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.
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.
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that TRPV1 contributes to the changes in both the behavioral phenotypes and the habenula activity in alcohol-withdrawn rats.
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.
The basal frequency of spontaneous excitatory postsynaptic currents and firing of lateral habenula neurons in alcohol-withdrawn rats was significantly increased.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= 11] vs. 6.7 ± 18.6% [n = 17], P = 0.027) and firing (by 23.4 ± 17.6% [n = 9] vs. 11.9 ± 16.3% [n = 12], P = 0.025) in Withdrawn rats than Naive rats. By contrast, the TRPV1 agonist capsaicin (3 μM) produced a weaker potentiation in Withdrawn than Naïve rats (spontaneous excitatory postsynaptic currents: by 203.6 ± 124.7% [n = 20] vs. 415.2 ± 424.3% [n = 15], P 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.
Enhanced TRPV1 function may contribute to increased glutamatergic transmission and activity of lateral habenula neurons, resulting in the aberrant behaviors during ethanol withdrawal.
Repeated cycles of excessive alcohol drinking and withdrawal induce a negative affective state and aberrant behaviors, including pain and anxiety.1 These psychiatric ailments act as negative reinforcers, promoting relapse drinking and the development of alcohol use disorders.2
Accumulating evidence indicates that the lateral habenula, an epithalamic structure in the brain, plays a crucial role in the aversive behaviors induced by many abused drugs, including alcohol.3–9 Recently we demonstrated increased nociception10,11 and anxiety- and depression-like behaviors12,13 in alcohol-withdrawn rats. These aberrant behaviors are concomitant with increased activity of, and glutamatergic transmissions to, lateral habenula neurons.12,13
The TRP channel, vanilloid type 1 or TRPV1, is the receptor for capsaicin14 and a nonselective cation channel that has been implicated in pain and behaviors associated with drugs of abuse such as anxiety.15 TRPV1 is in several brain regions including the habenula.16,17 Given that TRPV1 has been suggested to be able to facilitate glutamate transmission18 and is a target of alcohol,19 we hypothesized that lateral habenula TRPV1 contributes to the hyper-glutamatergic and hyperactivity of lateral habenula neurons as well as the aberrant behaviors in rats withdrawn from chronic alcohol exposure.
In this study, we test this hypothesis by assessing the role of TRPV1 in the electrophysiologic activity of lateral habenula neurons in epithalamic slices. We also measured the effect of pharmacologic manipulations of TRPV1 in the lateral habenula on pain and anxiety- and depression-like behaviors and relapse-like drinking behaviors in rats withdrawn from chronic intermittent ethanol drinking.
Materials and Methods
All experiments were performed in adult male Long Evans rats (n = 110 for electrophysiology and n = 280 for behaviors, 250–300 g in the start of the study). All procedures were performed by the National Institutes of Health guidance and approved by the Animal Care and Utilization Committee of Rutgers, the State University of New Jersey, New Jersey Medical School, Newark, New Jersey. Rats were maintained on a 12:12-h light:dark regiment (light on 20:00 with ad libitum food and water unless indicated otherwise; all experiments were carried out during the dark cycle). These rats were randomly divided into the alcohol- and water-drinking groups and were numbered and randomly assigned to experimental groups thereafter. The primary outcome was the change in alcohol-related behaviors and lateral habenula activity induced by pharmacologic manipulation of lateral habenula TRPV1 activity.
Brain Slice Preparation and Electrophysiology
Animals were euthanized under anesthesia using ketamine/xylazine (80 mg/10 mg/kg i.p.). Coronal epithalamic slices (250 μm thick) were cut at 10:00 on experiment day, from rats that were at 24-h withdrawal from the last alcohol-drinking session in the intermittent access to two-bottle free choice procedure for 8 weeks (ethanol-withdrawn), and from age-matched water drinking (Naïve) counterparts, in artificial cerebrospinal fluid (CSF) containing the following (in mM): 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2, 25 NaHCO3, 0.3 L-ascorbate, and 11 glucose, saturated with carbogen (95% O2/5% CO2), as described.22,23
Electrophysiologic events were recorded in warm (33°C) carbogenated artificial CSF (1.5 to 2.0 ml/min) from 11:00 to 18:00 h. Patch pipettes (6 to 8 MΩ) for voltage-clamp recordings contained (in mM): 140 Cs-methanesulfonate, 5 KCl, 2 MgCl2, 10 HEPES, 2 MgATP, 0.2 NaGTP, pH 7.2. Spontaneous firing was recorded by the loose-patch cell-attached technique. Spontaneous excitatory postsynaptic currents were recorded at a holding potential of −70 mV in the presence of gabazine (20 μM) and SCH50911 (10 μM) to block γ-aminobutyric acid type A and γ-aminobutyric acid type B receptors, respectively.
