Altered GABAA receptor-mediated synaptic transmission disrupts the firing of gonadotropin-releasing hormone neurons in male mice under conditions that mimic steroid abuse

Abstract

Gonadotropin-releasing hormone (GnRH) neurons are the central regulators of reproduction. GABAergic transmission plays a critical role in pubertal activation of pulsatile GnRH secretion. Self-administration of excessive doses of anabolic androgenic steroids (AAS) disrupts reproductive function and may have critical repercussions for pubertal onset in adolescent users. Here, we demonstrate that chronic treatment of adolescent male mice with the AAS 17alpha-methyltestosterone significantly decreased action potential frequency in GnRH neurons, reduced the serum gonadotropin levels, and decreased testes mass. AAS treatment did not induce significant changes in GABAA receptor subunit mRNA levels or alter the amplitude or decay kinetics of GABAA receptor-mediated spontaneous postsynaptic currents (sPSCs) or tonic currents in GnRH neurons. However, AAS treatment significantly increased action potential frequency in neighboring medial preoptic area (mPOA) neurons and GABAA receptor-mediated sPSC frequency in GnRH neurons. In addition, physical isolation of the more lateral aspects of the mPOA from the medially localized GnRH neurons abrogated the AAS-induced increase in GABAA receptor-mediated sPSC frequency and the decrease in action potential firing in the GnRH cells. Our results indicate that AAS act predominantly on steroid-sensitive presynaptic neurons within the mPOA to impart significant increases in GABAA receptor-mediated inhibitory tone onto downstream GnRH neurons, resulting in diminished activity of these pivotal mediators of reproductive function. These AAS-induced changes in central GABAergic circuits of the forebrain may significantly contribute to the disruptive actions of these drugs on pubertal maturation and the development of reproductive competence in male steroid abusers.

Figure 1.

AAS-dependent effects on AP firing in GnRH neurons from control and AAS-treated mice. Top, Three continuous minutes of recording in the loose-patch on-cell configuration demonstrating bursty AP firing from control and AAS-treated subjects from the same cohort depicting the differences of AP frequency; insets show individual APs within the burst. Display of responses on the left was scaled to match amplitudes of those on the right. Bottom left, AP frequency is decreased (*p = 2.03 × 10⁻⁴) in GnRH neurons from AAS-treated (gray; n = 18 cells) versus control (black; n = 22 cells) male mice. Bottom right, AAS treatment did not alter AP patterning in GnRH neurons. Inset, Representative autocorrelograms corresponding to irregular and bursty firing patterns.

Figure 2.

AAS-dependent effects on GABA_A receptor subunit and GnRH mRNA levels in GnRH neurons from control and AAS-treated mice. Data are presented as the 2^−ΔC_T, which indicate the average levels (relative to the housekeeping gene β-actin) of subunit mRNAs in GnRH neurons isolated from control (black; n = 10 mice) and AAS-treated (gray; n = 10 mice) mice for analysis of GABA_A receptor subunit mRNA levels and from a separate cohort of seven control and seven AAS-treated mice for analysis of GnRH mRNA levels.

Figure 3.

AAS-dependent effects on GABA_A receptor-mediated miniature and spontaneous PSCs in GnRH neurons. A, Averaged sPSCs recorded from GnRH neurons in slices isolated from control (black line) and AAS-treated (gray line) subjects (n = 12 neurons for control and n = 13 neurons for AAS-treated). B, Top, Representative sPSCs recorded from a GnRH neuron in a control and an AAS-treated mouse. Bottom, Average frequencies of sPSCs and mPSCs in GnRH neurons from control (black) and AAS-treated (gray) mice (data are from the same cells as shown in A). Asterisk indicates the sPSC frequency is greater in AAS condition (p = 0.014).

Figure 4.

AAS-dependent effects on AP firing in non-GnRH mPOA neurons. Left, AP frequency in mPOA neurons from control (black; n = 72 neurons) and AAS-treated (gray; n = 58 neurons) mice. Right, Representative autocorrelograms corresponding to bursty, regular, and irregular firing patterns and the percentages of neurons characterized by each firing pattern in control and AAS-treated mice. Asterisk indicates the AP frequency is greater in AAS condition (p = 0.015).

Figure 5.

Effects of acute interruption of inputs from the more lateral regions of the mPOA on GABA_A receptor-mediated sPSC and AP firing frequencies in GnRH neurons in control and AAS-treated mice. A, Illustration depicting the bilateral distribution of GnRH neurons (gray triangles) and the location of longitudinal cuts (as indicated by the dotted line and arrow) made to physically isolate these neurons from their upstream lateral afferents in the mPOA (right) and representative slice that shows the intact and disrupted mPOA (right). Asterisk indicates electrode tip poised above the GnRH neurons in disrupted mPOA. 3V, Third ventricle; AC, anterior commissure. B, Averaged data demonstrating sPSC frequency in GnRH neurons from control (black) and AAS-treated (gray) mice in recordings from intact and disrupted mPOAs. n values indicate numbers of neurons for each condition. Asterisk indicates that sPSC frequency was significantly greater (p = 0.023) in the intact mPOA than in the disrupted mPOA of AAS-treated mice. C, Averaged data demonstrating AP frequency in GnRH neurons from control (black) and AAS-treated (gray) mice in recordings from intact and disrupted mPOAs. n values indicate numbers of neurons for each condition. Asterisk indicates that sPSC frequency was significantly lower (p = 0.020) in the intact mPOA than in the disrupted mPOA of AAS-treated mice.

Figure 6.

Blocking GABA_A receptor function enhances GnRH neuron AP firing rate only in the intact mPOA from AAS-treated male mice. Recordings of APs were made from GnRH neurons under the same conditions as described in Figure 5 C before and after the addition of 100 μm PTX. Data from each group (control and AAS-treated) were normalized to their respective average AP frequency from disrupted mPOA in aCSF (100%). A, PTX did not alter AP firing in control subjects from either intact (n = 10; filled circles) or disrupted (n = 10; open circles) mPOA. B, Conversely, in AAS-treated subjects, AP frequency was significantly (*p = 0.019) increased with PTX application in GnRH neurons from the intact mPOA (n = 10; filled diamonds) but was without significant effect in the disrupted mPOA (n = 10; open diamonds). For AAS-treated animals, AP firing rates were comparable in slices in which GABAergic inputs were physically interrupted (disrupted POA) and in those in which the mPOA was intact, but GABA_A receptor-mediated transmission was blocked.