Elevation of Transient Receptor Potential Vanilloid 1 Function in the Lateral Habenula Mediates Aversive Behaviors in Alcohol-withdrawn Rats.

WHAT WE ALREADY KNOW ABOUT THIS TOPIC: Chronic alcohol use and withdrawal leads to increased pain perception, anxiety, and depression. These aberrant behaviors are accompanied by increased excitatory glutamatergic transmission to, and activity of, the lateral habenula neurons.Vanilloid type 1, or TRPV1, channels are expressed in the habenula and they facilitate glutamatergic transmission. Whether TRPV1 channel plays a role in habenula hyperactivity is not clear. WHAT THIS ARTICLE TELLS US THAT IS NEW: Glutamatergic transmission in the lateral habenula was inhibited by TRPV1 channel antagonists. In vivo, local administration of TRPV1 antagonists into the lateral habenula attenuated hyperalgesia, anxiety, and relapse-like drinking in rats who chronically consumed alcohol.The data suggest that enhanced TRPV1 channel function during withdrawal may contribute to aberrant behavior that promotes relapse alcohol consumption. BACKGROUND: Recent rat studies indicate that alcohol withdrawal can trigger a negative emotional state including anxiety- and depression-like behaviors and hyperalgesia, as well as elevated glutamatergic transmission and activity in lateral habenula neurons. TRPV1, a vanilloid receptor expressed in the habenula, is involved in pain, alcohol dependence, and glutamatergic transmission. The authors therefore hypothesized that TRPV1 contributes to the changes in both the behavioral phenotypes and the habenula activity in alcohol-withdrawn rats. METHODS: Adult male Long-Evans rats (n = 110 and 280 for electrophysiology and behaviors, respectively), randomly assigned into the alcohol and water (Naïve) groups, were trained to consume either alcohol or water-only using an intermittent-access procedure. Slice electrophysiology was used to measure spontaneous excitatory postsynaptic currents and firing of lateral habenula neurons. The primary outcome was the change in alcohol-related behaviors and lateral habenula activity induced by pharmacologic manipulation of TRPV1 activity. RESULTS: The basal frequency of spontaneous excitatory postsynaptic currents and firing of lateral habenula neurons in alcohol-withdrawn rats was significantly increased. The TRPV1 antagonist capsazepine (10 µM) induced a stronger inhibition on spontaneous excitatory postsynaptic currents (mean ± SD; by 26.1 ± 27.9% [n = 11] vs. 6.7 ± 18.6% [n = 17], P = 0.027) and firing (by 23.4 ± 17.6% [n = 9] vs. 11.9 ± 16.3% [n = 12], P = 0.025) in Withdrawn rats than Naive rats. By contrast, the TRPV1 agonist capsaicin (3 µM) produced a weaker potentiation in Withdrawn than Naïve rats (spontaneous excitatory postsynaptic currents: by 203.6 ± 124.7% [n = 20] vs. 415.2 ± 424.3% [n = 15], P < 0.001; firing: 38.1 ± 14.7% [n = 11] vs. 73.9 ± 41.9% [n = 11], P < 0.001). Conversely, capsaicin's actions in Naïve but not in Withdrawn rats were significantly attenuated by the pretreatment of TRPV1 endogenous agonist N-Oleoyldopamine. In Withdrawn rats, intra-habenula infusion of TRPV1 antagonists attenuated hyperalgesia and anxiety-like behaviors, decreased alcohol consumption upon resuming drinking, and elicited a conditioned place preference. CONCLUSIONS: Enhanced TRPV1 function may contribute to increased glutamatergic transmission and activity of lateral habenula neurons, resulting in the aberrant behaviors during ethanol withdrawal.

Electroacupuncture Attenuates Hyperalgesia in Rats Withdrawn from Chronic Alcohol Drinking via Habenular Mu Opioid Receptors.

