N6-substituated adenosine analog J4 attenuates anxiety-like behaviors in mice

Abstract

Rationale: Withdrawal from chronic alcohol exposure produces various physical and mental withdrawal symptoms. Activation of adenosine receptors is known to inhibit withdrawal-induced excitation. However, limited studies investigate how adenosine analogs may prove helpful tools to alleviate alcohol withdrawal-related affective behaviors.

Objectives: This study aimed to investigate the effects of J4 compared with saline using the mice vapor or voluntary ethanol drinking model on behavioral endpoints representing ethanol-withdrawal negative emotionality commonly observed during abstinence from chronic alcohol use.

Methods: We subjected C57BL/6 J mice to chronic intermittent ethanol (CIE) exposure schedule to investigate how 72-h withdrawal from alcohol alters affective-like behavior. Next, we determined how treatment with J4, a second-generation adenosine analog, influenced affective behaviors produced by alcohol withdrawal. Finally, we determined how J4 treatment alters voluntary ethanol drinking using the two-bottle-choice drinking paradigm.

Results: Our results show that 72-h withdrawal from chronic intermittent ethanol exposure produces limited affective-like disturbances in male C57BL/6 J mice exposed to 4 cycles ethanol vapor. Most importantly, J4 treatment irrespective of ethanol exposure decreases innate anxiety-like behavior in mice.

Conclusions: Withdrawal from chronic intermittent ethanol exposure and subsequent behavioral testing 72 h later produces minimal affective-like behavior. J4 treatment did however reduce marble-burying behavior and increased time spent in open arms of the elevated plus maze, suggesting J4 may be useful as a general anxiolytic.

Keywords: Adenosine; Elevated-plus maze; J4; Marble-burying; Two-bottle choice.

Figure 1.

(a) Schematic representation of chronic intermittent ethanol (CIE) schedule. Mice were exposed to EtOH vapor for 16 h/day for 4 consecutive days followed by a 72-h withdrawal period. This 7-day schedule of EtOH vapor exposure was repeated for 4 weeks. Upon the final removal from the EtOH vapor chamber on weeks 4, J4 was injected once daily (i.p) for 3 consecutive days during the 72-h withdrawal period. (b) Schematic representation of two-bottle choice voluntary drinking paradigm. Briefly, mice were allowed 24 h access to two bottles (water and EtOH). Mice were gradually escalated to 10% EtOH drinking in a stepwise fashion (3% to 6% to 10%) after which, the behavioral effects of J4 treatment on voluntary EtOH drinking, preference, and behavior following 72 h withdrawal determined.

Figure 2.

The effects of J4 treatment on locomotor activity were determined using open-field test (OFT). J4 treatment (0.1 mg/kg) significantly decreased total distance traveled during a 30 min open field session (a). Additionally, J4 treatment had no effect on vertical counts (b) or jump counts (c). n = 4/5 per group.

Figure 3.

Elevated plus maze assessment of J4 treatment following 72 h EtOH vapor withdrawal. i.p. administration of J4 (0.03 mg/kg) had no effect on frequency to enter open arms (a), latency to enter open arms (b), nor time spent in open arms (c) after a 5-minute assessment. Likewise, a 10-minute assessment of EPM activity provided similar results for frequency to enter open arms (d), Latency to enter open arms (e) and time spent in open arms (f). n = 10 per group.

Figure 4.

Marble burying task assessment following 72 h withdrawal from chronic intermittent EtOH vapor exposure (CIE). (a) Administration (i.p.) J4 (0.03 mg/kg) in CIE treated mice reduced the number of marbles buried after withdrawal. (b) Representative image depicting marble burying behavior (right panel). n = 10 per group

Figure 5.

Effects of J4 treatment on 10% ethanol drinking. Three consecutive days J4 treatment showed no differences in the amount of ethanol consumed (a) or the preference (b) for ethanol versus vehicle treated counterparts. n =11–13 per group.