Progesterone withdrawal increases the alpha4 subunit of the GABA(A) receptor in male rats in association with anxiety and altered pharmacology - a comparison with female rats

Abstract

Withdrawal from the neurosteroid 3alpha,5alpha-allopregnanolone after chronic administration of progesterone increases anxiety in female rats and up-regulates the alpha4 subunit of the GABA(A) receptor (GABA(A)-R) in the hippocampus. We investigated if these phenomena would also occur in male rats. Progesterone withdrawal (PWD) induced higher alpha4 subunit expression in the hippocampus of both male and female rats, in association with increased anxiety (assessed in the elevated plus maze) comparable to effects previously reported. Because alpha4-containing GABA(A)-R are insensitive to the benzodiazepine (BDZ) lorazepam (LZM), and are positively modulated by flumazenil (FLU, a BDZ antagonist), we therefore tested the effects of these compounds following PWD. Using whole-cell patch clamp techniques, LZM-potentiation of GABA ((EC20))-gated current was markedly reduced in CA1 pyramidal cells of male rats undergoing PWD compared to controls, whereas FLU had no effect on GABA-gated current in control animals but increased it in PWD animals. Behaviorally, both male and female rats were significantly less sensitive to the anxiolytic effects of LZM. In contrast, FLU demonstrated significant anxiolytic effects following PWD. These data suggest that neurosteroid regulation of the alpha4 GABA(A)-R subunit may be a relevant mechanism underlying anxiety disorders, and that this phenomenon is not sex-specific.

Fig. 1

PWD increases GABA_A-R α4 subunit levels in male and female rat hippocampus. A Representative Western Blot. This figure illustrates α4 subunit immunoreactivity (left panel, Lanes 1 and 2 —PWD implanted (female or male), lane 3 and 4 — sham implanted (female or male). GADPH control immunoreactivity protein (right panel) is indicated by arrows and does not change in any condition. B 24 hours after removal of a chronic P implant both male (indicated by +) and female (indicated by *) rats have significantly (p < 0.001) higher levels of the α4 subunit in isolated hippocampal membranes than sham-implanted rats. Data are represented as integrated optical densities relative to control. The numbers inside the bars are the sample size for each condition.

Fig. 2

Pharmacological changes after PWD. The response to GABA (10 μM, EC₂₀) in CA1 pyramidal neurons when applied in combination with LZM or FLU and analyzed using whole-cell patch clamp techniques. Results are expressed as the percent increase in peak GABA-gated current. A Reduction of LZM potentiation after PWD is not sex specific. LZM (100 μM) potentiates GABA-gated current in CA1 neurons isolated from sham-implanted animals, but there was almost total insensitivity to LZM potentiation in neurons isolated from male PWD rats, similar to previously reported data in female rats. For both panels A and B, female control — open bars, female PWD — light closed bars, male control —hatched bars, male PWD — closed bars. Statistical significance in this and the following graphs is indicated by (*) at p<0.01 when comparing PWD to sham-implanted controls. Sample size is indicated by numbers at the bottom of the bars in figure A and B. B Potentiation of GABA-gated current by FLU following PWD is not sex dependent. FLU (10 μM) is without effect on GABA-gated current in control rats, but significantly increases peak current following PWD in male rats as has been demonstrated in female rats. C Concentration curve of LZM in male control and male PWD rats. GABA-gated current is increased in male control rats (open circles) across a range of concentrations of LZM (0.1–100 μM) while the same concentrations do not effectively potentiate GABA-gated current after PWD (closed circles). For Figure C and D, sample size is indicated by the numbers beside the circles. D Concentration curve of FLU in male control and male PWD rats. GABA-gated current is unaffected in male control rats (open circles) across a range of concentrations of FLU (0.1–100 μM) while the same concentrations effectively potentiate GABA-gated current after PWD (closed circles).

Fig. 3

PWD Increases anxiety that is insensitive to LZM but is positively modulated by FLU in both sexes. A Anxiety levels — time open arm. Bars indicate the mean time spent in the open arm (sec) of the elevated plus maze for either male (closed bars) or female (shaded bars) animals. Sham-implanted rats (in this figure and Fig. 4) are denoted by the type of injection they received (i.e., vehicle, LZM or FLU). Sample sizes are indicated in Fig. 4. PWD significantly decreased time spent in the open arm in both sexes. Some subjects received injections of LZM (0.75 mg/kg), which is significantly anxiolytic (increases time spent in the open arm) in sham-implanted rats of both sexes. In contrast, LZM is not anxiolytic in animals undergoing PWD. FLU (20 mg/kg) significantly increased time in the open arm only in PWD animals. FLU-injected animals with sham implants were not significantly different than vehicle-injected animals. There were no significant effects of sex and no interaction of sex and either drug or implant condition. Significant effects (p<0.009) are indicated by (*), in comparison to sham implanted animals, by (+) in comparison to PWD animals, by (#) in comparison to LZM and by (−) in comparison to FLU. Full details, additional statistical comparisons and the relevant p values are enumerated in table 1. B Time outside the rail. LZM significantly increased the percent time beyond the rail of the open arm in females relative to vehicle-injected rats. LZM injections following PWD are significantly less anxiolytic than LZM injections alone for females with regard to time spent beyond the rail. Although FLU injections have no significant effect in sham-implanted rats, they significantly increase the time spent beyond the rail in both sexes following PWD, relative to vehicle-injected rats and relative to LZM injections during PWD. C Percent time open arm. PWD significantly decreased the percent time spent in the open arm (relative to closed arm) in both sexes. LZM significantly increased the percent time spent in the open in comparison to vehicle-treated, sham-implanted rats (vehicle) of both sexes. However, LZM is not significantly anxiolytic following PWD in either sex. FLU injections had no significant effects on the percent time spent in the open arm in sham-implanted rats. However, after PWD, FLU injections are anxiolytic relative to sham-implanted subjects of both sexes injected with either vehicle or FLU. D Open arm entries/total arm entries. PWD significantly decreased the percent open arm entries relative to total entries in both sexes. LZM significantly increased the percent open arm entries in comparison to vehicle-treated, sham-implanted rats (vehicle). In females, however LZM is significantly less anxiolytic following PWD in both sexes. FLU injections had no significant effects on the percent open arm entries in sham-implanted rats. However, after PWD, FLU injections were anxiolytic relative to sham-implanted subjects of both sexes injected with either vehicle or FLU.

Fig. 4

PWD does not alter locomotor activity. A Grid crosses. Indicates the total number of grid crosses in a ten-minute test session in the elevated plus maze in either male (closed bars) or female (shaded bars) rats. There were no significant effects of implant condition or drug treatment on locomotor activity. Females had a general higher activity level than males, which did not interact with either drug or implant condition. Sample size for this figure and for Fig. 3 is indicated by the numbers at the base of the bars. B Total entries. Although ANOVA tests indicate a significant effect of total arm entries in females, post hoc comparisons (enumerated in Table 1) reveal that the only significant effects were an increase of total entries in female PWD rats receiving FLU injections compared to PWD rats and to FLU-injected sham-implanted controls (vehicle).