Increased ethanol consumption and preference in mice lacking neurotensin receptor type 2

Abstract

Background: Neurotensin receptors (NTS) regulate a variety of the biological functions of neurotensin (NT) in the central nervous system. Although NT and neurotensin receptors type 1 (NTS1) are implicated in some of the behavioral effects of ethanol, the functional roles of neurotensin receptors type 2 (NTS2) in ethanol intoxication and consumption remain unknown. Here, we investigated behavioral effects mediated by NTS2 in response to ethanol, which are implicated in ethanol consumption and preference, using NTS2 null mice.

Method: First, we examined ethanol-induced locomotion, ataxia, hypnosis, and hypothermia in NTS2 null mice. Next, we measured ethanol consumption and preference in NTS2 null mice by giving them free choice between ethanol- and tap water-containing bottles. Then using a brain-permeable NT analog, NT69L, we examined the role of NTS2 in locomotor activity and ataxia. Finally, we examined the effect of NT69L on ethanol consumption and preference in NTS2 null mice.

Results: We found that NTS2 null mice appear less sensitive to the acute hypnotic effects of ethanol and consumed more ethanol compared to wild-type littermates in a 2-bottle choice experiment, even though ethanol-induced locomotion, ataxia, and hypothermia were similar between genotypes. Interestingly, the administration of NT69L for 4 consecutive days significantly reduced alcohol consumption and preference in wild-type littermates as well as in NTS2 null mice.

Conclusions: Our findings suggest that NTS2 regulates ethanol-induced hypnosis and ethanol consumption.

Fig. 1

Ethanol-induced locomotor activity and ataxia in NTS2 null and wild-type mice. (A) No significant differences in basal spontaneous locomotor activity were observed in NTS2 null mice compared to their wild-type littermates (n = 13 for each genotype). (B-C) Administration of 1.5 g/kg ethanol (i.p.) did not induce any significant changes in locomotor activity in wild-type mice (B) or NTS2 null mice (C) (n = 8 for each genotype). (D) Similarly, NTS2 null mice showed no significant reduction of ethanol-induced (1.5 g/kg) ataxia in a rotarod test compared to their wild-type littermates (n = 11 for each genotype). All data are expressed as mean ± SEM.

Fig. 2

Effect of acute, hypnotic doses of ethanol in NTS2 null and wild-type mice. (A) Reduced hypnotic effect of ethanol measured by the loss of the righting reflex (LORR) in NTS2 null mice, compared to wild-type littermates (n = 8 for each genotype and ethanol dose). There were significant effects of genotype (p = 0.034) and ethanol dose (p = 0.031), without a significant interaction between genotype and ethanol dose. (B-C) However, no significant differences in ethanol-mediated hypothermia after 3.2 g/kg (B) or 3.6 g/kg (C) ethanol administration were observed between NTS2 null (n = 9 for each ethanol dose) and their wild-type littermates (n = 11 for each ethanol dose). (D-E) Similarly, no significant difference in blood ethanol clearances after acute administration of 3.2 g/kg (D) or 3.6 g/kg (E) ethanol were observed in NTS2 null mice compared to their wild-type littermates (n = 10 for each genotype and ethanol dose). All data are expressed as mean ± SEM.

Fig. 3

Ethanol consumption and preference of NTS2 null and wild-type mice. (A-B) NTS2 null mice (n = 14) showed increased ethanol consumption (A) and preference (B) in a two-bottle choice experiment. *p < 0.05 compared to the wild-type mice (n = 13) at the same ethanol concentration (Tukey test). (C) No differences in genotype-associated taste preference (saccharin for sweet and quinine for bitter taste) of NTS2 null mice compared to wild-type littermates (n = 10 for each genotype). All data are expressed as mean ± SEM.

Fig. 4

NT69L-induced locomotor activity and ataxia in NTS2 null and wild-type mice. (A-B) 1.0 mg/kg NT69L (in saline, i.p.) induced a significant reduction in locomotor activation compared to their saline-treated groups both in wild-type mice (A) and NTS2 null mice (B) (n = 9 for wild-type mice and n = 6 for NTS2 null mice; *p < 0.05 by t-test). (C) NTS2 null mice showed an earlier activation of NT69L-mediated ataxia at 15 min after NT69L injection, but a similar NT69L-mediated ataxic compared to their wild-type littermates at 1.0 mg/kg NT69L from 30 min to 60 min after NT69L injection (n = 10 for each genotype). All data are expressed as mean ± SEM. *p < 0.05 compared to their saline-treated groups both in NTS2 null mice and wild-type littermates (Tukey test). #p < 0.05 compared to wild-type littermates (Tukey test).

Fig. 5

Effect of NT69L on ethanol consumption and preference in NTS2 null and wild-type mice. (A-B) Ethanol consumption (A) and preference (B) for ethanol are significantly reduced both in wild-type (n = 9) and NTS2 null mice (n = 8) during the NT69L-injection period. Post-injection period indicates 4 days immediately following the NT69L-injection period, whereas pre-injection period refers to 4 days prior to the NT69L-injection period. All injection periods were preformed while mice stably consumed 10% ethanol. All data are expressed as mean ± SEM. *p < 0.05 compared to the pre- or post-injection period as indicated (Tukey test).