Insula Dynorphin and Kappa Opioid Receptor Systems Regulate Alcohol Drinking in a Sex-Specific Manner in Mice.

Alcohol use disorder is complex and multifaceted, involving the coordination of multiple signaling systems across numerous brain regions. Previous work has indicated that both the insular cortex and dynorphin (DYN)/kappa opioid receptor (KOR) systems contribute to excessive alcohol use. More recently, we identified a microcircuit in the medial aspect of the insular cortex that signals through DYN/KOR. Here, we explored the role of insula DYN/KOR circuit components on alcohol intake in a long-term intermittent access (IA) procedure. Using a combination of conditional knock-out strategies and site-directed pharmacology, we discovered distinct and sex-specific roles for insula DYN and KOR in alcohol drinking and related behavior. Our findings show that insula DYN deletion blocked escalated consumption and decreased the overall intake of and preference for alcohol in male and female mice. This effect was specific to alcohol in male mice, as DYN deletion did not impact sucrose intake. Further, insula KOR antagonism reduced alcohol intake and preference during the early phase of IA in male mice only. Alcohol consumption was not affected by insula KOR knockout in either sex. In addition, we found that long-term IA decreased the intrinsic excitability of DYN and deep layer pyramidal neurons (DLPNs) in the insula of male mice. Excitatory synaptic transmission was also impacted by IA, as it drove an increase in excitatory synaptic drive in both DYN neurons and DLPNs. Combined, our findings suggest there is a dynamic interplay between excessive alcohol consumption and insula DYN/KOR microcircuitry.SIGNIFICANCE STATEMENT The insular cortex is a complex region that serves as an integratory hub for sensory inputs. In our previous work, we identified a microcircuit in the insula that signals through the kappa opioid receptor (KOR) and its endogenous ligand dynorphin (DYN). Both the insula and DYN/KOR systems have been implicated in excessive alcohol use and alcohol use disorder (AUD). Here, we use converging approaches to determine how insula DYN/KOR microcircuit components contribute to escalated alcohol consumption. Our findings show that insula DYN/KOR systems regulate distinct phases of alcohol consumption in a sex-specific manner, which may contribute to the progression to AUD.

Kappa opioid receptor and dynorphin signaling in the central amygdala regulates alcohol intake.

Excessive alcohol drinking has been shown to modify brain circuitry to predispose individuals for future alcohol abuse. Previous studies have implicated the central nucleus of the amygdala (CeA) as an important site for mediating the somatic symptoms of withdrawal and for regulating alcohol intake. In addition, recent work has established a role for both the Kappa Opioid Receptor (KOR) and its endogenous ligand dynorphin in mediating these processes. However, it is unclear whether these effects are due to dynorphin or KOR arising from within the CeA itself or other input brain regions. To directly examine the role of preprodynorphin (PDYN) and KOR expression in CeA neurons, we performed region-specific conditional knockout of these genes and assessed the effects on the Drinking in the Dark (DID) and Intermittent Access (IA) paradigms. Conditional gene knockout resulted in sex-specific responses wherein PDYN knockout decreased alcohol drinking in both male and female mice, whereas KOR knockout decreased drinking in males only. We also found that neither PDYN nor KOR knockout protected against anxiety caused by alcohol drinking. Lastly, a history of alcohol drinking did not alter synaptic transmission in PDYN neurons in the CeA of either sex, but excitability of PDYN neurons was increased in male mice only. Taken together, our findings indicate that PDYN and KOR signaling in the CeA plays an important role in regulating excessive alcohol consumption and highlight the need for future studies to examine how this is mediated through downstream effector regions.

Inhibitory transmission in the bed nucleus of the stria terminalis in male and female mice following morphine withdrawal.