Stereotaxic surgery was performed on rats after a stable baseline drinking level was reached, under anesthesia using ketamine/xylazine (80 mg/10 mg/kg i.p), as described.20 A bilateral guide cannula (C235G-3.0, 23 gauge; Plastics One, USA) was aimed 1 mm above the lateral habenula (in mm: AP: −3.85; ML: ±0.75; DV: −4.2 from the skull’s surface).12 One week after recovery from surgery, animals returned to drinking with the intermittent access to two-bottle free choice paradigm. Age-matched naïve rats also received surgery and drank only water. To conclude the study, histologic verification was completed as described.20 Supplementary Digital Content 1 (http://links.lww.com/ALN/B858) indicates the placement of cannula tips from animals that received surgery; rats with injection sites outside of the lateral habenula (n = 4) were not used in the data analysis.
Intra-lateral Habenula Microinjection
Microinjection was performed as described.13 Injectors were left in place for an additional 60 s to allow for diffusion. Behavioral experiments proceeded as noted in the following methods.
Common salts, 6, 7-Dinitroquinoxaline-2, 3-dione (DNQX), (2R)-amino-5-phosphonovaleric acid (AP5), capsazepine, N-Oleoyldopamine (OLDA), 2E-N-(2,3-Dihydro-1,4-benzodioxin-6-yl)-3-[4-(1,1-dimethylethyl)phenyl]-2-Propenamide (AMG9810), and capsaicin were purchased from Sigma-Aldrich (USA).
Thermal Pain Test
Thermal nociceptive thresholds were determined by measuring the paw withdrawal latency using the Hargreaves method.10,24 The paw withdrawal latency was defined as the length of time between the start of the light beam and the lift of the hind paw. To avoid tissue damage, a 20-s cut-off time was used. A comparatively lower paw withdrawal latency was interpreted as hyperalgesia, whereas a higher paw withdrawal latency was interpreted as analgesia. To better compare the effects between the baselines of Naïve and ethanol-withdrawn rats, the paw withdrawal latency (PWL) data were transformed to represent the percent of maximum possible effect, using the following formula:
Conditioned Place Paradigm
A separate group of Naïve and ethanol-withdrawn rats was trained for conditioned place preference or aversion to intra–lateral habenula artificial CSF, DNQX, capsazepine, or capsaicin to detect the presence of spontaneous pain.25 We established an unbiased procedure,26 consisting of three distinct phases: preconditioning, conditioning, and postconditioning. Each phase was conducted during the 24-hr withdrawal time.
Conditioned Place Paradigm Apparatus
We used a standard rat place preference apparatus (MED-CPP2-013C, Med Associates, Inc., USA), which consisted of two chambers (30L×20W×20H, in cm) separated by a guillotine door. Chambers differed by tactile floor cues (mesh vs. rod) and wall colors (white vs. black). Photo beam detectors identified the activity and position of each rat (MED-PC Software, Med Associates, Inc.).
Conditioned Place Paradigm Test Procedure
Phase I: Preconditioning (Day 1).
Animals were allowed free access to the apparatus for 15 min. Rats with a strong initial preference (greater than 75%) for one chamber were excluded from further analysis.
Phase II: Conditioning (Day 3).
During conditioning, one drug session and one artificial CSF session were performed, separated by 6 h. During this phase, the guillotine door was closed, and animals were confined in one chamber for 15 min immediately after intra–lateral habenula drug infusion.
Phase III: Postconditioning (Day 5).
For postconditioning, the guillotine door was opened, and the time spent in each chamber was recorded during the 15-min test period. The preference score was defined as the difference in time spent in the drug-paired chamber on the postconditioning day versus the preconditioning day. Positive or negative scores indicated preference or aversion, respectively.
Elevated Plus Maze Test
We used the elevated plus maze test to assess anxiety-like behaviors, as described.12,27 During the 5-min test, time spent in the open arms, number of entries into the open arms, and total distance traveled were recorded using Smart 3.0 (Pan lab Harvard Apparatus, Spain). Elevated plus maze testing took place 15 min after drug infusion. Each animal was exposed to the elevated plus maze test one time only.
Marble Burying Test
We also utilized the marble burying test to assess anxiety-like behaviors, as described.12 Following intra–lateral habenula injection, animals were placed into the test cage containing 20 marbles. The numbers of marbles buried in the bedding (to 2/3 their depth) after 30 min were counted.
Forced Swimming Test
We used a modified forced swimming test to measure depressive-like behaviors, in a transparent plastic tube (diameter = 24.5 cm, height = 51 cm), filled to 30 cm with water at 23 to 25°C. Each rat was placed in the tube of water for 15 min on the first session of the test and for 5 min on the test session 24 h later. Immobility was defined as the rat floating in the water without struggling and only making movements necessary to keep its head above water.
