Short-term exposure to a neuroactive steroid increases alpha4 GABA(A) receptor subunit levels in association with increased anxiety in the female rat

Abstract

Previous work from this laboratory has demonstrated that withdrawal from the neuroactive steroid 3alpha,5alpha-THP (3alpha-hydroxy-5alpha-pregnan-20-one) after 3-week exposure to its parent compound, progesterone (P), increases anxiety and produces benzodiazepine (BDZ) insensitivity in female rats. These events were linked to upregulation of the alpha4 subunit of the GABA(A) receptor (GABAR) in the hippocampus [Brain Res. 507 (1998) 91; Nature 392 (1998) 926; J. Neurosci. 18 (1998) 5275]. The present study investigates the role of shorter term hormone treatment on alpha4 subunit levels as well as relevant behavioral and pharmacological end-points related to GABAR function. After 2-3 days of P exposure, two- to threefold increases in alpha4 protein levels were observed, which declined to control values after 5-6 days of hormone exposure. This effect was due to the GABA-modulatory metabolite of P, 3alpha,5alpha-THP. alpha4 upregulation was inversely correlated with BDZ potentiation of GABA-gated current, assessed using whole cell patch clamp techniques on acutely isolated hippocampal pyramidal cells. A near total BDZ insensitivity was observed by 2-3 days of hormone exposure in association with the maximal increase in alpha4 levels. Up-regulation of the alpha4 GABAR subunit was also reflected by an increase in anxiety in the elevated plus maze. A significant decrease in open arm entries was observed after 72-h exposure to P, an effect which recovered by 6 days of P treatment. As demonstrated in vitro, alpha4 upregulation also resulted in a relative insensitivity to the anxiolytic actions of BDZ. These results suggest that short-term exposure to 3alpha,5alpha-THP produces changes in GABAR subunit composition similar to those that occur after chronic exposure and withdrawal from the steroid.

Fig. 1

Time course of increases in α4 GABAR subunit levels after P capsule implantation. (A) Statistical analysis of the data is represented as the integrated optical density of the immunoreactive bands and is expressed as a ratio of the control condition. Asterisks above 2 day, 3 day and WD indicate a statistically significant (P<0.05) increase above control levels. The numbers inside the bars are the individual n for each condition in experiments that were performed in triplicate. (B) Representative Western blot of α4 immunoreactivity across the first 5 days of hormone exposure showing typical values for the 67-kDa α4 subunit band. The levels of the 36-kDa control protein, GAPDH, do not change during hormone treatment.

Fig. 2

P-induced increases in α4 subunit levels are mediated by 3α,5α-THP. Representative Western blot demonstrating that the increase in the 67-kDa α4 subunit immunoreactivity seen after 48-h treatment with P (i.p. injection) is replicated by direct i.p. injection of 3α,5α-THP. Furthermore, it is also blocked by indomethacin injected simultaneously with P (P+Indo) which prevents conversion to 3α,5α-THP. The levels of the 36-kDa control protein, GAPDH, do not change during treatment (n=4 independent experiments performed in triplicate).

Fig. 3

Levels of α4 mRNA are increased following short-term exposure to 3α,5α-THP. A representative gel is presented indicating α4 mRNA levels (503 bp) in conjunction with the housekeeping mRNA GAPDH (657), following RT-PCR amplification. Forty-eight-hour exposure to 3α,5α-THP in vivo produced a significant increase in band density compared to control in the absence of significant changes in band density for the GAPDH control. No staining was apparent for the 0 DNA control (data not shown) (n=3 animals/group; performed in triplicate).

Fig. 4

BDZ potentiation of GABA-gated current is attenuated after 48-h exposure to P. Concentration–response curves illustrate responses of CA1 pyramidal neurons to GABA (10 μM), applied in combination with the BDZ, lorazepam (LZM, 0.1–100 μM), and assessed using whole cell patch clamp recording techniques. Results are expressed as a percent increase in peak GABA-gated current above levels generated by application of GABA alone. P exposure (2–3 days via s.c. implant) markedly reduces the BDZ potentiation of GABA-gated current normally observed under control conditions (upper left) (n=10–15 cells per group; *P<0.01 vs. control).

Fig. 5

Time course of measures of anxiety across the P exposure period. (A, left axis): time spent (s) in the open arm of the elevated plus maze is decreased after 3 days of P exposure (P3 day, 5 mg in oil, i.p.) compared to vehicle injected rats (C) (P3 day vs. C, *P<0.001), and returns to control levels after 6 days of P injections (P6 day). Lorazepam (LZM) significantly increases time spent in the open arm compared to vehicle, assessed in control animals (LZM vs. C, *P<0.001). In contrast, LZM does not increase time spent in the open arm after 3 days P injections (P3 day LZM) (LZM vs. P3 day LZM, ^∞P<0.001;). (A, right axis): P treatment for 21 days (P21 day) is similar to control conditions (C-WD) while P withdrawal (WD) significantly decreased time spent in the open arm compared to controls (PWD vs. C-WD, *P<0.02) or to P 21 day treatment (PWD vs. P21 day, ⁺P<0.006). Sample sizes are indicated in the bottom of the bar in panel and are the same as for all graphs in Fig. 1. (B) LZM significantly increases time spent beyond the rail compared to vehicle injections in control animals (LZM vs. C, *P<0.001), but does not do so following 3 days P injections (^∞P<0.001; LZM vs. P3 day LZM). (C) There is no significant effect of any treatment on locomotor activity assessed by number of grid crosses in the elevated plus maze.

Fig. 6

Summary diagram: The time course of P-induced changes in α4 levels, LZM response and anxiety. Illustration of α4 levels (on the left axis, ratio of control) and BDZ potentiation of GABA-gated current (in vitro LZM response, right axis, % control) reveals an inverse correlation, with significant changes noted 2–3 days after P exposure, and again following P withdrawal, compared to control values for both parameters. In conjunction with α4 upregulation, anxiety score (center axis, % control) increases at 3 days of P treatment (3 day) and again following P withdrawal (WD). Anxiety score is defined in the Methods. Error bars are omitted for clarity — for complete details, see Figs. 1, 4 and 6.