BACKGROUND: Hyperalgesia or increased sensitivity to pain is often found in alcoholics during alcohol withdrawal and may contribute to relapse drinking. Alternative therapies such as acupuncture and electroacupuncture (EA), through mechanisms involving opioid receptors, may reduce pain and substance dependence and withdrawal syndromes. The lateral habenula (LHb), an epithalamic structure rich in mu opioid receptors (MORs), is a critical target for both drugs of abuse and pain. We previously observed hyperalgesia in rats withdrawn from chronic ethanol (EtOH) drinking and found that EA at the acupoint Zusanli (ST36) reduced EtOH intake. This raised question of whether EA can alleviate hyperalgesia during alcohol withdrawal and, if so, whether the mechanism involves MORs in the LHb. METHODS: We trained male rats to drink EtOH using the intermittent access 20% EtOH 2-bottle free-choice drinking paradigm for 8 weeks, after which the alcohol supply was discontinued. We measured pain sensitivity using radiant heat (a light beam directed at the hind paw of rats) and compared the paw withdrawal latencies (PWLs) with and without EA at ST36. RESULTS: The PWLs were significantly shorter in rats at 24, 48, and 72 hours and 7 days after the discontinuation of EtOH when compared to EtOH-naïve rats. After a single administration of 2-Hz EA for 20 minutes at ST36, the PWLs at 24 hours after the withdrawal of EtOH were significantly greater than those of the sham group (2-Hz EA at the tail). Furthermore, the effect of EA on PWLs was significantly attenuated by bilateral intrahabenula infusion of the MOR antagonist naltrexone. CONCLUSIONS: These results suggest that EA can alleviate hyperalgesia during EtOH withdrawal through a mechanism involving MORs in the habenula. Based on this, EA could be of potential value as a therapy for hyperalgesia in alcohol dependence.

Pharmacological Manipulation of the Rostromedial Tegmental Nucleus Changes Voluntary and Operant Ethanol Self-Administration in Rats.

BACKGROUND: The aversive properties of ethanol (EtOH) that limit its intake are poorly understood. There is an increasing interest in the role of the rostromedial tegmental nucleus (RMTg), because it encodes aversion signals and inhibits motivated behaviors. It is also a major source of inhibitory GABAergic inputs to the midbrain dopamine neurons. Up to this time, the role of the RMTg in EtOH-drinking behaviors has not been well explored. METHODS: Male Long-Evans rats were trained either to drink EtOH under the intermittent 2-bottle-choice protocol or to self-administer EtOH in operant chambers under fixed-ratio-3 schedules. Changes in drinking behaviors induced by the bilateral infusion into the RMTg of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), an agonist of AMPA-type glutamate receptors, or muscimol, an agonist of GABAA receptors, were measured. RESULTS: Consumption and preference for EtOH, numbers of active lever pressing, and head entrance to the EtOH port were all significantly decreased upon activation of the RMTg by the infusion of AMPA, but were increased upon inhibition of the RMTg by the infusion of muscimol. By contrast, intra-RMTg infusion of these agents did not change sucrose consumption. CONCLUSIONS: These data show for the first time that EtOH-drinking and EtOH-seeking behaviors of rats changed inversely with RMTg function, supporting the idea that the RMTg plays a crucial role in EtOH-drinking behaviors.

Anxiety during alcohol withdrawal involves 5-HT2C receptors and M-channels in the lateral habenula.

