Effect of sigma-1 receptor antagonism on ethanol and natural reward seeking

Abstract

Sigma-1 (σ₁) receptors have been implicated in cognitive function, anxiety, depression, and the regulation of stress responses. Additionally, σ₁ receptors have been shown to participate in the behavioral and motivational effects of psychostimulants. Recent studies have demonstrated that σ₁ receptor antagonism prevents ethanol-induced conditioned place preference in mice and excessive drinking in alcohol-dependent or alcohol-preferring rats. Therefore, this study was designed to determine whether this role for σ₁ receptors extends to ethanol-seeking behavior using an animal model of relapse and tested whether the suppressant effect of a potent σ₁ receptor antagonist, BD1047, generalizes to natural reward-seeking behavior. Two separate groups of rats were trained to orally self-administer 10% (w/v) ethanol or a highly palatable reinforcer, 3%/0.125% (w/v) glucose/saccharin (SuperSac), in the presence of a discriminative stimulus (S⁺). Following extinction, during which the reinforcers and S⁺ were withheld, the presentation of the ethanol or SuperSac S⁺ produced comparable recovery of responding. BD1047 (1–20 mg/kg) produced similar behavioral effects on both ethanol S⁺- and SuperSac S⁺-induced reinstatement, with prevention of conditioned reinstatement only at the highest BD1047 dose. The present results show that σ₁ receptor blockade under the present conditions produces similar effects on conditioned reinstatement induced by ethanol- and SuperSac-related stimuli, suggestive of overlapping neural mechanisms that control ethanol and natural reward seeking.

Introduction

Cloned in 1996, the sigma 1 (σ₁) receptor is a 29 kDa, 223-amino-acid protein [1,2] expressed in regions of the brain that have been implicated in drug abuse and addiction (i.e., hippocampus, hypothalamus, ventral tegmental area, caudate putamen, nucleus accumbens, and amygdala) [3–5]. Sigma₁ receptors primarily reside at the mitochondrial-associated endoplasmic reticulum membrane (MAM) and display chaperone-like properties [6]. They modulate calcium flux through inositol triphosphate (IP3) receptors and regulate cellular survival through calcium modulation [6–8]. Sigma₁ receptors have been shown to translocate to cellular regions other than the MAM when they are activated or subjected to endoplasmic reticulum stress. Consequently, they have been proposed to be intracellular amplifiers and inter-organelle sensors for signal transduction [6–8].

Although the endogenous ligand of σ₁ receptors has yet to be fully identified, several lines of evidence suggest that neuroactive steroids, such as dehydroepiandrosterone (DHEA) and progesterone, act as potent endogenous σ₁ receptor modulators (e.g., for review, see [9]). Behaviorally, σ₁ receptors have been implicated in various physiological and pathophysiological mechanisms, such as cognitive function, anxiety, depression, the regulation of stress responses, neuroinflammation, neurodegenerative, and addictive disorders (e.g., [10,11]). Specifically related to addiction, σ₁ receptors have been proposed to be compelling targets for the development of novel pharmacotherapies to treat the effects of different drugs of abuse, including alcohol (for detailed review, see [11,12]). For example, σ₁ receptor antagonists attenuate ethanol-induced locomotor activity and block the acquisition of ethanol-induced conditioned place preference [13]. Moreover, σ₁ receptor antagonists have been shown to reduce operant ethanol self-administration and homecage drinking under conditions of high baseline intake in ethanol-dependent and genetically selected alcohol-preferring rats, decrease the motivation to work for ethanol on a progressive-ratio schedule of reinforcement, and attenuate the increased drinking associated with the alcohol deprivation effect [14,15].

To extend the understanding of the role of σ₁ receptors in addiction-relevant conditioned effects of ethanol, the effects of a σ₁ receptor antagonist, BD1047 [16,17], were examined on reinstatement of ethanol seeking induced by drug-related contextual stimuli. To establish whether BD1047 preferentially modifies drug-directed behavior or exerts general suppressant effects on motivated behavior, the effects of BD1047 were also tested on responding induced by stimuli conditioned to a potent conventional reinforcer [18–20].

Methods

Animals.

Seventy male Wistar rats (Charles River, Wilmington, MA; 200–250 g upon arrival) were housed 2–3 per cage in a temperature- and humidity-controlled vivarium on a reverse 12 h/12 h light/dark cycle with ad libitum access to food and water. All of the procedures were conducted in strict adherence to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of The Scripps Research Institute.

Drugs.

