Reversal of neurosteroid effects at alpha4beta2delta GABAA receptors triggers anxiety at puberty

Abstract

Puberty is characterized by mood swings and anxiety, which are often produced by stress. Here we show that THP (allopregnanolone), a steroid that is released as a result of stress, increases anxiety in pubertal female mice, in contrast to its anxiety-reducing effect in adults. Anxiety is regulated by GABAergic inhibition in limbic circuits. Although this inhibition is increased by THP administration before puberty and in adults, during puberty THP reduces the tonic inhibition of pyramidal cells in hippocampal region CA1, leading to increased excitability. This paradoxical effect of THP results from inhibition of alpha4betadelta GABAA receptors. These receptors are normally expressed at very low levels, but at puberty, their expression is increased in hippocampal area CA1, where they generate outward currents. THP also decreases the outward current at recombinant alpha4beta2delta receptors, and this effect depends on arginine 353 in the alpha4 subunit, a putative site for modulation by Cl-. Therefore, inhibition of alpha4beta2delta GABAA receptors by THP provides a mechanism for the generation of anxiety at puberty.

Figure 1

The neurosteroid THP decreases outward current gated by α4β2δ GABA_A receptors. (a) Representative traces showing the effects of 30 nM THP (right) on current gated by 1 μM GABA (EC₇₅), under conditions of outward Cl⁻ current (inward Cl⁻ flux, upper trace) and inward current (lower trace) for two δ-containing recombinant GABA_A receptor subtypes. The direction of Cl⁻current was reversed by varying internal Cl⁻ (upper trace, ECl = −70; lower trace, ECl = − 30 mV), but using a constant holding potential of −50 mV. (b) Mean effects of THP on outward and inward currents in response to 1 μM GABA (upper panel) or the GABA EC₂₀ (lower panel, α4β2δ, 0.1 μM; α4β2γ2, 5 μM; α1β2γ2, 10 μM; α5β3γ2, 5 μM) from 6 − 7 cells for each group (*P < 0.05 vs. the other receptor subtypes) (c) Current-voltage plots recorded under conditions of varying ECl (− 10, 0, 20 mV) in the presence or absence of 30 nM THP. Mean ± SEM for the slope conductance (gSlope) of the outward current (n = 7 – 8 cells for each group). (d) 30 nM THP effects on current generated by a voltage ramp over 400 ms. (Leak-subtracted current is presented as the average of 3 traces). (e) Effects of the inactive 3β-OH isomer of THP on outward GABA-gated current at α4β2δ GABA_A receptors (representative of 5 – 6 cells). (f) THP effects on desensitization of outward (upper trace) and inward (lower trace) current at α4β2δ receptors. This effect is representative of 6 cells for each group.

Figure 2

Arginine 353 in the α4 subunit is necessary for the direction-sensitive inhibition of α4β2δ GABA_A receptors by THP. (a) Alignment of the intracellular loop of α1 and α4 (H, human; M, mouse) subunits reveals limited identity (< 10%). *identical residues for all three. (The sequences for human and mouse α1 are identical.) Orange, residues to be mutated. (b) Representative traces showing the effect of 30 nM THP on GABA(10 μM)-gated current at the indicated mutated α4β2δ GABA_A receptors. Basic arginine (R351 or R353) residues in the α4 subunit were mutated to a neutral glutamine (Q) and/or a basic lysine (K). (c) Averaged data, Effects of 30 nM THP at α4β2δ receptors containing wild-type or mutant α4 subunits on outward GABA(1 μM)-gated current. (n = 4 – 5 cells for each group, *P < 0.05 versus wild-type α4β2δ). (d) Current-voltage plot recorded from α4[R353Q]β2δ GABA_A receptors before and after THP; ECl = −4.0 mV; predicted ECl = −3.8 mV, averaged from 5 cells for each point. (e) Summary diagram. Left, amino acid sequences 316-353 within the mouse α4 loop (basic residues, blue; mutated residues, orange). The two regions of the α4 loop with consecutive basic residues (316 – 318 and 351 – 353, in blue) were mutated as a group or singly to a neutral glutamine (Q in red) or to a basic lysine (K). Right, Effects of the indicated mutation on outward and inward GABA(10 μM)-gated current are indicated, as is the GABA EC₅₀ (Mean ± SEM). All mutations produced current of similar magnitude (100 – 200 pA; n = 5 – 6 cells for each group).

Figure 3

Increased expression of α4 and δ subunits on pyramidal cell dendrites of CA1 hippocampus at the onset of puberty. (a) Immunocytochemistry of α4 (upper panel) and δ (lower panel) GABA_A receptor subunits in stratum radiatum of CA1 hippocampus (40X magnification). Arrows point to immunolabeling along distal portions of dendrites. Pre-pubertal; Pre Pubertal, Pub. Calibration bar applies to all four panels. The insets show background labeling taken from the ventromedial hypothalamus, a region without detectable expression of these subunits. Representative of results from 5 – 6 mice for each group. (b)Electron micrograph of δ staining along the plasma membrane of the dendritic shaft (arrowhead) that is post-synaptic to an axon terminal as well as intracellularly (arrows). The long arrow points to an axon terminal that is likely to be GABAergic, based on the absence of postsynaptic density. (Representative of results from 5 pubertal mice.) (c) Western blot showing hippocampal expression of α4 and δ subunits after puberty and THP Wd compared to the pre-pubertal state. In one group, the decline in THP levels at puberty was prevented with 48 h administration of THP (10 mg kg⁻¹), Pub+THP. (d) Optical densities from Western blot results averaged from 6 hippocampi for each group normalized to the GAPDH control. *P < 0.05 versus Pre-pub for all graphs. (n = 3 – 4 animals for each group, performed in triplicate).