Anxiety disorders often co-occur with alcohol use disorders, but the mechanisms underlying this comorbidity remain elusive. Previously, we reported that rats withdrawn from chronic alcohol consumption (Post-EtOH rats) exhibited robust anxiety-like behaviors (AB), which were accompanied by neuronal hyperexcitability, and the downregulation of M-type potassium channels (M-channels) in the lateral habenula (LHb); and that serotonin (5-HT) stimulated LHb neurons via type 2C receptors (5-HT2CRs). Also, 5-HT2CR activation is known to inhibit M-current in mouse hypothalamic neurons. The present study investigated whether LHb 5-HT2CRs and M-channels contribute to AB in adult male Long-Evans rats. We used the intermittent-access to 20% ethanol two-bottle free-choice drinking paradigm to induce dependence. We measured AB with the elevated plus-maze, open-field, and marble-burying tests at 24 h withdrawal. We found that intra-LHb infusion of SB242084, a selective 5-HT2CR antagonist alleviated AB and reduced the elevated c-Fos expression in the LHb of Post-EtOH rats. By contrast, intra-LHb infusion of the selective 5-HT2CR agonist WAY161503 induced AB and increased c-Fos expression in the LHb in alcohol-naive but not Post-EtOH rats. Also, intra-LHb SB242084 significantly reduced self-administration of alcohol intake in the operant chambers. Furthermore, both 5-HT2CR protein levels and 5-HIAA/5-HT ratio was increased in the LHb of Post-EtOH rats. Finally, intra-LHb SB242084 increased LHb KCNQ2/3 membrane protein expression in Post-EtOH rats. Collectively, these results suggest that enhanced LHb 5-HT2CR signaling that interacted with M-channels triggers AB in Post-EtOH rats and that 5-HT2CRs may be a promising target for treating comorbid anxiety disorders in alcoholics.

Downregulation of M-channels in lateral habenula mediates hyperalgesia during alcohol withdrawal in rats.

Hyperalgesia often occurs in alcoholics, especially during abstinence, yet the underlying mechanisms remain elusive. The lateral habenula (LHb) has been implicated in the pathophysiology of pain and alcohol use disorders. Suppression of m-type potassium channels (M-channels) has been found to contribute to the hyperactivity of LHb neurons of rats withdrawn from chronic alcohol administration. Here, we provided evidence that LHb M-channels may contribute to hyperalgesia. Compared to alcohol naïve counterparts, in male Long-Evans rats at 24-hours withdrawal from alcohol administration under the intermittent access paradigm for eight weeks, hyperalgesia was evident (as measured by paw withdrawal latencies in the Hargreaves Test), which was accompanied with higher basal activities of LHb neurons in brain slices, and lower M-channel protein expression. Inhibition of LHb neurons by chemogenetics, or pharmacological activation of M-channels, as well as overexpression of M-channels' subunit KCNQ3, relieved hyperalgesia and decreased relapse-like alcohol consumption. In contrast, chemogenetic activation of LHb neurons induced hyperalgesia in alcohol-naive rats. These data reveal a central role for the LHb in hyperalgesia during alcohol withdrawal, which may be due in part to the suppression of M-channels and, thus, highlights M-channels in the LHb as a potential therapeutic target for hyperalgesia in alcoholics.

Low-dose ethanol excites lateral habenula neurons projecting to VTA, RMTg, and raphe.

It is unclear how social drinking can contribute to the development of addiction in susceptible individuals. However, alcohol's aversive properties are a well-known factor contributing to its abuse. The lateral habenula (LHb) is a key brain structure responding to various aversive stimuli, including those related to alcohol. We recently reported that ethanol at 10 mM or less that can be achieved by social drinking activates many LHb neurons and drives aversive conditioning. The current study sought to identify LHb circuits that are activated by a low-dose of ethanol using immunohistochemistry and anatomic tracing techniques on adult Sprague-Dawley rats. We showed here that an intraperitoneal injection of ethanol (0.25 g/kg), resulting in a blood ethanol concentration of 5.6 mM, significantly increased the number of cFos immunoreactive (IR) neurons in the LHb. Most of the ethanol-activated cFos-IR LHb neurons expressed vGluT2 (vesicular glutamate transporters 2, a marker of a glutamatergic phenotype). These LHb neurons projected to the ventral tegmental area (VTA), rostromedial tegmental nucleus (RMTg), and dorsal raphe. Moreover, injections of the anterograde tracer AAV-CaMKIIa-eGFP into the lateral hypothalamus produced a significant amount of labeled fibers with vGluT2 positive terminals on the ethanol-activated LHb cells. These results indicate that the LHb neurons stimulated by a low-dose of ethanol project to the VTA, RMTg, and dorsal raphe, and receive excitatory projections from the lateral hypothalamus. These neurocircuits may play a crucial role in mediating the initial aversive effects produced by a low-dose of ethanol.

