary antibody (mouse anti-RFP, 1:500; Rockland) in NDS. After washing the following day, tissue was incubated in the Alexa Fluor 594 secondary antibody (1:200; donkey anti-mouse; Jackson Immuno) in 0.1% Triton X-100 for 2 h. Tissue was then mounted on SuperFrost Plus slides and coverslipped with Prolong Diamond mounting media. Images were collected on a Keyence microscope at 4x, 10x, and 20x magnification.

QUANTIFICATION AND STATISTICAL ANALYSIS

All statistical analyses were conducted using SPSS software (v24) and GraphPad Prism 9 with a significance threshold set to 0.05. All significant main effects, factor interactions, t-statistics, and linear regression equations are reported in Table S1. Detailed descriptions of each experiment, including N, can be found in the method details section and statistical methods used for analysis are provided below.

ANOVA was used for analysis of the RNAscope data in Figure 1. The initial analysis included sex (male, female) as a between subjects variable, and A-P gradient (0.98, 0.74, 0.5, 0.26), D-V gradient (dLS, iLS, vLS), and probe (Pnoc, Oprl1) as within subjects factors. The main dependent variable was the percent of VGAT+ cells expressing the given probe for each ROI. Data were then collapsed as a function of sex for further analysis because there was neither a main effect or factor interaction with sex. Separate ANOVAs were used for D-V analysis using probe and A-P as a within subjects factors (Figures 1D–1F). ANOVA was also used for each probe analysis with A-P and D-V as within subjects factors (Figures 1G–1I). Sidak’s correction was used for post hoc analysis.

Fiber photometry experiments (Figure 2) were analyzed by ANOVA and included sex as a between subjects variable. Time (pre, post bout/stimulus average Z score) was the within subjects factor and average Z score during the pre and post bout interval was the main dependent variable. Post hoc analysis were planned comparisons as a function of bout (pre, post) using Sidak’s correction.

For electrophysiology experiments in Figure 3, ANOVA was used where sex and drinking history (water, alcohol) were between subjects variables, and current was a within-subjects factor. The dependent variables were input resistance (Ohm), capacitance (pF), resting membrane potential (mV), number of action potentials in a 1 min period, rheobase (pA), and action potentials per step. Sidak’s correction was used for post hoc analysis.

The optogenetic behavioral experiment (Figure 4) was analyzed by ANOVA. Sex and AAV (ChR2, mCherry) were between subjects factors and day (PreTest, Test) was a within subjects variable, with percent time spent on the laser-paired side serving as the main dependent variable. Data were collapsed across sex given the lack of main effect or factor interaction. Subsequent analysis included AAV and day as factors and percent time spent on the laser paired side was the dependent variable. Sidak’s correction was used for post hoc analysis. The chemogenetic experiment (Figure 4) was also analyzed by ANOVA and included sex and AAV (hM4Di, hM3Dq, and mCherry) as between subjects factors, drug treatment (vehicle, CNO) as a within subjects variable, and alcohol intake (g/kg), sucrose intake (mL/kg), distance traveled (cm), and time (s) spent in the center of an open field were the main dependent variables. Post hoc analysis were planned comparisons as a function of drug using Sidak’s correction.

ChR2-assisted circuit mapping studies (Figure 5) used both t test and ANOVA for analysis. Amplitude (pA) was analyzed by unpaired, two-tailed, t test to asses sex differences in single-pulse-evoked oIPSC. ANOVA was used paired-pulse analysis and included sex as a between subjects variable, time (first pulse, second pulse) as a repeated measure, and oIPSC (pA) was the main dependent variable. Sidak’s correction was used for post hoc analysis.

ANOVA was used for NOP genetic deletion studies (Figure 6) and included sex and AAV (Cre, GFP) as the main factors with alcohol intake (g/kg), sucrose intake (mL/kg), and water intake (mL/kg) serving as the dependent variables. Planned comparisons post hoc analysis was performed as a function of AAV using Sidak’s correction. In Figures S3–S5, two-tailed t-tests were used for NOP genetic deletion validation experiments and ANOVA was used to assess alcohol intake across weeks. ANOVA included sex and AAV as between subjects factors and time (Week 1, 2, 3) as a within subjects comparison. Post hoc analysis used Sidak’s correction for planned comparisons for AAV by week. Linear regression was used to determine the association between alcohol intake and BAC. ANOVA was used to assess BAC (mg/dL), distance traveled (cm), and body weight (g). Post hoc analysis used Sidak’s correction for planned comparisons as a function of AAV.

