The hormone progesterone is readily converted to 3alpha-OH-5alpha-pregnan-20-one (3alpha,5alpha-THP) in the brains of males and females. In the brain, 3alpha,5alpha-THP acts like a sedative, decreasing anxiety and reducing seizure activity, by enhancing the function of GABA (gamma-aminobutyric acid), the brain's major inhibitory neurotransmitter. Symptoms of premenstrual syndrome (PMS), such as anxiety and seizure susceptibility, are associated with sharp declines in circulating levels of progesterone and, consequently, of levels of 3alpha,5alpha-THP in the brain. Abrupt discontinuation of use of sedatives such as benzodiazepines and ethanol can also produce PMS-like withdrawal symptoms. Here we report a progesterone-withdrawal paradigm, designed to mimic PMS and post-partum syndrome in a rat model. In this model, withdrawal of progesterone leads to increased seizure susceptibility and insensitivity to benzodiazepine sedatives through an effect on gene transcription. Specifically, this effect was due to reduced levels of 3alpha,5alpha-THP which enhance transcription of the gene encoding the alpha4 subunit of the GABA(A) receptor. We also find that increased susceptibility to seizure after progesferone withdrawal is due to a sixfold decrease in the decay time for GABA currents and consequent decreased inhibitory function. Blockade of the alpha4 gene transcript prevents these withdrawal properties. PMS symptoms may therefore be attributable, in part, to alterations in expression of GABA(A) receptor subunits as a result of progesterone withdrawal.

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In the present study, we have characterized properties of steroid withdrawal using a pseudopregnant rat model. This paradigm results in increased production of endogenous progesterone from ovarian sources and as such is a useful physiological model. "Withdrawal" from progesterone induced by ovariectomy on day 12 of pseudopregnancy resulted in increased anxiety, as determined by a decrease in open arm entries on the elevated plus maze compared to control rats and pseudopregnant animals not undergoing withdrawal. Similar findings were obtained 24 hr after administration of a 5␣-reductase blocker to a pseudopregnant animal, suggesting that it is the GABA Amodulatory 3␣-OH-5␣-pregnan-20-one (3␣,5␣-THP) that produces anxiogenic withdrawal symptoms. Twenty-four hours after steroid withdrawal, the time constant for decay of GABA Agated current was also reduced sixfold, assessed using wholecell patch-clamp procedures on pyramidal neurons acutely dissociated from CA1 hippocampus. Thus, 3␣,5␣-THP withdrawal results in a marked decrease in total GABA A current, a possible mechanism for its anxiogenic, proconvulsant sequelae. In addition, 3␣,5␣-THP withdrawal resulted in insensitivity to the normally potentiating effect of the benzodiazepine lorazepam (LZM) on GABA A -gated Cl Ϫ current. This withdrawal profile is similar to that reported for other GABA A -modulatory drugs such as the benzodiazepines (BDZs), barbiturates, and ethanol. These changes were also associated with significant two and threefold increases in both the mRNA and protein for the ␣4 subunit of the GABA A receptor, respectively, in hippocampus. The pseudopregnancy paradigm may be a useful model for periods of endogenous 3␣,5␣-THP withdrawal such as premenstrual syndrome and postpartum or postmenopausal dysphoria, when increased emotional lability and BDZ insensitivity have been reported.

Transgenesis is a valuable methodology for studying gene expression patterns and gene function. It has recently become available for research on some parasitic nematodes, including Strongyloides stercoralis. Previously, we described a vector construct, comprising the promoter and 3′ UTR of the S. stercoralis gene Ss era-1 that gives expression of GFP in intestinal cells of developing F1 progeny. In the present study, we identified three new S. stercoralis promoters, which, in combination with the Ss era-1 3′ UTR, can drive expression of GFP or the red fluorescent protein, mRFPmars, in tissuespecific fashion. These include Ss act-2, which drives expression in body wall muscle cells, Ss gpa-3, which drives expression in amphidial and phasmidial neurons and Ss rps-21, which drives ubiquitous expression in F1 transformants and in the gonads of microinjected P0 female worms. Concomitant microinjection of vectors containing GFP and mRFPmars gave dually transformed F1 progeny, suggesting that these constructs could be used as co-injection markers for other transgenes of interest. We have developed a vector "toolkit" for S. stercoralis including constructs with the Ss era-1 3′ UTR and each of the promoters described above.

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