Variability in the benzodiazepine response of serotonin 5-HT1A receptor null mice displaying anxiety-like phenotype: evidence for genetic modifiers in the 5-HT-mediated regulation of GABA(A) receptors

Abstract

Benzodiazepines (BZs) acting as modulators of GABA(A) receptors (GABA(A)Rs) are an important group of drugs for the treatment of anxiety disorders. However, a large inter-individual variation in BZ sensitivity occurs in the human population with some anxiety disorder patients exhibiting diminished sensitivity to BZ and reduced density of GABA(A)Rs. The mechanism underlying BZ treatment resistance is not known, and it is not possible to predict whether an anxiety patient will respond to BZ. 5-hydroxytryptamine1A receptor (5-HT1AR) null mice (R-/-) on the Swiss-Webster (SW) background reproduce several features of BZ-resistant anxiety; they exhibit anxiety-related behaviors, do not respond to BZ, have reduced BZ binding, and have decreased expression of the major GABA(A)R subunits alpha1 and alpha2. Here, we show that R-/- mice on the C57Bl6 (B6) background also have anxiety phenotype, but they respond to BZ and have normal GABA(A)R subunit expression. This indicates that the 5-HT1AR-mediated regulation of GABA(A)R alpha subunit expression is subject to genetic modification. Hybrid SW/B6-R-/- mice also exhibit BZ-resistant anxiety, suggesting that SW mice carry a genetic modifier, which mediates the effect of the 5-HT1AR on the expression of GABA(A)Ralpha subunits. In addition, we show that this genetic interaction in SW mice operates early in postnatal life to influence the expression of GABA(A)R alpha subunits at the transcriptional level. These data indicate that BZ-resistant anxiety results from a developmental arrest of GABA(A)R expression in SW-R-/- mice, and a similar mechanism may be responsible for the BZ insensitivity of some anxiety patients.

Figure 6.

Altered mRNA stability does not account for the down regulation of the GABA_ARα₁ and α₂ subunit expression in the cortex of P28 SW-R^-/- mice. A, In actinomycin-D-treated samples, the half-lives of the α₁, α₂, and γ₂ subunit mRNAs are similar in R^+/+ and R^-/- mice. B, Identification of mRNP, monosome (mono), and polysome (poly light) pools separated by sucrose density gradient sedimentation. Distribution of ribosomal RNAs (top panel) and of Jerky and P0 proteins (bottom panel) from fractions 1-12 is shown. Fractions 13-23 were divided equally to poly intermediate and poly heavy pools. C, Association of α_1/2 subunit mRNAs with mRNP, monosome, and polysome pools detected by real-time RT-PCR. For each pooled sample, abundance in both R^-/- and R^+/+ mice is expressed relative to the R^+/+ mRNA pool (mean ± SE, n = 3; ^* indicates a significant difference between genotypes; p < 0.01; Fisher post hoc test).

Figure 1.

A-D, Anxiety-related behavior in 5-HT_1AR^-/- mice in the elevated plus maze (A, B) and open field (C, D). Percentage of time spent in the open arm (A) and percentage of open arm entries (B) in the elevated plus maze as well as percentage of time spent in the center (C) and entries to the center (D) in the open field are shown (mean ± SD). R^-/-mice (▪) on both the SW and B6 backgrounds exhibit increased anxiety-like behavior compared with their respective controls (R^+/+, □) (^*p < 0.05 and ^**p < 0.01; n = 6-10 per group). B6-R^+/+ mice also displayed more anxiety-like behavior than SW-R^+/+ mice in some measures (^†p < 0.05).

Figure 2.