Ethanol Withdrawal Drives Anxiety-Related Behaviors by Reducing M-type Potassium Channel Activity in the Lateral Habenula.

Alcohol use disorders (AUDs) and anxiety disorders (ADs) are often seen concurrently, but their underlying cellular basis is unclear. For unclear reasons, the lateral habenula (LHb), a key brain region involved in the pathophysiology of ADs, becomes hyperactive after ethanol withdrawal. M-type K+ channels (M-channels), important regulators of neuronal activity, are abundant in the LHb, yet little is known about their role in AUDs and associated ADs. We report here that in rats at 24 h withdrawal from systemic ethanol administration (either by intraperitoneal injection, 2 g/kg, twice/day, for 7 days; or intermittent drinking 20% ethanol in a two-bottle free choice protocol for 8 weeks), the basal firing rate and the excitability of LHb neurons in brain slices was higher, whereas the amplitude of medium afterhyperpolarization and M-type K+ currents were smaller, when compared to ethanol naive rats. Concordantly, M-channel blocker (XE991)-induced increase in the spontaneous firing rate in LHb neurons was smaller. The protein expression of M-channel subunits, KCNQ2/3 in the LHb was also smaller. Moreover, anxiety levels (tested in open field, marble burying, and elevated plus maze) were higher, which were alleviated by LHb inhibition either chemogenetically or by local infusion of the M-channel opener, retigabine. Intra-LHb infusion of retigabine also reduced ethanol consumption and preference. These findings reveal an important role of LHb M-channels in the expression of AUDs and ADs, and suggest that the M-channels could be a potential therapeutic target for alcoholics.

Inhibition of AMPA receptor and CaMKII activity in the lateral habenula reduces depressive-like behavior and alcohol intake in rats.

Depression is a well-known risk factor for developing relapse drinking, but the neuronal mechanisms underlying the interactions between depression and alcohol use disorders remain elusive. Accumulating evidence has associated depression with hyperactivity of the lateral habenula (LHb), an epithalamic structure in the brain that encodes aversive signals. Glutamate receptors contribute substantially to the excitability of LHb neurons. Glutamatergic synapses in LHb neurons largely express GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR) that can be modulated by Ca2+/calmodulin-dependent protein II (CaMKII). In the current study, we tested the hypothesis that withdrawal from repeated cycles of ethanol drinking triggers an increase in LHb AMPAR and CaMKII activity concomitant with depression-like symptoms, and their inhibitions bring a reduction in depressive-like behaviors and alcohol consumption. Western blotting revealed a higher level of phosphorylated AMPAR GluA1 subunit at a CaMKII locus (GluA1-Ser831) in the LHb of ethanol-withdrawn rats than that of age-matched naïve counterparts. In ethanol-withdrawn rats, pharmacological inhibition of LHb AMPAR activity significantly mitigated the depressive-like behavior and ethanol drinking and seeking behaviors, but affected neither sucrose intake nor locomotor activity; and inhibition of LHb CaMKII activity, or chemogenetic inhibition of LHb activity produced similar effects. Conversely, activation of LHb AMPARs induced depressive-like behaviors in ethanol-naïve rats. These results demonstrate that CaMKII-AMPAR signaling in the LHb exemplifies a molecular basis for depressive-like symptoms during ethanol withdrawal and that inhibition of this signaling pathway may offer a new therapeutic approach to address the comorbidity of alcohol abuse and depression.

Serotonin stimulates lateral habenula via activation of the post-synaptic serotonin 2/3 receptors and transient receptor potential channels.