The United States is experiencing an opioid crisis imposing enormous fiscal and societal costs and driving the staggering overdose death rate. While prescription opioid analgesics are essential for treating acute pain, cessation of use in individuals with a physical dependence induces an aversive withdrawal syndrome that promotes continued drug use to alleviate/avoid these symptoms. Additionally, repeated bouts of withdrawal often lead to an increased propensity for relapse. Understanding the neurobiology underlying withdrawal is essential for providing novel treatment options to alleviate physiological and affective components accompanying the cessation of opiate use. Here, we administered morphine and precipitated withdrawal with naloxone to investigate behavioral and cellular responses in C57BL/6J male and female mice. Following 3 days of administration, both male and female mice demonstrated sensitized withdrawal symptoms. Since the bed nucleus of the stria terminalis (BNST) plays a role in mediating withdrawal-associated behaviors, we examined plastic changes in inhibitory synaptic transmission within this structure 24 hours following the final precipitated withdrawal. In male mice, morphine withdrawal increased spontaneous GABAergic signaling compared with controls. In contrast, morphine withdrawal decreased spontaneous GABAergic signaling in female mice. Intriguingly, these opposing GABAergic effects were contingent upon activity-dependent dynamics within the ex vivo slice. Our findings suggest that male and female mice exhibit some divergent cellular responses in the BNST following morphine withdrawal, and alterations in BNST inhibitory signaling may contribute to the expression of behaviors following opioid withdrawal.

Ethanol-seeking behavior is expressed directly through an extended amygdala to midbrain neural circuit.

Abstinent alcohol-dependent individuals experience an enduring sensitivity to cue-induced craving and relapse to drinking. There is considerable evidence indicating that structures within the midbrain and extended amygdala are involved in this process. Individually, the ventral tegmental area (VTA) and the bed nucleus of the stria terminalis (BNST) have been shown to modulate cue-induced ethanol-seeking behavior. It is hypothesized that cue-induced seeking is communicated through a direct projection from the BNST to VTA. In the current experiments, an intersectional viral strategy was used in DBA/2J mice to selectively target and inhibit BNST projections to the VTA during a test of ethanol conditioned place preference (CPP). Inhibitory designer receptors exclusively activated by designer drugs (hM4Di DREADDs) were expressed in VTA-projecting BNST (BNST-VTA) cells by infusing a retrograde herpes-simplex virus encoding cre recombinase (HSV-Cre) into VTA and a cre-inducible adeno-associated virus encoding hM4Di (AAV-DIO-hM4Di) into BNST. Before testing the expression of preference, clozapine-N-oxide (CNO) was peripherally administered to activate hM4Di receptors and selectively inhibit these cells. Ethanol CPP expression was blocked by CNO-mediated inhibition of BNST-VTA cells. A follow-up study revealed this effect was specific to CNO activation of hM4Di as saline- and CNO-treated mice infused with a control vector (HSV-GFP) in place of HSV-Cre showed significant CPP. These findings establish a role for a direct BNST input to VTA in cue-induced ethanol-seeking behavior.

Periaqueductal gray/dorsal raphe dopamine neurons contribute to sex differences in pain-related behaviors.

Sex differences in pain severity, response, and pathological susceptibility are widely reported, but the neural mechanisms that contribute to these outcomes remain poorly understood. Here we show that dopamine (DA) neurons in the ventrolateral periaqueductal gray/dorsal raphe (vlPAG/DR) differentially regulate pain-related behaviors in male and female mice through projections to the bed nucleus of the stria terminalis (BNST). We find that activation of vlPAG/DRDA+ neurons or vlPAG/DRDA+ terminals in the BNST reduces nociceptive sensitivity during naive and inflammatory pain states in male mice, whereas activation of this pathway in female mice leads to increased locomotion in the presence of salient stimuli. We additionally use slice physiology and genetic editing approaches to demonstrate that vlPAG/DRDA+ projections to the BNST drive sex-specific responses to pain through DA signaling, providing evidence of a novel ascending circuit for pain relief in males and contextual locomotor response in females.

Alcohol drinking alters stress response to predator odor via BNST kappa opioid receptor signaling in male mice.

Maladaptive responses to stress are a hallmark of alcohol use disorder, but the mechanisms that underlie this are not well characterized. Here, we show that kappa opioid receptor signaling in the bed nucleus of the stria terminalis (BNST) is a critical molecular substrate underlying abnormal stress responses to predator odor following heavy alcohol drinking. Exposure to predator odor during protracted withdrawal from intermittent alcohol drinking resulted in enhanced prefrontal cortex (PFC)-driven excitation of prodynorphin-containing neurons in the BNST. Furthermore, deletion of prodynorphin in the BNST and chemogenetic inhibition of the PFC-BNST pathway restored abnormal responses to predator odor in alcohol-exposed mice. These findings suggest that increased corticolimbic drive may promote abnormal stress behavioral responses to predator odor during protracted withdrawal. Various nodes of this PFC-BNST dynorphin-related circuit may serve as potential targets for potential therapeutic mediation as well as biomarkers of negative responses to stress following heavy alcohol drinking.