All values in the text and figures indicate the mean ± SD. The sample sizes we used were based on previous publications,7,12 and no statistical power calculation was conducted before the study. All statistical calculations were carried out using Sigmaplot 14.0 (SYSTAT Software, USA). Baseline electrophysiologic data were recorded for 5 to 10 min, before drug superfusion, and during the washout. Because the basal excitatory postsynaptic current or firing frequency and amplitude varies in each cell, changes induced by each drug were calculated by the percent change. Data recorded during the initial control period were averaged and normalized to 100%. Before analysis, the distributions of the variables under study were examined using histograms. The use of parametric statistical analysis was deemed appropriate. The data of basal spontaneous excitatory postsynaptic currents or firings were analyzed using Student’s unpaired two-tailed t test between groups. The effects induced by drugs on spontaneous excitatory postsynaptic currents or firing were analyzed using a two-way ANOVA with “group” (Naïve vs. ethanol-withdrawn) as between-group factors and “dose” (Capsaicin: 0.01 to 30 µM; capsazepine or AMG9810: 0.1 to 100 μM; OLDA: 0.2 to 10 μM) or “pretreatment” (artificial CSF vs. 10μM N-Oleoyldopamine) as within-subject factor followed by the Tukey multiple comparisons test, as detailed in the figure legends. A Kolmogorov–Smirnov test was used to evaluate statistical significance for cumulative data. Dose–response data were fitted to the logistic equation: y=100xa/(xa+xoa), where y is the percentage change, x is the concentration of capsaicin, a is the slope parameter, and xo is the capsaicin concentration which induces a half-maximal change.
All the animals survived to the end of the behavior experiment, and there were no missing data except four rats with injection sites outside of the lateral habenula. Data from the behaviors were subject to a two-way ANOVA with “group” (Naïve vs. ethanol-withdrawn) as between-group factors and treatment as within-subject factor. Changes in pain threshold and ethanol-drinking data were subjected to a two-way repeated-measure ANOVA to extract significant main effect. Conditioned place test and hyperalgesia data were analyzed using a Pearson correlation. Statistical significance was declared at P < 0.05. Data were analyzed by experimenters who were blinded to the treatment history.
Elevation of TRPV1 Function Mediates Hyper-glutamatergic Transmission and Hyperactivity of Lateral Habenula Neurons during Ethanol Withdrawal
Consistent with a recent report,28 the frequency and amplitude of spontaneous excitatory postsynaptic currents in lateral habenula neurons were significantly enhanced in slices from ethanol-withdrawn (EtOH-WD) rats compared with Naïve rats (frequency: 2.3 ± 2.0 vs. 1.2 ± 1.0 Hz, t(58) = 2.71, P = 0.009, fig. 1, A, and B1–B2; amplitude: 22.9 ± 8.4 vs. 18.1 ± 8.7 pA, t(58) = 2.64, P = 0.011; fig. 1, C1–C2). The spontaneous excitatory postsynaptic currents were eliminated by the glutamate antagonists (2R)-amino-5-phosphonovaleric acid (AP5, 50 μM) plus DNQX (20 μM), indicating that these are events mediated by glutamate receptors. The spontaneous action potential firing frequency of lateral habenula neurons was also markedly higher in slices from ethanol-withdrawn rats (9.9 ± 7.2 vs. 6.8 ± 3.7 Hz, t(78) = 2.44, P =0.017; fig. 1, D and E). Moreover, bath application of AP5 (50 μM) plus DNQX (20 μM) significantly decreased the basal firing rate (main effect of Treatment F1,28 = 44.61, P < 0.001, two-way repeated measures ANOVA; fig. 1F1), and the inhibitions differed between groups (main effect of Group × Treatment interaction: F1,28 = 8.57, P = 0.007), with a greater reduction in ethanol-withdrawn rats (from 9.1 ± 4.8 to 5.6 ± 4.6 Hz, P < 0.001) than naïve rats (from 6.5 ± 3.7 to 5.1 ± 2.9 Hz, P = 0.013). Because the basal spontaneous excitatory postsynaptic current or firing rates varied among lateral habenula neurons of both groups of rats, we calculated the percent change before and during drug application for each cell in the following electrophysiologic experiments. As expected, glutamate antagonists induced a significantly stronger inhibition of spontaneous firing in ethanol-withdrawn than in Naïve rats (41.3 ± 33.7% vs. 20.3 ± 18.0%, t = 2.13, P = 0.042; fig. 1F2), indicating that the hyper-glutamatergic state contributes to lateral habenula hyperactivity during ethanol withdrawal.
