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Expression, functional properties, and clustering of alpha 1-, alpha 2-, and alpha 3-subunit containing GABA(A) receptors (GABA(A)Rs) were studied in dorsomedial SON neurons of the adult female rat supraoptic nucleus (SON) around parturition. We show that, although the decay time constant (tau(decay)) of GABAergic postsynaptic currents between and within individual recordings was very diverse, ranging from fast (i.e., alpha 1-like) to significantly slower (i.e., non-alpha 1-like), there was an overall shift towards slower decaying synaptic currents during the onset of lactation. This shift is not due to changes in mRNA expression levels, because real-time quantitative PCR assays indicated that the relative contribution of alpha 1, alpha 2, and alpha 3 remained the same before and after parturition. Also, changes in phosphorylation levels are not likely to affect the tau(decay) of postsynaptic currents. In alpha-latrotoxin (alpha-LTX)-induced bursts of synaptic currents from individual synapses, the tau(decay) of consecutive synaptic events within bursts was very similar, but between bursts there were large differences in tau(decay). This suggested that different synapses within individual SON neurons contain distinct GABA(A)R subtypes. Using multilabeling confocal microscopy, we examined the distribution of postsynaptic alpha 1-, alpha 2-, and alpha 3-GABA(A)Rs, based on colocalization with gephyrin. We show that the three GABA(A)R subtypes occurred either in segregated clusters of one subtype as well as in mixed clusters of two or possibly even three receptor subtypes. After parturition, the density and proportion of clusters containing alpha 2- (or alpha 3-), but not alpha1-GABA(A)Rs, was significantly increased. Thus, the functional synaptic diversity at the postsynaptic level in dorsomedial SON neurons is correlated with a differential clustering of distinct GABA(A)R subtypes at individual synapses.

During the female reproductive cycle, hypothalamic oxytocin (OT) neurons undergo sharp changes in excitability. In lactating mammals, bursts of electrical activity of OT neurons result in the release of large amounts of OT in the bloodstream, which causes milk ejection. One hypothesis is that OT neurons regulate their own firing activity and that of nearby OT neurons by somatodendritic release of OT. In this study, we show that OT neuron activity strongly reduces inhibitory synaptic transmission to these neurons. This effect is blocked by antagonists of both adenosine and OT receptors and is mimicked by OT application. Inhibition of solubleN-ethylmaleimide-sensitive factor attachment protein receptor complex formation by tetanus toxin completely blocked the stimulation-induced reduction in inhibitory input, as did the calcium chelator BAPTA. During lactation, the readily releasable pool of secretory vesicles in OT cell bodies was doubled, and calcium currents were upregulated. This resulted in an increased inhibition of GABAergic synaptic transmission by somatodendritic release during lactation compared with the adult virgin stage. These results demonstrate that somatodendritic release is augmented during lactation, which is a novel form of plasticity to change the strength of synaptic transmission.

In this study, we investigate how neurosteroid sensitivity of GABAAreceptors (GABAARs) is regulated. We examined this issue in neurons of the supraoptic nucleus (SON) of the rat and found that, during parturition, the GABAARs become insensitive to the neurosteroid allopregnanolone attributable to a shift in the balance between the activities of endogenous Ser/Thr phosphatase and PKC. In particular, a constitutive endogenous tone of oxytocin within the SON after parturition suppressed neurosteroid sensitivity of GABAARs via activation of PKC. Vice versa before parturition, during late pregnancy, application of exogenous oxytocin brings the GABAARs from a neurosteroid-sensitive mode toward a condition in which the receptors are not sensitive. This indicates that there may be an inverse causal relationship between the extent to which the GABAAR or one of its interacting proteins is phosphorylated and the neurosteroid sensitivity of the GABAAR. Neurosteroid sensitivity was not affected by changes in subunit composition of GABAARs known to occur concurrently in these cells.

We here investigated inhibitory synapse turnover in the adult brain using the hypothalamic supraoptic nucleus where new synapses form during different physiological conditions, in particular on oxytocin neurons largely controlled by GABAergic inputs and locally released oxytocin. Patch clamp recordings and ultrastructural analysis of the nucleus in acute slices from late gestating rats showed that oxytocin and estrogen promoted rapid formation of inhibitory synapses. Thus, after 2-h exposure to a combination of oxytocin and 17-β estradiol, the frequency of miniature inhibitory postsynaptic currents was significantly enhanced. Since their amplitude and presynaptic GABA release probability were unmodified, this indicated an increased number of synapses. Electron microscopy confirmed increased densities of symmetric, putative GABAergic synapses within 2-h exposure to the peptide or steroid, effects which were reversible and oxytocin receptor mediated. Our observations thus offer direct evidence that hypothalamic GABAergic microcircuitries can undergo rapid and functional remodeling under changing neuroendocrine conditions. © 2006 Elsevier Inc. All rights reserved.

Nongenomic gonadal steroid feedback to oxytocin containing neurons in the supraoptic nucleus of the hypothalamus is mediated via the neurosteroid allopregnanolone (3alpha-OH-DHP) that acts as an allosteric modulator of the postsynaptic GABA(A) receptors. We found evidence to support the idea that neurosteroids not only potentiate GABA(A) receptor function but also prevent its suppression by PKC. In addition, we found that neurosteroid sensitivity of GABA(A) receptor itself is dependent on the balance between endogenous phosphatase and PKC activity and not, as previously suggested, on subunit composition changes of the GABA(A) receptor. These data imply that native GABA(A) receptors are only sensitive to 3alpha-OH-DHP if there is endogenous phosphatase activity. In contrast, when, due to endogenous release of oxytocin in the hypothalamus, the intracellular balance is shifted from high phosphatase activity toward a higher level of PKC-dependent phosphorylation, this leads to 3alpha-OH-DHP-insensitivity of the GABA(A) receptors. How the regulatory mechanisms of the GABA(A) receptor physiology for the hypothalamus may also account for alterations in GABA transmission observed in other brain areas is discussed.

Oxytocin neurons in the supraoptic nucleus (SON) exhibit marked neuronal plasticity during each reproductive cycle. We have previously shown that this neuronal plasticity includes GABA A receptor subunit switching around the time of parturition. Here we focus on additional plasticity in short-term regulatory mechanisms of postsynaptic receptor function before and after parturition, i.e. alterations in metabotropic and allosteric modulation of GABA A receptor activity. Both short- and long-term regulation of the GABA A receptor function affects the electrical behaviour of the oxytocin neurons (Brussaard and Herbison, 2000) ; however, their causal linkage until recently remained unclear. Non-genomic gonadal steroid feedback to oxytocin neurons is mediated via the neurosteroid allopregnanolone (3a-OH-DHP) that is an allosteric modulator of postsynaptic GABA A receptors. We recently found evidence to support the idea that (1) neurosteroids not only potentiate GABA A receptor function but also prevent its suppression by PKC (Brussaard et al., 2000), and (2) that neurosteroid sensitivity of GABA A receptor is not regulated by subunit switching, but instead, is dependent on the balance between endogenous phosphatase and PKC activity (Koksma et al., 2002) . Thus, before pregnancy, the GABA A receptors are sensitive to 3α-OH-DHP, due to a constitutively high level of phosphatase activity. At parturition, endogenous release of oxytocin within the SON shifts the intracellular balance towards a higher level of phosphorylation, leading to 3α-OH-DHP insensitivity of the GABA A receptors. Here we discuss the putative mechanisms underlying these changes in receptor physiology, their causal relations and the functional significance for the hormonal output.