The connection between stress and alcohol use is highly complex. On one hand, there is the idea of having a drink to 'steady the nerves'. On the other hand, in alcoholics, abnormal responses to stress often accompany heavy drinking. In this case, it remains unknown whether stress cause excessive drinking, or vice versa. Areas of the brain that normally help respond to stress work differently in long-term, heavy drinkers. One example is a structure called the bed nucleus of the stria terminalis (BNST), which is over-active in anxiety disorders and is also associated with some of the symptoms of alcohol withdrawal. The mechanism behind both problems is thought to be a specific 'signaling system' that is activated by a small molecule called dynorphin. Previous research into the effects of dynorphin was performed either in the context of alcoholism or of anxiety disorders, but it was not known if there was a connection between the two. Therefore, Hwa et al. wanted to determine how prolonged alcohol use might affect responses to stress, and whether dynorphin signaling plays a role. To model long-term alcohol use in the laboratory, a group of mice was given free access to alcohol every other day, ensuring that they developed the mouse equivalent of a drinking habit. After six weeks, these 'heavy drinkers' went through a period of abstinence, mimicking alcohol withdrawal. Then, the mice were stressed by exposing them to a chemical that smelled like a fox, one of the mice's predators in the wild. When mice smell predators, they normally respond by fleeing from the area and digging up debris to defend itself. As expected, the control mice in this study, which did not drink alcohol, did just that. In contrast, the heavy drinkers largely ignored the predator scent by not digging and even spent time hanging around the area that smelled like the predator. Blocking dynorphin-induced signaling in the alcoholic mice, either using a drug or by deleting the gene that codes for dynorphin, reset the stress response to normal, allowing these mice to avoid the predator and dig as normal. Furthermore, measuring the electrical activity in the brain revealed that the BNST was abnormally active in alcohol-drinking mice, driven by signals from another part of the brain, the prefrontal cortex. This reveals part of the circuitry in the brain responsible for the connection between alcohol withdrawal and the stress response. These results shed new light on the biological mechanisms underpinning the relationship between alcohol use and stress. In the future, these could be used to determine why heavy drinking can overlap with anxiety disorders, or to develop new treatments that would help recovering alcoholics cope better with stress.

The kappa opioid receptor modulates GABA neuron excitability and synaptic transmission in midbrainprojections from the insular cortex.

As an integrative hub, the insular cortex (IC) translates external cues into interoceptive states that generate complex physiological, affective, and behavioral responses. However, the precise circuit and signaling mechanisms in the IC that modulate these processes are unknown. Here, we describe a midbrain-projecting microcircuit in the medial aspect of the agranular IC that signals through the Gαi/o-coupled kappa opioid receptor (KOR) and its endogenous ligand dynorphin (Dyn). Within this microcircuit, Dyn is robustly expressed in layer 2/3, while KOR is localized to deep layer 5, which sends a long-range projection to the substantia nigra (SN). Using ex vivo electrophysiology, we evaluated the functional impact of KOR signaling in layer 5 of the IC. We found that bath application of dynorphin decreased GABA release and increased glutamate release on IC-SN neurons, but did not alter their excitability. Conversely, dynorphin decreased the excitability of GABA neurons without altering synaptic transmission. Pretreatment with the KOR antagonist nor-BNI blocked the effects of dynorphin in IC-SN neurons and GABA neurons, indicating that the changes in synaptic transmission and excitability were selectively mediated through KOR. Selective inhibition of IC GABA neurons using a KOR-derived DREADD recapitulated these effects. This work provides insight into IC microcircuitry and indicates that Dyn/KOR signaling may act to directly reduce activity of layer 5 GABA neurons. In turn, KOR-driven inhibition of GABA promotes disinhibition of IC-SN neurons, which can modulate downstream circuits. Our findings present a potential mechanism whereby chronic upregulation of IC Dyn/KOR signaling can lead to altered subcortical function and downstream activity.