To determine whether TRPV1 plays a role in the hyper-glutamatergic and hyperactivity state of lateral habenula neurons of ethanol-withdrawn rats, we examined the effects of capsazepine, a TRPV1 antagonist, on spontaneous excitatory postsynaptic currents and firings in lateral habenula neurons. Bath application of capsazepine (0.1 to 100 μM) dose-dependently inhibited the spontaneous excitatory postsynaptic current frequency (main effect of Dose: F3, 76 = 5.59, P = 0.002; fig. 2, A1–A2) and firing rate (F3, 67 = 9.62, P < 0.0001; fig. 2, C1–C2) in slices from ethanol-withdrawn rats, but not Naïve rats. Thus, 10 μM capsazepine induced significantly greater reduction on glutamate transmission (by 26.1 ± 27.9% in ethanol-withdrawn rat vs. 6.7 ± 18.6% in naïve rats, main effect of Group: F1,76 = 5.06, P = 0.027) and neuronal activity (by 23.4 ± 17.6% in ethanol-withdrawn rats vs. 11.9 ± 16.3% in naïve rats, main effect of Group: F1,67 = 5.28, P = 0.025) during ethanol withdrawal. No Group × Dose interaction was found (spontaneous excitatory postsynaptic currents: F3,76 = 0.62, P = 0.606; Firing: F3,67 = 1.90, P = 0.139). Because capsazepine could also inhibit other ion channels and receptors,29–31 we repeated the experiments with AMG9810 (AMG, 0.1 to 100 μM), a more selective TRPV1 antagonist. AMG9810 also induced a much stronger inhibition in ethanol-withdrawn than in Naïve rats on spontaneous excitatory postsynaptic current frequency (Group: F1,61 = 4.88, P = 0.031; Dose: F3,61 = 10.83, P < 0.0001; fig. 2, B1–B2) and firing rate (Group: F1,55 = 7.96, P = 0.007; Dose: F3,55 = 5.81, P = 0.002; fig. 2, D1–D2). No significant difference was detected between the Group × Dose interaction (spontaneous excitatory postsynaptic currents: F3, 61 = 0.97, P = 0.413, Firing: F3, 55 = 1.67, P = 0.183).
Furthermore, neither capsazepine (F3,76 = 0.41, P = 0.743; fig. 2A3) nor AMG9810 (F3,61 = 0.14, P = 0.939; fig. 2B3) at all the doses tested significantly altered the spontaneous excitatory postsynaptic current amplitude. These data indicate that lateral habenula TRPV1 channels are tonically activated under physiologic and pathologic conditions. More importantly, lateral habenula TRPV1 basal activity is elevated in ethanol-withdrawn rats.
Capsaicin-induced Enhancement of Glutamatergic Transmission and Activity of Lateral Habenula Neurons Is Weaker in Ethanol-withdrawn Rats
To further assess the effects of TRPV1 on synaptic plasticity during ethanol withdrawal, we examined the effect of the agonist capsaicin on the electrophysiologic properties of lateral habenula neurons. Bath perfusion of capsaicin (0.01 to 30 μM) concentration-dependently increased the frequency of spontaneous excitatory postsynaptic currents in lateral habenula neurons in both Naïve (fig. 3A) and ethanol-withdrawn (fig. 3B) rats (main effect of Dose: F6,208 = 7.95, P < 0.001; fig. 3C), with EC50s of 0.2 and 0.6 μM, respectively. We also detected a marked increase in cumulative probability of frequencies of spontaneous excitatory postsynaptic currents, suggesting an increase in presynaptic glutamate release by capsaicin application (fig. 3, A3 and B3, left panels). Importantly, capsaicin’s action was significantly greater in Naïve than in ethanol-withdrawn rats (203.6 ± 124.7% in ethanol-withdrawn rats vs. 415.2 ± 424.3% in naïve rats induced by 3 μM capsaicin, main effect of Group: F1, 208 = 28.20, P < 0.001; fig. 3C).Though capsaicin-induced increases in spontaneous excitatory postsynaptic current frequency were accompanied by a higher incidence of larger spontaneous excitatory postsynaptic currents (Kolmogorov-Smirnov test, fig. 3, A3 and B3, right panels), the mean amplitude did not significantly change in all the doses tested in either Naïve or ethanol-withdrawn rats (main effect of Group: F1, 208 = 1.12, P = 0.291; Dose: F6, 208 = 1.95, P = 0.075; fig. 3D). No significant difference was detected between the group × dose interaction (frequency: F6, 208 = 0.88, P = 0.508, amplitude: F6, 208 = 0.82, P = 0.558).
Capsaicin also concentration-dependently increased lateral habenula neuronal activity (main effect of Dose: F6, 142 = 19.78, P < 0.0001), with EC50s of 1.7 and 2.3 μM, respectively, in Naïve and ethanol-withdrawn groups. Additionally, capsaicin’s action was greater in Naïve than in ethanol-withdrawn rats (Group: F1, 142 = 42.0, P < 0.001; Group × Dose interaction: F6, 142 = 2.86, P = 0.012; 38.1 ± 14.7% in ethanol-withdrawn rats vs. 73.9 ± 41.9% in naïve rats induced by 3 μM capsaicin, post hoc P = 0.009; fig. 3, E and F). These data indicate that the lateral habenula neurons of ethanol-withdrawn rats are less sensitive to capsaicin.