Figure 4

THP inhibits tonic GABAergic current recorded from the hippocampal slice at puberty. (a) Outward current recorded from CA1 hippocampal pyramidal cells in the slice by whole-cell patch clamp (ECl = −70 mV, −50 holding potential, pipet solution, K-gluconate; bath, 200 nM gabazine to block synaptic current and 2 mM kynurenic acid to block excitatory current). Pre-pubertal, Pre-pub; pubertal, Pub. Inset, THP effects on the inward tonic current at puberty. (b) THP-evoked change in outward and inward tonic current, Averaged data. THP withdrawal, THP Wd. (mean ± SEM, n = 8 – 12 cells for each group). (c) Tight-seal cell-attached current-clamp recording of the holding potential during dendritic application of the GABA agonist gaboxadol (5 μM) to the stratum radiatum. (Representative of cells from 5 pubertal mice). (d) Perforated patch voltage-clamp recordings from the soma of a CA1 pyramidal cell of the post-synaptic response to bath applied THP. Inset, the change in access resistance determined from the current response to a 10 mV step before and after perforation. (Bath, 1 μM TTX and 1 μM GABA; also 200 nM gabazine and L-65,708, to block synaptic current and α5-GABA_A receptors, respectively; 10 μM CGP 55845, 5 mM TEA and 50 μM kynurenic acid to block GABA_B receptors, K+ channels and excitatory amino acid receptors, respectively.) (e) Averaged data, n = 5 cells for each group. *P < 0.01 versus pre-THP, **P < 0.001 versus Pre-pub.

Figure 5

THP increases excitability of hippocampal pyramidal cells at the onset of puberty. (a) Current-voltage plots, The difference current recorded before and after bath application of 120 μM gabazine (pipet solution, cesium-methanesulfonate; bath contains 1 μM TTX, 1 μM GABA and 50 μM L-655,708). (b) Averaged slope conductance (gSlope, assessed from −60 to −40 mV; n = 5 – 6 cells for each group). *P < 0.05 versus pre-THP, **P < 0.05 versus Pre-pub. (c) Tight-seal cell-attached voltage-clamp recordings from the soma (−40 mV) of CA1 hippocampal pyramidal cells. (d) THP effects on spiking, averaged data. (n = 5 cells for each group). *P < 0.05 versus Pre-THP; **P < 0.05 versus Prepub for all graphs.

Figure 6

THP lowers the current threshold for spiking of pyramidal cells at the onset of puberty. (a)Whole cell current clamp recordings conducted from CA1 hippocampal pyramidal cells. Voltage responses recorded in response to increasing 0.3 nA current injection (−1 nA, initial current) for slices recorded before puberty (Pre-pub), or at puberty in wild-type (Pub) or δ^−/− (Pub . δ^−/− ) mice. (The THP trace lacks the 800 pA current trace for ease of comparison.) Inset, spiking at threshold, 800 pA, pre-THP; 500 nA THP in a non-spiking pubertal cell. In some cases, Ih was blocked with 20 μM Zd 7288 (Pub + Zd 7288). .Red trace, equivalent current injection, threshold for the less excitable state. Blue trace, equivalent current injection, threshold for the more excitable state.) (b) Mean ± SEM averaged from 7 – 8 cells for each group. Current threshold to spiking, I threshold; voltage threshold to spiking, Vm threshold; spike frequency, No. of spikes; action potential amplitude, AP amplitude; action potential half-width, AP half-width. Spike frequency was assessed at the minimum current required to produce spiking in both pre- and post-THP traces. *P < 0.05 versus Pre-pub.

Figure 7

THP paradoxically increases anxiety after the onset of puberty. (a) Alterations in anxiety produced by stress or injection of THP (10 mg kg⁻¹, i.p.) are presented as a percentage change in open arm time in the elevated plus maze compared to mean values from a sham control group, identical to the experimental group (age- and genotype-matched) except for the indicated treatment (stress or THP). In order to test the role of THP release in the stress response, in some cases the inactive 3β-OH isomer of THP (stress + 3β-OHTHP) or finasteride were pre-administered. Replacement THP (10 mg kg⁻¹, intraperitoneally, in oil, for three days) was also administered to prevent the decline in THP at puberty. n = 6 – 9 mice for each group, *P < 0.05 versus control, **P < 0.05 versus Pre-pub. (b) Open arm time (Mean ± SEM) for all control groups not subjected to restraint stress.