Neuroactive steroids such as the progesterone metabolite allopregnanolone act as positive allosteric modulators at GABA_A receptors (Harrison et al. 1987). Changes in steroid levels can modulate GABA_A receptor subunit expression and it has been shown that abrupt withdrawal from chronic progesterone treatment in vivo and in vitro is associated with increased levels of the α4 subunit in hippocampal, cortical and cerebellar tissue (Smith et al. 1998; Follesa et al. 2001). Whether the normal cyclical variations in progesterone levels that occur in females influence GABA_A receptor subunit expression is not known. In the present study we have investigated immunostaining for α4 and δ GABA_A receptor subunits in neurones in the periaqueductal grey matter (PAG), a region involved in producing panic-like anxiety states, in female rats at different stages of the oestrous cycle and in male rats.

Twenty female and five male 200Ð240 g urethane-anaesthetised (0.5Ð0.7 ml 100 g^-1 20 % solution I.P.) Wistar rats were used. The hormonal status of the females was determined from vaginal cytology. All rats were humanely killed by perfusion with 100 ml heparinised (100 U ml^-1) 165 mM NaCl at 30 °C followed by 200 ml 4 % paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7 at 20 °C. The brain was removed and cryoprotected in 30 % sucrose in PB at 4 °C overnight. Frozen coronal sections of midbrain 40 mm thick were processed to reveal immunoreactivity for α4 and δ GABA_A receptor subunits using antibodies SC-7355 N-19 and SC-7367 C-20, respectively (Santa Cruz Biotechnology).

α4 and δ subunit-immunopositive neurones were present throughout the PAG at all rostro-caudal levels. α4 subunit-stained cells were polygonal (> 75 %) fusiform or triangular with a mean diameter, measured along the longest axis, of 14.6 ± 0.4 mm (S.E.M.). δ subunit-stained cells were similar in shape and size (mean diameter 15.5 ± 0.5 mm). The density of immunopositive cells was counted within the dorsal (D), dorsolateral (DL), lateral (L) and ventrolateral (VL) sectors of the PAG. In late dioestrus, when progesterone levels fall sharply, the number of α4 immunoreactive cells increased significantly above the numbers counted at other stages of the cycle or in male rats. The increase was greatest in the DL sector. δ subunit-immunopositive cells in the DLPAG did not change significantly during the oestrous cycle.

Thus increases in α4 subunit expression in the PAG when progesterone levels fall could contribute to the increases in anxiety experienced on some women during the late luteal phase of the menstrual cycle (equivalent to late dioestrus).

This work was supported by the British Heart Foundation.

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All procedures accord with current UK legislation.