Changes in properties and neurosteroid regulation of GABAergic synapses in the supraoptic nucleus during the mammalian female reproductive cycle

Abstract

GABAA receptor‐mediated synaptic innervation of oxytocin neurones in the supraoptic nucleus (SON) was analysed in adult female rats going through their first reproductive cycle by recording the spontaneous inhibitory postsynaptic currents (sIPSCs) at six stages of female reproduction. During pregnancy we observed a reduction in the interval between monoquantal sIPSCs. The synaptic current amplitude, current decay and neurosteroid sensitivity of postsynaptic GABAA receptors observed at this stage were not distinguishable from those measured in virgin stage SON. Upon parturition an increase in monoquantal synaptic current decay occurred, whereas potentiation by the progesterone metabolite allopregnanolone (3α‐OH‐DHP) was suppressed. Throughout a substantial part of the lactation period the decay of synaptic currents remained attenuated, whilst the potentiation by 3α‐OH‐DHP remained suppressed. Several weeks after the end of lactation sIPSC intervals, their current decay velocity as well as the potentiation by 3α‐OH‐DHP were restored to pre‐pregnancy levels, which is indicative of the cyclical nature of synaptic plasticity in the adult SON. Competitive polymerase chain reaction (PCR) analysis showed that virgin animals expressed α1 and α2 GABAA receptor subunit mRNA at a relative ratio of 2 : 1 compared with β‐actin. After pregnancy both α1 and α2 subunit mRNA levels were transiently increased, although at a relative ratio of 1 : 4, in line with the hypothesis that α2 plays a large role in postsynaptic receptor functioning. During post‐lactation both α subunits were downregulated. We propose that synaptic remodelling in the SON during pregnancy includes changes in the putative number of GABA release sites per neurone. At parturition, and during the two consecutive weeks of lactation, a subtype of postsynaptic GABAA receptors was observed, distinct from the one being expressed before and during pregnancy. Synaptic current densities, calculated in order to compare the impact of synaptic inhibition, showed that, in particular, the differences in 3α‐OH‐DHP potentiation of these two distinct GABAA receptor subtypes produce robust shifts in the impact of synaptic inhibition of oxytocin neurones at the different stages of female reproduction.

Steroid modulation of GABAA receptor-mediated transmission in the hypothalamus: Effects on reproductive function

“…Initial reports indicated that a shift in the relative expression levels of GABA A receptor α 1 and α 2 subunit mRNAs in these neurons as female rodents transit from late pregnancy through parturition and to a lactating state results in a concomitant decrease in the sensitivity of these neurons to modulation by allopregnanolone, thus promoting disinhibition and enhanced firing of oxytocin neurons (Fénelon and Herbison, 1996a;Brussaard et al, 1997;Brussaard et al, 1999; for review Brussaard and Herbison, 2000;Herbison, 2001). While effects of antisense knockdown of α 2 subunit expression in neocortical slices demonstrated the expected acceleration of synaptic current decay kinetics (Brussaard et al, 1997), SON slices were not tested with anti-sense constructs, nor were antisense-treated cortical slices assayed for differences in the sensitivity to allosteric modulation by allopregnanolone to test directly if changes in α subunit expression resulted in concomitant changes in allopregnanolone potentiation.…”

Progesterone-metabolite prevents protein kinase C-dependent modulation of gamma -aminobutyric acid type A receptors in oxytocin neurons

“…Allopregnanolone (5␣-pregnan-3␣-ol-20-one), abbreviated here as 3␣-OH-DHP, was obtained from Research Biochemicals and dissolved in DMSO at 10 mM and further diluted in external solution before application. The lipophylicity of this substance prevents accurate estimates of the final concentration of 3␣-OH-DHP at the site of action, as has been extensively discussed previously (4,5). Staurosporine, TPA (phorbol 12-myristate 13-acetate, PMA), and the inactive form of PMA, 4-␣-PMA, were all from Research Biochemicals and dissolved as stocks in DMSO at 10 mM.…”

“…The spiking frequency of these neurons is under control of a GABAergic input (1)(2)(3). The postsynaptic ␥-aminobutyric acid type A (GABA A ) receptors that mediate this input are susceptible to allosteric interaction with the neurosteroid allopregnanolone 3␣-OH-DHP during some stages of the female reproductive cycle, in particularly during pregnancy (4,5). In addition, somatodendritic release of oxytocin within the SON acting on autoreceptors (6) decreases the fast synaptic inhibition of the magnocellular cells, via suppression of postsynaptic GABA A receptor activity in a Ca 2ϩ -dependent manner (7,8).…”

“…Electrodes had a tip resistance of around 2 M⍀ and uncompensated series resistance Ͻ 12 M⍀ (which usually was compensated for 70%). The external solution contained 125 mM NaCl, 25 mM NaHCO 3 , 3 mM KCl, 5 -OVT (Bachem; data not shown). Allopregnanolone (5␣-pregnan-3␣-ol-20-one), abbreviated here as 3␣-OH-DHP, was obtained from Research Biochemicals and dissolved in DMSO at 10 mM and further diluted in external solution before application.…”

“…Methods Dissection and Recordings. Juvenile male (21-24 days postnatal) or adult female Wistar rats, either after 20 days of pregnancy (P20) or on the first day after parturition (PPD1) were decapitated, and 400-m-thick coronal hypothalamus slices incorporating the middle portion of the SON were prepared as described (4,5,7) by using a Leica (Nussloch, Germany) vibratome slicer. Spontaneous GABAergic synaptic currents were recorded at room temperature (20°C) at a holding potential of Ϫ70 mV with an Axopatch 200A amplifier (Axon Instruments, Foster City, CA) in the whole-cell voltage clamp mode (see refs.…”