Capsaicin-induced Enhancement of Glutamatergic Transmission and Activity of Lateral Habenula Neurons Is Weaker in the Presence of the TRPV1 Endogenous Agonist N-Oleoyldopamine
To test whether the decreased capsaicin response in ethanol-withdrawn rats is attributable to an elevated endogenous TRPV1 activity, we pretreated slices with TRPV1 endogenous agonist N-Oleoyldopamine (OLDA, 0.2 or 10 µM)32 for 6 to 8 min before the co-application of capsaicin (3 μM; fig. 4, A and B). High (10 μM) but not low (0.2 μM) concentration of OLDA significantly enhanced spontaneous excitatory postsynaptic current frequency in Naïve but not in ethanol-withdrawn rats (main effect of Dose: F1,54 = 11.01, P = 0.002; Group: F1,54 = 7.83, P = 0.007; fig. 4, A–C). No difference was found for group × dose interaction (F1,54 = 0.09, P = 0.762).
Notably, capsaicin (3 μM)–induced enhancement on spontaneous excitatory postsynaptic current frequency was significantly decreased with Oleoyldopamine (10 μM) pretreatment compared with Oleoyldopamine-free solution in Naïve rats (main effect of Pretreatment: F1, 61 = 6.58, P = 0.013), but not in ethanol-withdrawn rats (main effect of Group: F1, 61 = 8.0, P = 0.006; Pretreatment × Group interaction: F1, 61 = 0.66, P = 0.419; fig. 4, A, B, and E). These chemicals did not change the mean spontaneous excitatory postsynaptic current amplitude (Oleoyldopamine alone or Oleoyldopamine plus capsaicin; fig. 4, A, B, and D). Furthermore, Oleoyldopamine dose-dependently accelerated the firing in Naïve rats (F1, 60 = 7.47, P = 0.008; fig. 4, F and G) but not in ethanol-withdrawn rats, thus producing a stronger excitation in Naïve rats (main effect of Group: F1, 60 = 4.06, P = 0.049; Group × Dose interaction: F1, 60 = 1.18, P = 0.281). In the presence of Oleoyldopamine (10 μM), capsaicin (3 μM)–induced increase in firing was significantly attenuated in Naïve rats but not in ethanol-withdrawn rats (main effect of Pretreatment: F1, 46 = 14.13, P < 0.001; Group: F1, 46 = 10.43, P = 0.002; Group × Pretreatment interaction: F1, 46 = 1.48, P = 0.231; fig. 4H). These results support the view that an elevated endogenous TRPV1 activity may contribute to the blunted response to capsaicin in ethanol-withdrawn rats.
Development of Hyperalgesia during Withdrawal from the Intermittent Access to Two-bottle Free Choice Drinking Paradigm
All rats survived after the experiments and all data were analyzed as intended, thus there were no missing values for our behavior experiments. We next examined whether changes in lateral habenula TRPV1 in ethanol-withdrawn rats contribute to the changes in nociception. To characterize the changes in nociception, we measured the paw withdrawal latency to thermal stimuli at 24-hr withdrawal from the last drinking session after the 24-session (8-week) drinking period. There was a significant interaction between the group (Naïve vs. ethanol-withdrawn) and time (baseline vs. eight-week) (two-way repeated measures ANOVA: F1, 272 = 52.58, P < 0.0001; fig. 5A). The paw withdrawal latency was significantly reduced after 24 drinking sessions in ethanol-withdrawn rats (post hoc P < 0.001, eight-week vs. baseline), suggesting that chronic intermittent ethanol consumption and withdrawal induce hyperalgesia.
Inhibition of Lateral Habenula TRPV1 Reduces Hyperalgesia in Ethanol-withdrawn Rats
To investigate the role of lateral habenula TRPV1 on pain during ethanol withdrawal, we examined the paw withdrawal latency 10 min after intra–lateral habenula injection of capsazepine (60 nmol) or capsaicin (6 nmol). To better compare the drug’s effects between the groups, we converted the paw withdrawal latency to the percentage of maximum possible effect (see Materials and Methods section).
Intra–lateral habenula capsazepine or capsaicin produced a significant increase in the percent of maximum possible effect of the paw withdrawal latency in the ethanol-withdrawn, but not the Naive rats, confirmed by two-way ANOVA (main effect of Group: F1,74 = 134.12, P < 0.0001; Treatment: F3,74 = 36.14, P < 0.0001; Group × Treatment interaction: F3,74 = 15.72, P < 0.0001; fig. 5B) followed by Tukey post hoc test (both P < 0.001, Naïve vs. ethanol-withdrawn; both P < 0.001 capsazepine/capsaicin vs. artificial CSF). These results suggest that both positive and negative modulations of lateral habenula TRPV1 function can reduce hyperalgesia of ethanol-withdrawn rats. Additionally, intra–lateral habenula AMG9810 significantly decreased the paw withdrawal latency in ethanol-withdrawn rats (Group × Treatment interaction: F1,36 = 87.75, P < 0.001; see Supplementary Digital Content 2, panel A, http://links.lww.com/ALN/B859).