Ethanol-induced conditioned place preference and aversion differentially alter plasticity in the bed nucleus of stria terminalis.

Contextual cues associated with drugs of abuse, such as ethanol, can trigger craving and drug-seeking behavior. Pavlovian procedures, such as place conditioning, have been widely used to study the rewarding/aversive properties of drugs and the association between environmental cues and drug seeking. Previous research has shown that ethanol as an unconditioned stimulus can induce a strong conditioned place preference (CPP) or aversion (CPA) in rodents. However, the neural mechanisms underlying ethanol-induced reward and aversion have not been thoroughly investigated. The bed nucleus of the stria terminalis (BNST), an integral part of the extended amygdala, is engaged by both rewarding and aversive stimuli and plays a role in ethanol-seeking behavior. Here, we used ex-vivo slice physiology to probe learning-induced synaptic plasticity in the BNST following ethanol-induced CPP and CPA. Male DBA/2 J mice (2-3 months old) were conditioned using previously reported ethanol-induced CPP/CPA procedures. Ethanol-induced CPP was associated with increased neuronal excitability in the ventral BNST (vBNST). Conversely, ethanol-induced CPA resulted in a significant decrease in spontaneous glutamatergic transmission without alterations in GABAergic signaling. Ethanol-CPA also led to a significant increase in the paired-pulse ratio at excitatory synapses, suggestive of a decrease in presynaptic glutamate release. Collectively, these data demonstrate that the vBNST is involved in the modulation of contextual learning associated with both the rewarding and the aversive properties of ethanol in mice.

Predator odor increases avoidance and glutamatergic synaptic transmission in the prelimbic cortex via corticotropin-releasing factor receptor 1 signaling.

Acute exposure to a salient stressor, such as in post-traumatic stress disorder, can have lasting impacts upon an individual and society. To study stress in rodents, some naturalistic methods have included acute exposure to a predator odor, such as the synthetic fox odor 2,4,5, trimethyl-3-thiazoline (TMT). These experiments explore the stress-related behaviors and cortical activity induced by TMT exposure in adult male C57BL/6J mice and the influence of the stress neuropeptide corticotropin-releasing factor (CRF) on these responses. Compared to H2O and a novel odorant, vanilla, mice exposed to TMT in the home cage showed increased avoidance and defensive burying indicative of evident stress responses. Consistent with stress-induced activation of the medial prefrontal cortex (mPFC), we found that the prelimbic (PL) and infralimbic (IL) subregions of the mPFC had elevated c-Fos immunolabeling after TMT and vanilla compared to H2O. Slice physiology recordings were performed in layers 2/3 and 5 of the PL and IL, following TMT, vanilla, or H2O exposure. In TMT mice, but not vanilla or H2O mice, PL layers 2/3 showed heightened spontaneous excitatory post-synaptic currents and synaptic drive, suggesting TMT enhanced excitatory transmission. Synaptic drive in PL was increased in both TMT and H2O mice following bath application of 300 nM CRF, but only H2O mice increased excitatory currents with 100 nM CRF, suggesting dose-effect curve shifts in TMT mice. Further, systemic pretreatment with the CRF-R1 antagonist CP154526 and bath application with the CRF-R1 antagonist NBI27914 reduced excitatory transmission in TMT mice, but not H2O mice. CP154526 also reduced stress-reactive behaviors induced by TMT. Taken together, these findings suggest that exposure to TMT leads to CRF-R1 driven changes in behavior and changes in synaptic function in layer 2/3 neurons in the PL, which are consistent with previous findings that CRF-R1 in the mPFC plays an important role in predator odor-related behaviors.

Stress-Induced Alterations of Norepinephrine Release in the Bed Nucleus of the Stria Terminalis of Mice.