Ethyl alcohol was dissolved in tap water to a concentration of 10% w/v for self-administration. N-(2-[3,4-dichlorophenyl]ethyl)-N-methyl-2-(dimethylamino)ethylamine (BD1047; Tocris, Minneapolis, MN) was dissolved in sterile water and administered intraperitoneally (IP) in a volume of 1 ml/kg immediately before the behavioral testing.

Apparatus.

The animals were trained and tested in standard 29 × 24 × 19.5 cm operant conditioning chambers (Med Associates, St. Albans, VT) located inside ventilated sound-attenuating cubicles. Each chamber was equipped with a 0.15 ml drinking reservoir positioned 4 cm above the grid floor in the center of the front panel of the chamber and two retractable levers (6 cm above the grid floor) located 4.5 cm on either side of the drinking reservoir. Auditory stimuli were presented via a speaker mounted on the rear wall. Responses on the right (active) lever resulted in activation of the infusion pump for 0.5 s, resulting in the delivery of 0.1 ml of the liquid reinforcer into the drinking reservoir. Responses on the left (inactive) lever were recorded but had no other scheduled consequences. A computer controlled the delivery of fluids, presentation of auditory stimuli, and recording of behavioral data.

Pretraining.

All of the rats were pretrained to self-administer SuperSac (3% glucose and 0.125% saccharin w/v) for 5 days in daily 30 min sessions on a continuous reinforcement schedule. Starting on day 6, the rats were divided into two groups. One group continued to self-administer SuperSac for 9 additional days. For the other group, the training continued with 10% (w/v) ethanol added to the SuperSac solution for 4 days. During the following 4 training days, glucose was removed from the solution, followed by the removal of saccharin after the completion of 14 total training days (see [18–20]). Two rats were lost at an early stage of the experiment because of health complications, one rat did not acquire SuperSac self-administration, and one rat stopped self-administering when 10% ethanol was added to the SuperSac solution, reducing the number of animals to 66 (EtOH group, final n = 34; SuperSac group, final n = 32).

Conditioned Reinstatement.

Conditioning Procedure.

Following pretraining, ethanol (10% w/v) or SuperSac availability was made contingent on the presence of a discriminative stimulus (S^D) that signaled the availability (S⁺) or nonavailability (S^—) of ethanol or SuperSac. Each S^D consisted of a compound olfactory stimulus (banana or anise extract; McCormick, Hunt Valley, MD) plus auditory stimulus using continuous white noise (70 dB) or a continuous tone (7 kHz, 70 dB). Banana/white noise served as the S⁺, and anise/continuous tone served as the S^—. The first two conditioning sessions were conducted under the S⁺ condition, followed on the third day by an S^— session. Thereafter, the sequence of S⁺ and S⁻ conditions was determined randomly, and daily training continued for a total of 20 sessions (10 S⁺ and 10 S^—).

Extinction.

After the conditioning sessions were completed, the rats were subjected to 30 min extinction conditions. Extinction sessions began by extending the levers without presentation of the S^D. Responses at the previously active lever activated the syringe pump motor only and did not result in the delivery of ethanol or SuperSac. Extinction sessions were conducted once daily until a criterion of ≤ 5 responses/session over three consecutive extinction sessions was met.

Reinstatement Tests:

The test sessions were identical to those during conditioning, with the exception that ethanol or SuperSac was not available. The day after the last day of extinction, the animals were presented with the S^—. Two days later, the effects of BD1047 (0–20 mg/kg) were tested on S⁺-induced reinstatement (n = 7–10 animals/dose).

Statistical Analysis.

Differences in responding at the active lever between the respective reward and non-reward conditions during the last day of the training/conditioning phase were analyzed by paired t-tests, and comparisons of the number of days of extinction and ethanol S⁺- or SuperSac S⁺-induced reinstatement (under vehicle conditions) were analyzed by unpaired t-tests. Differences in the number of responses between the extinction and reinstatement phases in the EtOH and SuperSac groups were analyzed separately by one-way within-subjects analysis of variance (ANOVA). The effects of BD1047 on reinstatement responses were analyzed separately for the EtOH and SuperSac groups by one-way between-subjects ANOVA. Significant results in the ANOVAs were followed by Protected Least Significant Difference post hoc tests or pairwise comparisons.

Results

Ethanol.

