gamma-Aminobutyric acid-type A receptor deficits cause hypothalamic-pituitary-adrenal axis hyperactivity and antidepressant drug sensitivity reminiscent of melancholic forms of depression

Abstract

Background: The gamma-aminobutyric acid (GABA) Type A receptor deficits that are induced by global or forebrain-specific heterozygous inactivation of the gamma2 subunit gene in mouse embryos result in behavior indicative of trait anxiety and depressive states. By contrast, a comparable deficit that is delayed to adolescence is without these behavioral consequences. Here we characterized gamma2-deficient mice with respect to hypothalamic-pituitary-adrenal (HPA) axis abnormalities and antidepressant drug responses.

Methods: We analyzed the behavioral responses of gamma2(+/-) mice to desipramine and fluoxetine in novelty suppressed feeding, forced swim, tail suspension, and sucrose consumption tests as well as GABA(A) receptor deficit- and antidepressant drug treatment-induced alterations in serum corticosterone.

Results: Baseline corticosterone concentrations in adult gamma2-deficient mice were elevated independent of whether the genetic lesion was induced during embryogenesis or delayed to adolescence. However, the manifestation of anxious-depressive behavior in different gamma2-deficient mouse lines was correlated with early onset HPA axis hyperactivity during postnatal development. Chronic but not subchronic treatment of gamma2(+/-) mice with fluoxetine or desipramine normalized anxiety-like behavior in the novelty suppressed feeding test. Moreover, desipramine had antidepressant-like effects in that it normalized HPA axis function and depression-related behavior of gamma2(+/-) mice in the forced swim, tail suspension, and sucrose consumption tests. By contrast, fluoxetine was ineffective as an antidepressant and failed to normalize HPA axis function.

Conclusions: Developmental deficits in GABAergic inhibition in the forebrain cause behavioral and endocrine abnormalities and selective antidepressant drug responsiveness indicative of anxious-depressive disorders such as melancholic depression, which are frequently characterized by HPA axis hyperactivity and greater efficacy of desipramine versus fluoxetine.

Figure 1. The γ2^+/− mice show increased anxiety-related behavior that is sensitive to chronic desipramine and fluoxetine

A, Separate groups of mice were subject to 28-day chronic treatment with vehicle, desipramine or fluoxetine, respectively, followed by analysis in the NSFT at 12 weeks of age. Vehicle-treated γ2^+/− vs. WT mice showed increased latency to feed (n = 8-16, p < 0.05 in both experiments, ANOVA, Dunnett's test), consistent with previous results (28). Chronic treatment of γ2^+/− mice with fluoxetine (20 mg/L in drinking water, left panel) or desipramine (480 mg/L in drinking water, right) had anxiolytic like effects in that both drugs reduced the latency to initiate feeding compared to vehicle controls (fluoxetine, n = 16, p < 0.01; desipramine, n = 10-11, p < 0.01; ANOVA, Dunnett's test). By contrast, drug treatment had no effect on WT mice (n = 8-13, p > 0.05 for either drug). B, Subchronic fluoxetine (5 days, 20 mg/L) vs. vehicle increased the mean latency of γ2^+/− mice to initiate feeding (n = 14-16, p < 0.05, ANOVA, Dunnett's test) and thus had an anxiogenic-like effect. By contrast, subchronic treatment of γ2^+/− mice with desipramine (480 mg/L) vs. vehicle had no effect on the mean latency to initiate feeding (n = 13-16, p > 0.05, ANOVA, Dunnett's test). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2. The γ2^+/− mice show antidepressant-like responses to desipramine but not fluoxetine in the FST and TST

A-D, Antidepressant drug- or vehicle-treated γ2^+/− and WT mice were subjected to the FST (A, C) and TST (B, D) at 13 and 14 weeks of age, corresponding to drug treatment for 35 and 42 days, respectively. (A) In the FST, vehicle-treated γ2^+/− vs. WT mice started to float sooner and spent more time immobile (n = 26-29, mean time to first immobility, p < 0.001; immobility, p < 0.001, ANOVA, Fisher's test). Desipramine (240 and 480 mg/L) vs. vehicle increased the time to first immobility (p < 0.05 and p < 0.001, resp.) and reduced the total immobility of γ2^+/− mice [n = 14-29, p < 0.01 (240 mg/ml), p < 0.001 (480 mg/ml)]. No significant drug effects were observed in γ2^+/− mice treated with 120 mg/L desipramine, nor with any desipramine concentration in WT mice. (B) In the TST, desipramine (240 mg/L) vs. vehicle reduced the immobility of γ2^+/− mice [(t)19 = 2.26, n = 10-11, p < 0.05) but had no significant effect on WT mice [t(23) = 0.98, n = 13, p(ns)]. Simple comparison of vehicle-treated γ2^+/− and WT mice revealed a tendency towards greater immobility in γ2^+/− mice [t(21) = 1.82, p = 0.08]. (C, D) Fluoxetine (20 mg/L, 35 days) paradoxically reduced the time to first immobility of WT mice in the FST (C) (n = 12-14, p < 0.001, ANOVA, Fisher's test) and lacked antidepressant like effects, irrespective of test, in γ2^+/− mice. Vehicle-treated γ2^+/− vs. WT littermates analyzed in parallel showed the depressive-like phenotype also evident in (p < 0.01)(A, B).

