Neurosteroid administration and withdrawal alter GABAA receptor kinetics in CA1 hippocampus of female rats

Abstract

Withdrawal from the GABA-modulatory steroid 3alpha-OH-5alpha-pregnan-20-one (3alpha,5alpha-THP) following exposure of female rats to the parent compound progesterone (P) produces a syndrome characterized by behavioural excitability in association with up-regulation of the alpha4 subunit of the GABA(A) receptor (GABAR) in the hippocampus. Similar changes are seen after 48 h exposure to its stereoisomer, 3alpha,5beta-THP. Here, we further characterize the effects of P withdrawal on GABAR kinetics, using brief (1 ms) application of 5-10 mm GABA to outside-out patches from acutely isolated CA1 hippocampal pyramidal cells. Under control conditions, GABA-gated current deactivated biexponentially, with tau(fast) = 12-19 ms (45-60% of the current), and tau(slow) = 80-140 ms. P withdrawal resulted in marked acceleration of deactivation (tau(fast) = 3-7 ms and tau(slow) = 30-100 ms), as did 48 h exposure to 3alpha,5beta-THP (tau(fast) = 5-8 ms; tau(slow) = 40-120 ms). When recombinant receptors were tested in HEK-293 cells, a similar acceleration in tau(fast) was observed for alpha4beta2delta and alpha4beta2gamma2 GABARs, compared to alpha1beta2gamma2 and alpha5beta2gamma2 receptors. In addition, tau(slow) was also accelerated for alpha4beta2delta receptors, which are increased following steroid withdrawal. As predicted by the Jones-Westbrook model, this change was accompanied by reduced receptor desensitization as well as an acceleration of the rate of recovery from rapid desensitization. A theoretical analysis of the data suggested that steroid treatment leads to receptors with a greater stability of the bound, activatable state. This was achieved by altering multiple parameters, including desensitization and gating rates, within the model. These results suggest that fluctuations in endogenous steroids result in altered GABAR kinetics which may regulate neuronal excitability.

Figure 1. Progesterone withdrawal accelerates the deactivation of GABA-gated current

A, representative traces showing responses to brief (∼ 1 ms) pulses of GABA (10 mm) recorded from outside-out patches of CA1 hippocampal pyramidal cells following progesterone withdrawal (P Wd), 48 h 3α,5β-THP (48 h THP) or sham conditions (Control). Each trace represents the average of 6–10 individual traces. (Fits are shown next to full traces.) The deactivation rate is best described as a biexponential decay, with a τ_fast in the range of 10–22 ms and a τ_slow of 80–145 ms for the control recordings. Following P withdrawal (P Wd), in Group I 60% of the current deactivated with a τ_fast of 3–6 ms (mean = 4.88 ± 0.61 ms), and a τ_slow of 80–120 ms (mean = 87.0 ± 12.0 ms). In Group II, 40% of the current recorded deactivated with a τ_fast of 3–7 ms, and a τ_slow of 30–40 ms. Forty-eight hours of treatment with 3α,5β-THP produced similar acceleration in deactivation times. Note that in both populations, τ_fast is significantly faster than control values, while in Group II τ_slow is also significantly faster than control. Average peak amplitude was unaffected by prior steroid treatment. The top trace indicates the open tip junctional current. (These results are representative of those recorded from 20 to 30 patches/group.) Inset: amplified traces illustrate an accelerated τ_fast following P Wd compared to control. Inset, representative open tip junction potential for a control recording. B, distribution of values for τ_fast and τ_slow for control (Con, left panels), progesterone withdrawal (P Wd, middle panels), and 48 h treatment with 3α,5β-THP (48 h THP, right panels). Values for τ_slow display a bimodal distribution for P Wd and 48 h THP conditions. All other distributions display a single mode.

Figure 2. Kinetics of recombinant GABA_A receptor isoforms

Representative current traces (A) and averaged values (B) illustrate the different kinetics exhibited by recombinant α1β2γ2, α4β2γ2, α5β2γ2 or α4β2δ GABAR recorded from HEK-293 cells using whole cell or outside-out patch recording techniques. Both α4-containing GABAR isoforms deactivate with a faster τ_fast than α1 or α5-containing GABAR. (These results are averaged from 6–8 cells/group, *P < 0.05 versusα1β2γ2.)