At the end of the conditioning phase, the rats showed stable ethanol self-administration and a significant reduction of responding during non-reward sessions (t₃₃ = 10.3, p < 0.001; Fig. 1A). Following the initiation of the extinction contingency, the rats required an average of 9.2 ± 0.3 (mean ± SEM) sessions to reach the criterion (Fig. 1B). During subsequent reinstatement tests, the ethanol S⁺ but not non-reward-associated S^— elicited a recovery of responding in BD1047 vehicle-treated rats (p < 0.05, pairwise comparison after ANOVA: F_2,14 = 10.4, p < 0.001; Fig. 1B). When BD1047 was tested against the ethanol S⁺, a main effect for BD1047 dose was found (ANOVA: F_3,33 = 8.5, p < 0.001), but a significant reduction of ethanol S⁺-induced reinstatement was detected only at the highest dose tested (20 mg/kg; p < 0.001, Fisher PLSD test; Fig. 1B). Responses at the inactive lever remained low (≤ 4 responses) throughout testing and were not modified by BD1047 (data not shown).

Figure 1.

(A) Active lever responses during conditioning sessions in the presence of stimuli paired with ethanol (EtOH/S⁺) availability vs. non-availability (Non-Reward/S^—). ***p < 0.001, vs. EtOH/S⁺. (B) Extinction (EXT) responses and responses during an initial reinstatement test in the presence of the stimulus paired with reward non-availability (S^—) and reinstatement responses in the presence of stimuli previously associated with ethanol availability (S⁺) in vehicle-treated rats (0) and across doses of BD1047. ***p < 0.001, vs. 0 mg/kg; ⁺p < 0.05, vs. EXT and S^— following ANOVAs; 3 mg/kg, F_2,12 = 4.0, p < 0.05; 10 mg/kg, F_2,16 = 6.3, p < 0.01.

SuperSac.

The rats showed stable SuperSac self-administration and a significant reduction of responding during non-reward sessions (t₃₁ = 22.5, p < 0.001; Fig. 2A). The animals required an average of 10.5 ± 0.4 sessions to reach the extinction criterion (Fig. 2B), which was significantly longer than what was observed for the EtOH group (t₆₄ = −2.3, p < 0.05). The SuperSac S⁺ but not non-reward-associated S^— elicited a recovery of responding in BD1047 vehicle-treated rats (p < 0.05, pairwise comparison after ANOVA: F_2,16 = 6.8, p < 0.05; Fig. 2B) that was identical to the recovery of responding induced by the ethanol S⁺ (t₁₅ = −1.8, p > 0.05; Fig. 1B and 2B for comparison). Similar to what was observed in the case of ethanol S⁺-induced reinstatement, BD1047 significantly reduced SuperSac S⁺-induced reinstatement to extinction levels only at the 20 mg/kg dose (p < 0.001; PLSD test following ANOVA: F_3,31 = 5.9, p < 0.01; Fig. 2B). Responses at the inactive lever remained low (≤ 4 responses) throughout testing and were not modified by BD1047 (data not shown).

Figure 2.

(A) Active lever responses during conditioning sessions in the presence of stimuli paired with SuperSac availability (SuperSac/S⁺) vs. non-availability (Non-Reward/S^—). ***p < 0.001, vs. SuperSac/S⁺. (B) Extinction (EXT) responses and responses during an initial reinstatement test in the presence of the stimulus paired with reward non-availability (S^—) and reinstatement responses in the presence of stimuli previously associated with SuperSac availability (S⁺) in vehicle-treated rats (0) and across BD1047 doses. ***p < 0.001, vs. 0 mg/kg; ⁺p < 0.05, vs. EXT and S^— following ANOVAs; 3 mg/kg, F_2,12 = 6.9, p < 0.01; 10 mg/kg, F_2,14 = 7.3, p < 0.01.

Discussion

The present study shows that BD1047 reduced reinstatement induced by an ethanol-related contextual stimulus or a stimulus conditioned to a palatable conventional reinforcer (SuperSac) similarly. The data confirm that antagonizing σ₁ receptors prevents ethanol-seeking behavior using a conditioned reinstatement model, similar to observations in an earlier study that used an ethanol-induced conditioned place preference procedure in mice [13]. The results also show that pharmacological blockade of σ₁ receptors did not dissociate the motivational effects of ethanol- vs. natural reward-related stimuli.

The reason BD1047 produced identical behavioral effects on both ethanol and SuperSac seeking is not presently clear. However, one tentative explanation for the nonselective effect of BD1047 may be similarities in the neurocircuits that regulate ethanol intake and the overconsumption of palatable food. For example, hypothalamic peptides regulate feeding and ethanol drinking (e.g., [21] and [22] for review), such that overlapping neurocircuitry may exist that mediates both ethanol and natural reward (e.g., [23,24]). Sigma₁ receptors are expressed in the nucleus accumbens, ventral tegmental area, hypothalamus, and hippocampus [3–5], structures with known roles in normal motivated behavior, drug abuse, and addiction. Therefore, the antagonism of σ₁ receptors within these brain regions may indiscriminately prevent ethanol or SuperSac seeking.

