Galanin receptor subtype 2 (GalR2) null mutant mice display an anxiogenic-like phenotype specific to the elevated plus-maze

Abstract

The neuropeptide galanin has been implicated in anxiety-related behaviors, cognition, analgesia, and feeding in rodents. Neuromodulatory actions of galanin are mediated by three G-protein coupled receptors, GalR1, GalR2, and GalR3. The present study investigates the role of the GalR2 receptor by evaluating behavioral phenotypes of mice with a targeted mutation in the GalR2 gene. A three-tiered behavioral phenotyping approach first examined control measures of general health, body weight, neurological reflexes, sensory abilities and motor function. Mice were then assessed on several tests for cognitive and anxiety-like behaviors. GalR2 null mutants and heterozygotes were not significantly different from wildtype littermates on two cognitive tests previously shown to be sensitive to galanin manipulation: acquisition of the Morris water maze spatial task, and trace cued and contextual fear conditioning, an emotional learning and memory task. Two independent cohorts of GalR2 null mutant mice demonstrated an anxiogenic-like phenotype in the elevated plus-maze. No genotype differences were detected on several other measures of anxiety-like behavior. The discovery of an anxiogenic phenotype specific to the elevated plus-maze, similar to findings in GalR1 null mutants, highlights the potential therapeutic efficacy of targeting GalR1 and GalR2 receptors in treating anxiety disorders.

Figure 1

Anxiogenic-like phenotype of GalR2 on the elevated plus maze. Two independent cohorts of GalR2 −/− displayed an anxiogenic-like phenotype compared to their +/+ littermates in the elevated plus-maze. GalR2 −/− mice spent significantly * less time in the open arms (1a) and made fewer entries into the open arms (b) than +/+ mice. The −/− mice in experiment 1 made significantly * more entries into the closed arms (c), while total arm entries was similar across genotypes (d) suggesting that less exploration of open arms did not reflect lower overall exploratory behavior. The genotypes did not differ on total arm entries (1c). For the figures in graphs 1a–d Cohort 1 N = 22 +/+, 23 +/−, 19 −/−; Cohort 2 N = 14 +/+, 12 +/−, 17 −/−. * p < .05 as compared to +/+.

Figure 2

Light ↔ dark exploration, open field exploration and stress-induced hyperthermia. The genotypes did not differ significantly on the number of transitions between the light and dark compartments (a) or in the percentage of time spent in the dark compartment (b) demonstrating similar responding across the genotypes in the light ↔ dark exploration test. For figures 2a, b N = +/+ = 22, +/− = 23 and −/− = 17. There was no significant effect of genotype on horizontal activity (c) or total distance traveled (d) in the open field exploration test. There was a significant effect of sex on both of these measures indicating that females traveled significantly shorter distances than males. GalR2 −/− mice showed decreased center exploration time (e) consistent with an anxiogenic-like phenotype that did not reach statistical significance (p > .05) N = +/+ = 38, +/− = 41, −/− = 36. There was no significant effect of genotype on the increase in body temperature in the second rectal probe measurement as compared to the baseline measurement (f). Handling and prior temperature probe measurement significantly increased the body temperature of all genotypes. N = +/+ = 10, +/− = 10, −/− = 13.

Figure 2

Light ↔ dark exploration, open field exploration and stress-induced hyperthermia. The genotypes did not differ significantly on the number of transitions between the light and dark compartments (a) or in the percentage of time spent in the dark compartment (b) demonstrating similar responding across the genotypes in the light ↔ dark exploration test. For figures 2a, b N = +/+ = 22, +/− = 23 and −/− = 17. There was no significant effect of genotype on horizontal activity (c) or total distance traveled (d) in the open field exploration test. There was a significant effect of sex on both of these measures indicating that females traveled significantly shorter distances than males. GalR2 −/− mice showed decreased center exploration time (e) consistent with an anxiogenic-like phenotype that did not reach statistical significance (p > .05) N = +/+ = 38, +/− = 41, −/− = 36. There was no significant effect of genotype on the increase in body temperature in the second rectal probe measurement as compared to the baseline measurement (f). Handling and prior temperature probe measurement significantly increased the body temperature of all genotypes. N = +/+ = 10, +/− = 10, −/− = 13.

Figure 3

Hotplate, tail flick, and body weight. There was no significant genotype difference in latency to respond on the hotplate test (a) Cohort 1 N = +/+ = 22, +/− = 23, −/− = 18. GalR2 −/− mice in Cohorts 1 and Cohort 2 N = +/+ = 15, +/− = 27, −/− = 19 showed similar tail flick response latencies compared to +/+ controls (b). All genotypes increased in weight across time (c). GalR2 −/− males (Cohort 1) were significantly heavier than +/− mice at 28 and 32 weeks of age.GalR2 +/− females (Cohort 1) were significantly heavier than −/− at all ages and significantly heavier than +/+ littermates at 16 and 32 weeks. No significant differences in body weights were detected in Cohort 2 mice.

Figure 4

Trace cued contextual fear conditioning. GalR2 −/− mice showed similar levels of freezing as wildtype littermates in all phases of the trace cued fear conditioning test: Post training levels (a); contextual test 24 hours later (b) and during the novel context auditory cued test 48 hours post training (c). N = +/+ = 10, +/− =10, −/− = 12.

Figure 5

Morris water maze. Latency to locate the visible and hidden platforms during Morris water maze acquisition was similar for all genotypes (a). All genotypes demonstrated spatial learning during the probe trial by spending significantly more time in the trained quadrant (b). All genotypes made significantly more platform crossings in the trained quadrant (c) than in the other three quadrants. N = +/+ = 22, +/− = 20, −/− = 18.