Stress can drive adaptive changes to maintain survival during threatening stimuli. Chronic stress exposure, however, may result in pathological adaptations. A key neurotransmitter involved in stress signaling is norepinephrine. Previous studies show that acute stress elevates norepinephrine levels in the bed nucleus of the stria terminalis (BNST), a critical node regulating anxiety and upstream of stress responses. Here, we use mice expressing channelrhodopsin in norepinephrine neurons to selectively activate terminals in the BNST, and measure norepinephrine release with optogenetics-assisted fast-scan cyclic voltammetry (FSCV). We demonstrate that while corticosterone habituates to chronic restraint stress, cFos activation of medullary norepinephrine neurons shows equivalent activation under both acute and chronic stress conditions. Mice exposed to a single restraint session show an identical optically stimulated norepinephrine release profile compared to that of unexposed mice. Mice experiencing 5 days of restraint stress, however, show elevated norepinephrine release across multiple stimulation parameters, and reduced sensitivity to the α2-adrenergic receptor (AR) antagonist idazoxan. These data are the first to examine norepinephrine release in the BNST to tonic and phasic stimulation frequencies, and confirm that repeated stress alters autoreceptor sensitivity.

DREADD Agonist 21 Is an Effective Agonist for Muscarinic-Based DREADDs in Vitro and in Vivo.

Chemogenetic tools such as designer receptors exclusively activated by designer drugs (DREADDs) are routinely used to modulate neuronal and non-neuronal signaling and activity in a relatively noninvasive manner. The first generation of DREADDs were templated from the human muscarinic acetylcholine receptor family and are relatively insensitive to the endogenous agonist acetylcholine but instead are activated by clozapine-N-oxide (CNO). Despite the undisputed success of CNO as an activator of muscarinic DREADDs, it has been known for some time that CNO is subject to a low rate of metabolic conversion to clozapine, raising the need for alternative chemical actuators of muscarinic-based DREADDs. Here we show that DREADD agonist 21 (C21) (11-(1-piperazinyl)-5H-dibenzo[b,e][1,4]diazepine) is a potent and selective agonist at both excitatory (hM3Dq) and inhibitory (hM4Di) DREADDs and has excellent bioavailability, pharmacokinetic properties, and brain penetrability. We also show that C21-induced activation of hM3Dq and hM4Di in vivo can modulate bidirectional feeding in defined circuits in mice. These results indicate that C21 represents an alternative to CNO for in vivo studies where metabolic conversion of CNO to clozapine is a concern.

Involvement of ventral tegmental area ionotropic glutamate receptors in the expression of ethanol-induced conditioned place preference.

The ventral tegmental area (VTA) is a well-established neural substrate of reward-related processes. Activity within this structure is increased by the primary and conditioned rewarding effects of abused drugs and its engagement is heavily reliant on excitatory input from structures upstream. In the case of drug seeking, it is thought that exposure to drug-associated cues engages glutamatergic VTA afferents that signal directly to dopamine cells, thereby triggering this behavior. It is unclear, however, whether glutamate input to VTA is directly involved in ethanol-associated cue seeking. Here, the role of intra-VTA ionotropic glutamate receptor (iGluR) signaling in ethanol-cue seeking was evaluated in DBA/2J mice using an ethanol conditioned place preference (CPP) procedure. Intra-VTA iGluRs α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPAR)/kainate and N-methyl-d-aspartate (NMDAR) were blocked during ethanol CPP expression by co-infusion of antagonist drugs 6,7-dinitroquinoxaline-2,3-dione (DNQX; AMPA/kainate) and d-(-)-2-Amino-5-phosphonopentanoic acid (AP5; NMDA). Compared to aCSF, bilateral infusion of low (1 DNQX+100 AP5ng/side) and high (5 DNQX+500 AP5ng/side) doses of the AMPAR and NMDAR antagonist cocktail into VTA blocked ethanol CPP expression. This effect was site specific, as DNQX/AP5 infusion proximal to VTA did not significantly impact CPP expression. An increase in activity was found at the high but not low dose of DNQX/AP5. These findings demonstrate that activation of iGluRs within the VTA is necessary for ethanol-associated cue seeking, as measured by CPP.

The bed nucleus of the stria terminalis regulates ethanol-seeking behavior in mice.

