Estrous cycle variations in GABA(A) receptor phosphorylation enable rapid modulation by anabolic androgenic steroids in the medial preoptic area

Abstract

Anabolic androgenic steroids (AAS), synthetic testosterone derivatives that are used for ergogenic purposes, alter neurotransmission and behaviors mediated by GABA(A) receptors. Some of these effects may reflect direct and rapid action of these synthetic steroids at the receptor. The ability of other natural allosteric steroid modulators to alter GABA(A) receptor-mediated currents is dependent upon the phosphorylation state of the receptor complex. Here we show that phosphorylation of the GABA(A) receptor complex immunoprecipitated by β(2)/β(3) subunit-specific antibodies from the medial preoptic area (mPOA) of the mouse varies across the estrous cycle; with levels being significantly lower in estrus. Acute exposure to the AAS, 17α-methyltestosterone (17α-MeT), had no effect on the amplitude or kinetics of inhibitory postsynaptic currents in the mPOA of estrous mice when phosphorylation was low, but increased the amplitude of these currents from mice in diestrus, when it was high. Inclusion of the protein kinase C (PKC) inhibitor, calphostin, in the recording pipette eliminated the ability of 17α-MeT to enhance currents from diestrous animals, suggesting that PKC-receptor phosphorylation is critical for the allosteric modulation elicited by AAS during this phase. In addition, a single injection of 17α-MeT was found to impair an mPOA-mediated behavior (nest building) in diestrus, but not in estrus. PKC is known to target specific serine residues in the β(3) subunit of the GABA(A) receptor. Although phosphorylation of these β(3) serine residues showed a similar profile across the cycle, as did phosphoserine in mPOA lysates immunoprecipitated with β2/β3 antibody (lower in estrus than in diestrus or proestrus), the differences were not significant. These data suggest that the phosphorylation state of the receptor complex regulates both the ability of AAS to modulate receptor function in the mPOA and the expression of a simple mPOA-dependent behavior through a PKC-dependent mechanism that involves the β(3) subunit and other sites within the GABA(A) receptor complex.

Figure 1. Changes in GABA_A receptors across the estrous cycle

(A) Top: representative Western blot illustrating signals corresponding to phosphoserine (pSer) in tissue harvested from individual animals in diestrus (D), proestrus (P) and estrus (E). Lysates were immunoprecipitated with a monoclonal antibody directed against the β₂/β₃ subunits of the GABA_A receptor prior to electrophoresis and Western blotting. Bottom: Following incubation with antibody directed against pSer, the blot was stripped and re-probed with the antibody directed against β₃. (B) Top: representative Western blot illustrating the signal for phosphoserine408/409 in the β₃ subunit (pβ₃) and the β₃ subunit (β₃) (Bottom) in mPOA tissue harvested from adolescent male (M) and female (F) mice. All signals corresponded to a molecular weight of 58 kDa. (C) Averaged data from female mice in diestrus (n = 7), proestrus (n = 7) and estrus (n = 9) corresponding to the signal for pSer normalized to that of β₃. (D) Averaged data from female mice in diestrus (n = 9), proestrus (n = 10) and estrus (n = 11) corresponding to the signal for pβ₃ normalized to that of β₃. Assessment with the antibody was also made for a second group of animals (7 in diestrus, 7 in proestrus, and 9 in estrus) with a different lot of this antibody with comparable results (data not shown). (E) Levels of GABA_A receptor subunit mRNAs in the mPOA. Data are presented as the 2^−ΔC_T values, which indicate the average levels of subunit mRNAs relative to 18S in tissue from diestrous (n = 9), proestrous (n = 10) and estrous (n = 10) mice. P values were: α₁: 0.28; α₂: 0.37; α₅: 0.23; β₁: 0.46; β₂: 0.37; β₃: 0.52; γ₁: 0.36; γ₂: 0.41; δ: 0.93; ε: 0.17.

Figure 2. Allosteric modulation by AAS of GABA_A receptor-mediate synaptic responses in the mPOA in diestrus and estrus

Representative (A) sIPSCs and (B) mIPSCs from a single mPOA neuron of a diestrus mouse illustrating reversible potentiation of the peak current by acute application of 1 μM 17α-MeT. Data illustrating effect on peak currents for (C) sIPSCs (n = 15) and (D) mIPSCs (n = 10) in individual mPOA neurons from diestrus females with addition of 1 μM 17α-MeT to the bath. Representative (E) sIPSCs and (F) mIPSCs from a single mPOA neuron of an estrus female illustrating no effect of acute application of 1 μM 17α-MeT. Data illustrating effect on peak currents for (G) sIPSCs (n = 10) and (H) mIPSCs (n = 9) in individual mPOA neurons from estrous females with addition of 1 μM 17α-MeT to the bath.

Figure 3. Effect of PKC inhibition on

allosteric modulation by AAS of GABA_A receptor-mediate synaptic responses in the mPOA in the diestrous mouse. (A) Representative sIPSCs from a single mPOA neuron of a diestrous mouse illustrating the absence of an effect of acute application of 1 μM 17α-MeT when 200nM was present in the pipette solution. (B) Data illustrating effect on peak currents for sIPSCs in individual mPOA neurons from diestrous mice with addition of 1 μM 17α-MeT to the bath and 200 nM calphostin intracellularly. (C) Average peak current amplitudes for mIPSCs in mPOA neurons of diestrous mice prior to (Control), during (17α-MeT), and following (Wash) exposure to 1 μM 17α-MeT (n = 15).

Figure 4. Effects of acute systemic exposure to 17α-MeT on behaviors mediated by neural circuits that include the mPOA

(A) Average data of nest scores and remaining nestlet material (see Section 2.5.1) for diestrous (n = 7 for each cycle stage) and estrous (n = 7 for each cycle stage) mice given a single injection of either oil or 7.5 mg/kg 17α-MeT. (B) Average marble burying scores assess during a 30 min period 30 min following an acute injection of oil or 17α-MeT in diestrus (n = 8 for each cycle stage) and estrus (n = 6 for each cycle stage) mice. (C) Number of entries into the open and closed arms, as well at total number of entries (left) and fraction of total time in the open, closed and center parts of the elevated plus maze for adult female mice (unstaged) assessed during a 5 min period 30 min following injection of oil (n = 20) or 17α-MeT (n = 20). In (A), * indicates p < 0.04.