There is growing interest on the role of the lateral habenula (LHb) in depression, because it closely and bilaterally connects with the serotoninergic raphe nuclei. The LHb sends glutamate efferents to the raphe nuclei, while it receives serotoninergic afferents, and expresses a high density of serotonin (5-HT) receptors. Recent studies suggest that 5-HT receptors exist both in the presynaptic and postsynaptic sites of LHb neurons, and activation of these receptors may have different effects on the activity of LHb neurons. The current study focused on the effect of 5-HT on the postsynaptic membrane. We found that 5-HT initiated a depolarizing inward current (I((5-HTi))) and accelerated spontaneous firing in ∼80% of LHb neurons in rat brain slices. I((5-HTi)) was also induced by the 5-HT uptake blocker citalopram, indicating activity of endogenous 5-HT. I((5-HTi)) was diminished by 5-HT(2/3) receptor antagonists (ritanserin, SB-200646 or ondansetron), and activated by the selective 5-HT(2/3) agonists 1-(3-Chlorophenyl) piperazine hydrochloride or 1-(3-Chlorophenyl) biguanide hydrochloride. Furthermore, I((5-HTi)) was attenuated by 2-Aminoethyl diphenylborinate, a blocker of transient receptor potential channels, and an IP3 receptor inhibitor, indicating the involvement of transient receptor potential channels. These results demonstrate that the reciprocal connection between the LHb and the 5-HT system highlights a key role for 5-HT stimulation of LHb neurons that may be important in the pathogenesis of depression.

Dysfunction in amygdala-prefrontal plasticity and extinction-resistant avoidance: A model for anxiety disorder vulnerability.

Individuals exhibiting an anxiety disorder are believed to possess an innate vulnerability that makes them susceptible to the disorder. Anxiety disorders are also associated with abnormalities in the interconnected brain regions of the amygdala and prefrontal cortex (PFC). However, the link between anxiety vulnerability and amygdala-PFC dysfunction is currently unclear. Accordingly, the present study sought to determine if innate dysfunction within the amygdala to PFC projection underlies the susceptibility to develop anxiety-like behavior, using an anxiety vulnerable rodent model. The inbred Wistar Kyoto (WKY) rat was used to model vulnerability, as this strain naturally expresses extinction-resistant avoidance; a behavior that models the symptom of avoidance present in anxiety disorders. Synaptic plasticity was assessed within the projection from the basolateral nucleus of the amygdala (BLA) to the prelimbic cortical subdivision of the PFC in WKY and Sprague Dawley (SD) rats. While WKY rats exhibited normal paired-pulse plasticity, they did not maintain long-term potentiation (LTP) as SD rats. Thus, impaired plasticity within the BLA-PL cortex projection may contribute to extinction resistant avoidance of WKY, as lesions of the PL cortex in SD rats impaired extinction of avoidance similar to WKY rats. Treatment with d-cycloserine to reverse the impaired LTP in WKY rats was unsuccessful. The lack of LTP in WKY rats was associated with a significant reduction of NMDA receptors containing NR2A subunits in the PL cortex. Thus, dysfunction in amygdala-PFC plasticity is innate in anxiety vulnerable rats and may promote extinction-resistant avoidance by disrupting communication between the amygdala and prefrontal cortex.

Chronic intermittent voluntary alcohol drinking induces hyperalgesia in Sprague-Dawley rats.

The mechanisms of hyperalgesia in alcoholics are not completely clear, and the development of animal models would therefore be necessary in investigating the underlying changes. Several studies including our own have demonstrated that the intermittent access to 20% ethanol two-bottle choice procedure (IA2BC) promotes escalation of drinking, and induces physical dependence in the Sprague-Dawley (SD) rat, one of the strains most commonly used in preclinical alcohol research. In this study, we investigated whether the IA2BC procedure could produce hyperalgesia in SD rats. We show here that, the SD rats in the IA2BC procedure significantly escalated their drinking within 8 weeks, which is consistent with other studies. Starting from 8 weeks of repeated chronic drinking, the mechanical and thermal sensitivity was significantly increased. During withdrawal, there were noticeable physical dependence signs, including tail stiffness and lower limb flexion, which started at 4 hours and lasted for more than 3 days after ethanol removal. Importantly, during withdrawal, the mechanical and thermal sensitivity was further increased, which started at 12 hours and lasted for more than seven days after ethanol removal. These results suggest that utilizing the SD rat under the IA2BC procedure could be a useful animal model with heuristic value for exploring the mechanisms underlying hyperalgesia induced by chronic alcohol abuse.