Our electrophysiologic data indicate that activation of TRPV1 can enhance α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated glutamatergic transmission, we, therefore, investigated the role of AMPARs in the lateral habenula in the pain response. Consistent with our recent report,13 intra–lateral habenula injection of AMPAR antagonist DNQX (10 μM, 200 nl per side) induced a significant increase in the percent of maximum possible effect to the thermal stimuli in both ethanol-withdrawn (P < 0.001, DNQX vs. artificial CSF) and Naïve (P = 0.003) rats, and was significantly greater in the ethanol-withdrawn rats (P < 0.001, Naïve vs. ethanol-withdrawn; fig. 5B). These data indicate that lateral habenula AMPARs play a critical role in nociception, and enhanced glutamate signaling could be responsible for the hyperalgesia during ethanol withdrawal.
Correlation between Place Conditioning and Hyperalgesia
Next, we examined the effect of pharmacologic inhibition of lateral habenula TRPV1 channels on the spontaneous pain using a conditioned place paradigm. In terms of the place conditioning score, there was a main effect of group (F1,67 = 85.50, P < 0.0001, fig. 5C), a main effect of treatment (F3,67 = 28.15, P < 0.001), and a significant interaction between group and treatment (F3,67 = 21.79, P < 0.001). Ethanol-withdrawn (post hoc P < 0.001, capsazepine vs. artificial CSF), but not Naïve rats (P = 0.941), spent significantly longer time in the chamber paired with an intra–lateral habenula infusion of capsazepine. Thus, capsazepine induced a conditioned place preference in ethanol-withdrawn rats (P < 0.001 vs. Naïve). Additionally, intra–lateral habenula DNQX elicited a strong conditioned place preference in both ethanol-withdrawn (P < 0.001 vs. artificial CSF) and Naïve (P = 0.007) rats and was significantly stronger in the ethanol-withdrawn group (P = 0.001 vs. Naive).
To determine whether spontaneous and evoked pain are correlated, we plotted the place conditioning score against the percent of maximum possible effect obtained from the same group of rats (fig. 5, D and E). The data indicated a strong, positive correlation between the conditioned place paradigm score and the percent of maximum possible effect of DNQX (fig. 5D) or capsazepine (fig. 5E) in ethanol-withdrawn rats. In Naïve rats, there was a strong, positive correlation between the conditioned place paradigm score and the percent of maximum possible effect of DNQX (fig. 5D), suggesting that inhibition of lateral habenula AMPARs can have an analgesic and rewarding effect. Overall, these results suggest that lateral habenula TRPV1 contributes to both evoked and spontaneous pain in ethanol-withdrawn rats.
Lateral Habenula TRPV1 Channels Contribute to Anxiety-like Behaviors
Recently, we have shown that lateral habenula inhibition can alleviate the elevated anxiety in ethanol-withdrawn rats.8 In this study, we examined the effect of pharmacologic manipulation of lateral habenula TRPV1 channels on the anxiety levels of rats using the elevated plus maze.28 As expected, we observed elevated anxiety levels in ethanol-withdrawn rats reflected by the reduction in time spent in the open arms (main effect of Group × Treatment interaction: F2,34 = 14.72, P < 0.001; P = 0.004 for post hoc comparisons, Naïve vs. ethanol-withdrawn; fig. 6, A and B) and in open arm entries (Group × Treatment interaction: F2,34 = 8.85; P < 0.001; P < 0.001 for post hoc comparisons; fig. 6C) in the elevated plus maze. In the ethanol-withdrawn group, intra–lateral habenula capsazepine significantly increased the time spent (main effect of Treatment: F2,34 = 9.43, P < 0.001; P < 0.001 vs. artificial CSF for post hoc comparisons; fig. 6B) and the number of entries (F2,34 = 7.45, P = 0.002; P = 0.002 for post hoc comparisons; fig. 6C) to the open arms, indicating that antagonism of lateral habenula TRPV1 reduces anxiety-like behaviors. Importantly, capsazepine did not alter the total distance traveled in the elevated plus maze (main effect of Treatment: F2,27 = 1.43, P = 0.257, fig. 6D), suggesting that locomotor activity was not altered. These results were also consistent with pharmacologic manipulation of the lateral habenula with alternate antagonist AMG9810 (see Supplementary Digital Content 2, panels B–D, http://links.lww.com/ALN/B859).
There was a significant effect of interaction between group and drug treatment in the marble burying test (F2,42 =14.14, P < 0.001; fig. 6E). Naïve rats receiving artificial CSF infusion buried significantly fewer marbles, compared with ethanol-withdrawn rats receiving artificial CSF infusion (post hoc P = 0.001), further confirming enhanced anxiety levels in ethanol-withdrawn rats. Moreover, ethanol-withdrawn animals receiving intra–lateral habenula capsazepine (P < 0.001 vs. artificial CSF) or capsaicin (P = 0.03 vs. artificial CSF) buried significantly fewer marbles, whereas Naïve rats treated with intra–lateral habenula capsaicin buried significantly more marbles (P < 0.001 vs. artificial CSF). Together, these results suggest that lateral habenula TRPV1 contributes to the anxiety-like behaviors in rats and that lateral habenula TRPV1 function is altered after repeated cycles of ethanol drinking and withdrawal.