Anxiolytic effect of diazepam in the elevated plus maze. A-F, Mean (±SD) percentage of open arm time and entries for SW (A, D), B6 (B, E), and hybrid SW/B6 (C, F) R^+/+ and R^-/- mice. The anxiolytic effect of the BZ diazepam (0.2, 1 mg/kg), compared with vehicle-injected controls (V), was evident in all R^+/+ mice and in B6-R^-/-mice (^*p < 0.05; ^**p < 0.01; n = 12-16 per group). SW-R^-/- (A, D) and SW/B6-R^-/- (C, F) mice were not responsive. G, H, The sedative effect of the BZ diazepam (1, 3, 10 mg/kg) detected as a decrease in locomotor activity (distance traveled times 10³ cm/hr) is also displayed. Diazepam (10 mg/kg) (BZ) resulted in significant reduction in locomotor activity in SW-R^+/+ (G), B6-R^+/+, and B6-R^-/- (H) but not in SW-R^-/- (G) mice (^*p < 0.05; ^**p < 0.01; n = 8 -12 per group). Decreased locomotor activity of SW R^-/- (G, lane V) and B6-R^-/- (H, lane V) compared with their background-matched controls was also detected (^††p < 0.01; ^†††p < 0.001).

Figure 3.

GABA_AR subunit mRNA expression in 5-HT_1AR^+/+ and R^-/-mice. A-D, GABA_AR α₁, α₂, and γ₂ subunit mRNA abundance in frontal cortex (A, C) and amygdala (B, D) was measured by real-time RT-PCR. Levels in R^-/- for each gene are expressed relative to the corresponding strain background R^+/+ levels (A,B). In C and D, expression levels in R^+/+ mice on different backgrounds are directly compared with each other. Levels in B6- and hybrid SW/B6-R^+/+ are expressed relative to SW R^+/+ level (dotted line, 1). Significant changes in expression are indicated (^*p < 0.05; mean ± SE; n = 3).

Figure 4.

GABA_AR subunit protein expression in 5-HT_1AR^-/- mice. Protein levels were estimated by semiquantitative Western blot analysis. A, Representative blots showing increasing amounts of total protein (10, 20, and 40 μg) from frontal cortex and amygdala samples were probed with specific antibodies against the α₁ and α₂ subunits. B, Examples of quantification of the intensity of α₁ immunolabeling in the cortex of SW, B6, and hybrid SW/B6 mice (R^+/+, ○;R^-/-, •). C, D, Relative subunit expression level is displayed as the ratio of R^-/- to R^+/+ labeling intensity for each strain (^*p < 0.05; mean ± SE; n = 2 two independent blots).

Figure 5.

Developmental arrest of GABA_ARα₁ andα₂ subunit expression in frontal cortex of SW-R^-/- mice. A, Developmental time course of subunit mRNA accumulation in SW-R^+/+ mice measured by real-time RT-PCR. For each gene, expression at each stage is relative to P28 value (mean ± SE; n = 3). B, GABA_AR subunit mRNA abundance in R^-/- at each postnatal stage is expressed relative to R^+/+ level (^*p < 0.05; t test; ±SE; n = 6). C, Semiquantitative Western blot analysis of GABA_AR subunit levels expressed as the ratio of R^-/-:R^+/+ mice (^*p < 0.05; mean ± SE; n = 6). D, Developmental expression of the presynaptic marker synaptophysin and the postsynaptic PSD-95 are not altered in SW-R^-/- compared with SWR^+/+ mice. Mean (±SE) relative intensity of immunoblot labeling is expressed relative to P28 (n = 3).

Figure 7.

Summary of the interaction between 5-HT_1AR and GABA_AR α subunits in the amygdala of SW and B6 mice. In SW, the 5-HT_1AR has a positive effect (yellow arrows) on α subunit transcriptional activation. In B6, the 5-HT_1AR has no effect or an inhibitory action (yellow line) on α subunit mRNA expression. At the translational or post-translational level (blue arrows), there is also positive feedback regulation in B6 (blue line) but not in SW mice. Inactivation of the 5-HT_1AR in SW mice therefore results in a down regulation of α_1/2 subunits, altered assembly of functional GABA_ARs (green lines), which are not responsive to BZ. The expression of GABA_AR α subunits is unchanged in B6-R^-/- mice. Gray shading represents changes in subunit expression (the same level as background represents no change) and the diameter of circles scales with the magnitude of the change. For simplicity, genetic background-dependent differences in subunit expression are not taken into account.