Figure 3. Desensitization in response to episodic agonist application is attenuated following progesterone withdrawal

A, representative traces illustrate responses of pyramidal cells to trains of 1 ms GABA (10 mm) pulses applied at frequencies of 2, 8, 20 or 50 Hz. Following progesterone withdrawal (P Wd), desensitization developed at higher agonist application frequencies than seen for control, first apparent at 8 Hz and reaching a maximum of 18% at 50 Hz application frequencies. In contrast, under control conditions, desensitization was apparent with 2 Hz GABA pulses (200 ms interpulse interval) and reached an 84% maximum desensitization at a 50 Hz application. B, deactivation following 50 Hz GABA application was (P < 0.001) faster following P withdrawal (38.2 ± 4.3 ms) compared to control (110.2 ± 5.6 ms). (n = 12–16 cells/group).

Figure 4. Desensitization in response to prolonged agonist exposure is attenuated following steroid treatment

Representative traces illustrate significant attenuation in both the rate and degree of desensitization of GABA response following 48 h treatment with 3α,5β-THP (48 h THP). Desensitization kinetics (τ_fast, τ_slow) were determined for 5 s exposure to GABA (5 mm) using outside-out patches of membrane from acutely isolated CA1 hippocampal pyramidal cells following steroid treatment. Inset: deactivation following this prolonged exposure period was also significantly (P < 0.01) faster following steroid pretreatment (THP, average weighted τ= 20.45 ± 9.2 ms) compared to control (CON, average weighted τ= 148.11 ± 18.8 ms). (n = 20–25 patches/group).

Figure 5. Recovery from fast desensitization is accelerated following progesterone withdrawal

A, superimposed currents gated by paired 1 ms pulses of 10 mm GABA, at varying interpulse intervals (20– 2000 ms) are depicted for control, progesterone withdrawal (P Wd) or 48 h treatment with 3α,5β-THP (48 h THP). Following both steroid protocols, the extent of fast desensitization was reduced, and the rate of recovery from this desensitized state was accelerated compared to control as determined by the amplitude of the second GABA response relative to the first. B, the percentage recovery of current amplitude for the second GABA response relative to the first represents recovery from the fast desensitized state (D_fast), and is plotted as a function of the interpulse interval for averaged datapoints from both groups. Percentage recovery was calculated as ((Amp_test− Onset_test)/(Amp_init− Onset_test)) × 100, where Amp_init is the amplitude of the initial GABA response, Amp_test is the amplitude of the second (test) GABA response, and Onset_test is the value of the current at the onset of the second response. In all cases, the amplitude of the initial response was normalized to its maximal value during the experiment to account for variability in current. Each point represents the average from 8 to 10 different patches from 5 to 6 animals. The rate of recovery was best fitted by a biexponential equation, which was markedly faster following the steroid treatment protocols compared to control (P < 0.05). (n = 8–10 samples per point).

Figure 6. Computer simulation of GABAR gating following steroid treatment

A simplified biliganded model (A, upper part, centre), based on Jones & Westbrook (1995) and Celentano & Wong (1994) was modified to simulate the data from this study. It includes one open state and two desensitized states, with rate constants (table, upper right) derived from single channel data, modified from other models and approximated from desensitization data from the present study. A, Model I results in simulated current with deactivation and desensitization kinetics similar to that from control hippocampal pyramidal cell patches. Incorporation of a more rapid rate of recovery from the fast desensitized state (↑r_f) markedly accelerated τ_slow (Model II, inset), consistent with the Jones-Westbrook model, but failed to modify τ_fast. Additionally, incorporation of ↑α and ↓β to replicate single channel properties of δ-containing GABAR (Model III) replicated one subpopulation of currents following steroid treatment, with acceleration in both τ_fast and τ_slow. The second population of currents recorded following steroid treatment (faster τ_fast only) was simulated by additionally incorporating a ↓k_off (Model IV). Bottom panel, simulations resulted in markedly different rates and extent of desensitization, comparable to those obtained from control (75% desensitization) and steroid-treated animals (36% desensitization). B, rate of recovery from fast desensitization using a paired pulse protocol. Models I and III simulate the relative differences between control and P Wd data, respectively. Left, simulated traces. (The current response to the second agonist application is truncated.) Right, percentage recovery from the fast desensitized state, estimated as a biexponential decay.