Lateral Habenula TRPV1 Is Involved in Depression-like Behaviors
The latency of first immobility time (F2,30 = 63.01, P < 0.0001; fig. 6F) and total immobility time (F2,30 = 37.17, P < 0.0001; fig. 6G) of the forced swimming test were tested using a two-way ANOVA and showed a significant main effect of group × treatment interaction. Ethanol-withdrawn rats had a significantly shorter latency to first immobility (post hoc P < 0.001) and a longer immobility time (P < 0.001) than Naïve rats (fig. 6, F and G), indicating that ethanol-withdrawn rats displayed depression-like behaviors. In parallel, intra–lateral habenula infusion of capsaicin substantially shortened the latency to first immobility and prolonged the total immobility time, thus producing depression-like behaviors in Naïve rats (both P < 0.001 vs. artificial CSF), without changing either the latency to first immobility (P = 0.996) or the total immobility time (P = 0.817) in ethanol-withdrawn rats. Conversely, intra–lateral habenula infusion of capsazepine had no eﬀect on both factors in ether Naïve or ethanol-withdrawn rats. These results suggest that activation of lateral habenula TRPV1 is sufficient to induce depression-like behaviors, but lateral habenula TRPV1 is not involved in the depression-like behaviors during ethanol withdrawal.
Inhibition of Lateral Habenula TRPV1 Channels Decreases Relapse-like Ethanol Consumption
Because the aversive responses during withdrawal may contribute to relapse drinking, we next investigated the impact of lateral habenula TRPV1 channels on ethanol consumption when the rats resumed drinking after 24 h of withdrawal from the last ethanol session. Intra–lateral habenula infusion of capsazepine or capsaicin significantly decreased ethanol intake, which was paralleled by a significant decrease in preference for ethanol after 30-min and 2-h access to ethanol (main effect of Treatment: F2,53 = 59.16, P < 0.001; fig. 7A), and a significant increase in water intake at 2 h (F2,53 = 61.45, P < 0.001; fig. 7C), thus reducing ethanol preference (F2,53 = 102.67, P < 0.001; fig. 7B). The total fluid intake was also slightly but significantly reduced (F2,53 =32.36, P < 0.001; fig. 7D).
Although TRPV1 has a well-established role in the pain response, studies are now uncovering its role in psychiatric disorders including drug addiction. This study investigated the role of lateral habenula TRPV1 channels in the aberrant behaviors occurring during ethanol withdrawal. We observed that the frequency of spontaneous firing and spontaneous excitatory postsynaptic currents of lateral habenula neurons were increased in slices from animals withdrawn from repeated ethanol administration. Importantly, the frequency of these events was reduced by TRPV1 antagonists capsazepine and AMG9810. The reduction was stronger in ethanol-withdrawn than naïve animals. Furthermore, aversive behaviors such as increased nociception sensitivity and anxiety-like behaviors in withdrawn rats were alleviated by intra–lateral habenula capsazepine. This treatment also produced a significant place preference and reduced ethanol consumption and preference. These results suggest that enhanced TRPV1 function may contribute to these aversive behaviors during withdrawal.
We have previously reported increased frequency of spontaneous excitatory postsynaptic currents and firing of lateral habenula neurons28 as well as phosphorylated AMPAR expression in the lateral habenula of ethanol-withdrawn rats,13 and inhibition of lateral habenula AMPARs reduced anxiety-like behaviors, as well as alcohol intake.13 Here, we report that inhibition of lateral habenula AMPARs elicited both an analgesic effect and place preference, suggesting that lateral habenula AMPARs contribute to nociception and reward.
Before this study, the role of TRPV1 in the lateral habenula was unknown. To understand lateral habenula TRPV1 function, we first used the competitive antagonist capsazepine.33 Capsazepine significantly reduced spontaneous excitatory postsynaptic current frequency, suggesting a presynaptic effect,18 although a postsynaptic effect may also be involved. This idea is consistent with previous reports suggesting that activation of TRPV1 increases the influxes of Na+ and Ca2+ that facilitate depolarization and release of neurotransmitters,34 including glutamate.18,35,36 Notably, capsazepine might also inhibit the TRPM8 channel,29,30 voltage-gated calcium channels,31 and nicotinic acetylcholine receptors. However, the effects of capsazepine that we have reported here may act through TRPV1, because the more selective TRPV1 antagonist AMG9810 elicited a similar effect.
Remarkably, in lateral habenula neurons of ethanol-withdrawn rats compared with Naïve rats, spontaneous firing was faster, and capsazepine’s inhibition was stronger, suggesting that enhanced TRPV1 function may contribute to lateral habenula hyperactivity. Also, capsazepine suppressed spontaneous firing and spontaneous excitatory postsynaptic currents, suggesting that lateral habenula TRPV1 is tonically activated37,38 (but see Musella et al.18 ). The selective TRPV1 agonist capsaicin enhanced spontaneous excitatory postsynaptic currents and firing of lateral habenula neurons, but this enhancement was much weaker in ethanol-withdrawn animals, suggesting a higher level of TRPV1 function during ethanol withdrawal. This view is supported by the data showing that capsaicin-induced enhancement was reduced in the presence of the endogenous agonist N-Oleoyldopamine in the lateral habenula neurons of Naïve but not withdrawn rats. However, the smaller effect of capsaicin in ethanol-withdrawn animals conflicts with studies in other states which show that when TRPV1 function is enhanced (i.e., inflammatory hyperalgesia) capsaicin responses are increased. The mechanism underlying this confliction is currently unknown. This difference may suggest that withdrawal from chronic intermittent alcohol administration induces a specific change in lateral habenula TRPV1.