Figure 3. The γ2^+/− mice show antidepressant-like responses to desipramine but not fluoxetine in the SCT

A. Time course of experiment: Antidepressant drug treatment was initiated on Day 1 (8 weeks of age) and was switched to daily p.o. injections starting on the 22^nd day (fluoxetine, 4 mg/kg; desipramine, 30 mg/kg). Vehicle treated mice were handled identically but injected with water. The mice were singly housed starting on the 24^th day of drug treatment and trained to drink sucrose (10%) for three days starting on the 25^th day. Water consumption was measured over a period of three days from the 28^th to the 30^th day, and sucrose consumption was measured over 22 h on the last day of a four-day sucrose consumption period. B. Sucrose consumption: Vehicle-treated γ2^+/− mice consumed less sucrose than WT littermates (n = 13-15; p < 0.05, Fisher's test), indicating an anhedonia-like phenotype of γ2^+/− mice. Chronic treatment with desipramine had antidepressant-like effects in that it increased the sucrose consumption of both WT and γ2^+/− mice (n =11-15; p < 0.01 for both comparisons). By contrast, fluoxetine reduced the sucrose consumption of WT mice (n =14-15; p < 0.05) and had no effect on γ2^+/− mice. Similar genotype and treatment effects but with lower levels of significance were evident for sucrose consumption on day 31-33 (not shown). C. Water consumption assessed by pairwise comparisons of vehicle and drug treated groups was unaltered (p > 0.05 for all comparisons). However, desipramine treated mice showed a genotype independent reduction in water consumption (p < 0.05). D. Normalization of sucrose consumption data in (A) for water consumption in (B) reproduced the anhedonia like phenotype and accentuated the antidepressant drug effects observed without normalization.

Figure 4. Cort levels are elevated in γ2 subunit-deficient mice

A, B, Serum Cort levels in 8-week old female (A, B) and male (B) γ2^+/− and WT controls were measured at baseline and 7.5, 12.5, 30 or 60 min after a 5-min forced swim stressor. Cort levels of γ2^+/− vs. WT littermates were elevated both in behaviorally naïve animals (baseline) and after 60 min recovery from a 5-min stressor, and this effect was seen in both females and males (n = 9, p < 0.001 for all comparisons). Stress-induced Cort levels 7.5, 12.5 and 30 min after the end of the stressor were not different between genotypes (n = 3, p > 0.05 for all comparisons). C, Cort concentrations at 8 weeks of age of both Emx1Cre X fγ2/+ mice with an ‘early’ deficit and CaMKIICre2834 X fγ2/+ with a ‘late’ deficit were elevated compared to respective littermate fγ2/+ controls, both at baseline and after recovery from stress and in both male and female mice (n = 3-6, p < 0.001 for all comparisons, data shown for females only). D, Cort concentrations of γ2^+/− mice compared to WT littermates (mixed sex) were unaltered at P14 (n = 8,12) and P17 (n = 6,10; p > 0.05 in both cases) but markedly increased two days after weaning at P23 (n = 8, p < 0.05). By contrast, CaMKIICre2834 X fγ2/+ remained indistinguishable from littermate controls (CaMKIICre2834 and fγ2/+) until at least P23 (n = 7,9, p = 0.95). Values represent means ± SEMs (two sample two-tailed t-tests). For numerical values see Table S6 in Supplement 1.

Figure 5. HPA axis function of γ2^+/− mice is normalized by desipramine but not fluoxetine

A. Desipramine (240 mg/L, 28 days) vs. vehicle reduced the elevated Cort levels of γ2^+/− mice (p < 0.05), while WT mice were unaffected (n = 5-7, p > 0.05, ANOVA, Dunnett test). B. Fluoxetine (20 mg/L, 28 days) increased Cort levels of WT mice (n = 9, p < 0.05, ANOVA, Dunnett's test) and had no effect in γ2^+/− mice (n = 9, p > 0.05). Basal Cort levels were elevated in vehicle-treated γ2^+/− vs. WT mice (n = 9, p < 0.001) as evident by a strong tendency also in (A) (p = 0.07). Veh, vehicle; Des, desipramine; FLX, fluoxetine.