Systemic and LS microinjection experiments (Figure 7) were analyzed by ANOVA and included sex as a between subjects factor, drug (Vehicle, SB-612111) as a within subjects factor, and the main dependent variable was alcohol intake (g/kg). Planned comparisons post hoc analysis was performed as a function of drug using Sidak’s correction. NOP slice electrophysiology experiments were also analyzed by ANOVA and included sex and antagonist (SB-612111, SB-612111+nociceptin) as a between subjects factor, agonist (aCSF, nociceptin) as a within subjects factor, and membrane potential (mV) and change in membrane potential (mV) were the dependent variables. Post hoc analysis were planned comparisons as a function of drug.

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-RFP (mouse) Monoclonal Antibody	Rockland	Cat # 200-301-379
Alexa Fluor® 594 AffiniPure™ Donkey Anti-Mouse IgG (H + L)	Jackson ImmunoResearch	Cat# 715-585-150
Bacterial and virus strains
pGP-AAV-syn-FLEX-jGCaMP7f-WPRE	Addgene	Cat# 104492-AAV8
pAAV-hSyn-DIO-mCherry	Addgene	Cat# 50459-AAV8
pAAV-hSyn-DIO-hM4D(Gi)-mCherry	Addgene	Cat# 44362-AAV8
pAAV-hSyn-DIO-hM3D(Gq)-mCherry	Addgene	Cat# 44361-AAV8
AAV8-EF1a-hSyn-DIO-synaptophysin-mCherry	MIT Viral Gene Transfer Core	N/A
pAAV-EF1a-double floxed-hChR2(H134R)-mCherry-WPRE-HGHpA	Addgene	Cat# 20297-AAV8
AAV8-hSyn-cre-GFP	UNC Vector Core	N/A
AAV8-hSyn-GFP	UNC Vector Core	N/A
Chemicals, peptides, and recombinant proteins
Clozapine N-oxide (CNO) dihydrochloride	HelloBio	Cat# HB6149
QX-314 bromide (N-Ethyllidocaine bromide)	Abcam	Cat# ab120117
Picrotoxin	Abcam	Cat# ab120315
CNQX disodium salt	Abcam	Cat# ab120044
DL-AP5 sodium salt	Abcam	Cat# ab120271
SB 612111 hydrochloride	Tocris	Cat# 3573
Nociceptin	Tocris	Cat# 0910
Critical commercial assays
RNAscope™ Multiplex Fluorescent V2 Assay	ACD Bio	Cat# 323110
TSA Vivid™ Fluorophore Kit 520	Biotechne	Cat# 7523
TSA Vivid™ Fluorophore Kit 570	Biotechne	Cat# 7526
TSA Vivid™ Fluorophore Kit 650	Bioteche	Cat# 7527
RNAscope™ Probe- Mm-Slc32a1	ACD Bio	Cat# 319191
RNAscope™ Probe- Mm-Pnoc-C2	ACD Bio	Cat# 437881-C2
RNAscope™ Probe- Mm-Oprl1-C3	ACD Bio	Cat# 514301-C3
Experimental models: Organisms/strains
C57BL/6J	The Jackson Laboratory	Strain #:000664 RRID:IMSR_JAX:000664
STOCK Pnoctm1.1(cre)Mrbr/J	The Jackson Laboratory	Stock# 034278 RRID:IMSR_JAX:034278 Parker et al. 2019
B6.129(Cg)-Oprl1tm1.1Mrbr/J	The Jackson Laboratory	Stock# 036308 RRID:IMSR_JAX:036308 Parker et al. 2019
Software and algorithms
GraphPad Prism 9	GraphPad	N/A
IBM SPSS Statistics 24	IBM	N/A
MATLAB	Mathworks	N/A
QuPath 0.5.1	QuPath	N/A
Easy Electrophysiology v2.5	Easy Electrophysiology Ltd	N/A
pClamp 10.7	Molecular Devices	N/A
Ethovision XT	Noldus	N/A
Accuscan, Fusion v6.5	Omnitech Electronics	N/A

Highlights.

• LS^Pnoc are sensitive to a history of binge drinking and regulate alcohol intake

• LS^Pnoc form GABAergic synapses in the LS, LH, and SuM

• Genetic deletion of NOP from LS^Pnoc terminal fields decreases alcohol drinking

• Systemic or intra-LS NOP antagonist decreases alcohol intake