Drug-associated stimuli are considered important factors in relapse to drug use. In the absence of drug, these cues can trigger drug craving and drive subsequent drug seeking. One structure that has been implicated in this process is the bed nucleus of the stria terminalis (BNST), a chief component of the extended amygdala. Previous studies have established a role for the BNST in cue-induced cocaine seeking. However, it is unclear if the BNST underlies cue-induced seeking of other abused drugs such as ethanol. In the present set of experiments, BNST involvement in ethanol-seeking behavior was assessed in male DBA/2J mice using the conditioned place preference procedure (CPP). The BNST was inhibited during CPP expression using electrolytic lesions (Experiment 1), co-infusion of GABAA and GABAB receptor agonists muscimol and baclofen (M+B; Experiment 2), and activation of inhibitory designer receptors exclusively activated by designer drugs (hM4Di-DREADD) with clozapine-N-oxide (CNO; Experiment 3). The magnitude of ethanol CPP was reduced significantly by each of these techniques. Notably, infusion of M+B (Exp. 2) abolished CPP altogether. Follow-up studies to Exp. 3 showed that ethanol cue-induced c-Fos immunoreactivity in the BNST was reduced by hM4Di activation (Experiment 4) and in the absence of hM4Di, CNO did not affect ethanol CPP (Experiment 5). Combined, these findings demonstrate that the BNST is involved in the modulation of cue-induced ethanol-seeking behavior.

Effects of dopamine receptor antagonists on the acquisition of ethanol-induced conditioned place preference in mice.

RATIONALE: Studies support differential roles of dopamine receptor subfamilies in the rewarding and reinforcing properties of drugs of abuse, including ethanol. However, the roles these receptor subfamilies play in ethanol reward are not fully delineated. OBJECTIVE: To examine the roles of dopamine receptor subfamilies in the acquisition of ethanol-induced conditioned place preference (CPP), we pretreated animals systemically with antagonist drugs selective for dopamine D1-like (SCH-23390) and D2-like (raclopride) receptors prior to ethanol conditioning trials. METHODS: Effects of raclopride (0-1.2 mg/kg) and SCH-23390 (0-0.3 mg/kg) on the acquisition of ethanol-induced CPP were examined in DBA/2J mice (experiments 1 and 2). Based on significant effects of SCH-23390, we then determined if SCH-23390 (0.3 mg/kg) produced a place preference on its own (experiment 3). To evaluate whether SCH-23390 impaired learning, we used a conditioned place aversion (CPA) paradigm and pretreated animals with SCH-23390 (0-0.3 mg/kg) before conditioning sessions with LiCl (experiment 4). RESULTS: Whereas raclopride (0-1.2 mg/kg) did not affect acquisition, SCH-23390 (0.1-0.3 mg/kg) impaired the development of ethanol-induced CPP. SCH-23390 (0.3 mg/kg) did not produce place preference when tested alone and SCH-23390 (0.1-0.3 mg/kg) did not perturb the acquisition of LiCl-induced CPA. CONCLUSIONS: Our results support a role for dopamine D1-like but not D2-like receptors in ethanol's unconditioned rewarding effect as indexed by CPP. Blockade of D1-like receptors did not affect aversive learning in this procedure.

Effects of the novel cannabinoid CB1 receptor antagonist PF 514273 on the acquisition and expression of ethanol conditioned place preference.

The centrally expressed cannabinoid receptor (CB1) has been considered a potential therapeutic target in treating alcoholism. Though CB1 receptors have been shown to modulate primary and conditioned ethanol reward, much of this research employed animal models that require ethanol ingestion or oral routes of administration. This is problematic considering CB1 antagonist drugs have high anorectic liability and have been used clinically in the treatment of obesity. Therefore, the present study examined CB1 antagonism in DBA/2J mice using an unbiased ethanol-induced conditioned place preference (CPP) procedure, a paradigm that does not require ethanol ingestion. To evaluate the role of CB1 receptors in primary ethanol reward, the highly potent and selective novel CB1 antagonist 2-(2-chlorophenyl)-3-(4-chlorophenyl)-7-(2,2-difluoropropyl)-6,7-dihydro-2H-pyrazolo[3,4-f][1,4]oxazepin-8(5H)-one (PF 514273) was administered 30 min before place preference conditioning with a fixed dose of ethanol (acquisition). To evaluate the role of CB1 receptors in ethanol-conditioned reward, PF 514273 was administered 30 min before place preference testing (expression). Although PF 514273 reduced ethanol-stimulated and basal locomotor activity, it did not perturb the acquisition or expression of ethanol-induced CPP. Results from the present study appear inconsistent with other studies that have demonstrated a role for CB1 antagonism in ethanol reward using oral administration paradigms. Our findings suggest that CB1 antagonism may have greater involvement in consummatory behavior than ethanol reward.