Another possible explanation for the same behavioral effects of BD1047 on both ethanol and SuperSac seeking can be gleaned from neuroanatomical findings on the hippocampal distribution of σ₁ receptors. Particularly relevant, σ₁ receptors are highly expressed in the hippocampus [3], a brain site with an established role in the occasion-setting action of contextual stimuli (e.g., [25]). Therefore, one may hypothesize that the σ₁ receptor antagonist in the present study interfered with the processing of reward (ethanol and SuperSac)-related contextual information at the hippocampal level, reducing its motivational impact. However, caution must be taken with this interpretation because BD1047 was injected peripherally, a nonspecific route of administration commonly used to test the effects of σ₁ receptor ligands. Therefore, a more comprehensive understanding of the pharmacological profile of BD1047 relevant to reinstatement and relapse is needed. Additionally, assessments of the specific links between the effects of σ₁ receptor manipulations in anatomically distinct brain regions on ethanol vs. SuperSac seeking will remain for future research.

The data presented here differ somewhat from a previous report that tested the effects of BD1047 on conditioned reinstatement of cocaine seeking vs. conditioned reinstatement of another highly palatable conventional reinforcer, sweetened condensed milk (SCM) [26]. That study reported that BD1047 selectively prevented conditioned reinstatement of cocaine-seeking behavior over SCM-seeking behavior at a dose of 20 mg/kg. An equivalent dose of BD1047 was effective in the present study but also blocked SuperSac seeking. The discrepancy between the present findings on the effects of BD1047 on SuperSac conditioned reinstatement vs. the earlier findings of lack of effects on SCM seeking may be related to the different conditioning procedures in these studies. In the earlier study [26], the rats were trained using identical contingencies for both cocaine and SCM (i.e., fixed-ratio 1 [FR1] schedule with a 20 s timeout), whereas in the present study, the reinforcement contingency for ethanol and SuperSac was a continuous schedule of reinforcement (i.e., FR1 and no timeout). Therefore, one possibility is that under these latter contingencies, conditioned reinstatement of natural reward-seeking behavior was more sensitive to the inhibitory effects of BD1047 [26]. Another possibility may be related to the quality (i.e., composition) of the conventional reinforcers in the two studies. Importantly, the main difference between SuperSac and SCM is the presence of fat in the SCM solution. The fat component combined with the sweetness of SCM may have created an additional factor for a strong association between SCM and the cues during conditioning. This stronger association may have translated into more resistant conditioned reinstatement to the σ₁ receptor antagonist’s effect.

Notably, although the results strongly suggest that the behavioral effects measured here are mediated by σ₁ receptors, one limitation of the present study is the lack of reversal data for the BD1047 effects using a specific σ₁ receptor agonist, such as 2-morpholin-4-ylethyl 1-phenylcyclohexane-1-carboxylate (PRE-084) or igmesine [12]. To confirm that BD1047’s effects are mediated through a direct interaction with σ₁ receptors, BD1047’s behavioral effects would have to be reversed when co-administered with a specific σ₁ receptor agonist. This will be an area for further investigation.

The antagonism of σ₁ receptors is particularly efficient in preventing ethanol drinking in animals that are postdependent and genetically predisposed to high ethanol intake [14,15,27]. Knowing that neuroadaptive changes, such as the increased expression of σ₁ receptors, occur following drug exposure (for detailed review, see [11,12]) and considering the importance of relapse prevention in postdependent individuals, an important question that remains to be answered is whether the effects of σ₁ receptor blockade on ethanol conditioned reinstatement increase in animals with a history of ethanol dependence. The efficacy of σ₁ receptor antagonists is expected to increase in postdependent animals (i.e., their effects will appear at lower doses because of increased σ₁ receptor expression), a hypothesis to be tested in the future.

Conclusion

The present findings show that σ₁ receptor antagonism using BD1047 prevented both ethanol and SuperSac seeking following the same dose-effect function. The data, together with previously published reports, suggest that σ₁ receptors participate in the regulation of reward-seeking behavior in general.

Abbreviations:

BD1047

N-(2-[3,4-dichlorophenyl]ethyl)-N-methyl-2-(dimethylamino) ethylamine dihydrobromide

σ₁R

sigma-1 receptor