Procedures based on classical and operant conditioning principles in rodents have been established and validated to study nociception and evaluate the effects of analgesics. They are an important addition to the traditional stimulus-evoked pain measurements.39–41 The conditioned place paradigm procedure has been used to study ongoing or spontaneous pain,25 centered around the concept that the attenuation of ongoing pain is rewarding. Therefore, we investigated the effect of capsazepine with this procedure. Indeed, capsazepine elicited a significant place preference in ethanol-withdrawn but not in Naïve rats, suggesting capsazepine attenuated aversiveness of ethanol withdrawal. The AMPAR antagonist DNQX elicited a strong place preference in both Naive and withdrawn animals, suggesting that inhibition of lateral habenula AMPARs is rewarding. The conditioned place paradigm data are consistent with the data of the paw withdrawal latency to thermal stimuli in both groups of rats. Together, these results suggest that inhibition of lateral habenula–TRPV1 could reduce aversiveness induced by withdrawal, which would make lateral habenula TRPV1 a potential therapeutic target for those experiencing alcohol use disorders.
Anxiety is an early withdrawal symptom that develops after cessation of ethanol consumption in alcoholics.41,42 Although some people may use alcohol to relieve anxiety, the negative effects of this type of self-medication quickly outweigh any of the short-term positive relief one may experience.41–44 Therefore, from a medical standpoint, it is best to treat both alcohol abuse and anxiety simultaneously, as addressing only alcohol abuse will allow anxiety to reoccur, leading to relapse drinking as a means of coping with the issue. In our study, inhibition of lateral habenula TRPV1 channels with capsazepine or AMG9810 significantly reduced anxiety-like behaviors measured in the elevated plus maze or marble burying test, but not depression-like behaviors measured in the forced swimming test. Importantly, inhibition of lateral habenula TRPV1 significantly reduced relapse-like drinking. In summary, the data suggest that targeting TRPV1 channels in the lateral habenula could reduce both pain and anxiety-like behaviors resulting from ethanol withdrawal and potentially curb relapse to alcohol.
To our knowledge, there is no information regarding what percent of the lateral habenula neurons are TRPV1-positive. However, our electrophysiologic data showed that more than 80% of lateral habenula neurons in naïve rats (88 of 101 neurons on spontaneous excitatory postsynaptic currents, and 63 of 76 neurons on firing) responded to TRPV1 agents, suggesting that TRPV1 is expressed in the majority of the lateral habenula neurons. About 95% of lateral habenula neurons are glutamatergic.45 Lateral habenula neurons receive glutamatergic projections from the basal ganglia and prefrontal cortex, and project to midbrain monoaminergic nuclei. Destinations include the dopaminergic ventral tegmental area and substantia nigra, γ-aminobutyric acid–mediated rostromedial tegmental nucleus, serotonergic raphe, and encephalin-producing periaqueductal grey.9,46 This study showed that the activation of TRPV1 increased glutamatergic transmission and activity of lateral habenula neurons. Future studies are needed to investigate the sources of these glutamatergic inputs and the projections of the lateral habenula neurons activated by TRPV1.
In conclusion, we observed that lateral habenula TRPV1 function increased during ethanol withdrawal, which may contribute to increased glutamate transmission and activity of lateral habenula neurons, as well as hyperalgesia and anxiety-like behaviors. Inhibition of lateral habenula TRPV1 alleviated these aberrant behaviors (fig. 8). Thus, lateral habenula TRPV1 channels could be a novel therapeutic target against alcohol use disorders.
The authors acknowledge Tibor Rohacs, M.D., Ph.D., Department of Pharmacology, Physiology, and Neuroscience, Rutgers – New Jersey Medical School (Newark, New Jersey) for reviewing and offering constructive feedback on the manuscript; Priscilla White, M.D., Department of Anesthesiology, Rutgers – New Jersey Medical School for editing the manuscript; and Jing Li, M.D., Ph.D., and Seungwoo Kang, Ph.D., Department of Anesthesiology, Physiology, and Pharmacology, Rutgers – New Jersey Medical School, and Ying Li, Ph.D., Department of Pharmacology, Physiology, and Neuroscience, Rutgers – New Jersey Medical School, for comments on the manuscript.
Supported by grant Nos. AA021657 and AA022292 from the National Institutes of Health (Bethesda, Maryland; to Dr. Ye).
The authors declare no competing interests.