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.

GABA A receptor-mediated inhibition depends on the maintenance of intracellular ClϪ concentration ([Cl Ϫ ] in ) at low levels. In neurons in the developing CNS, [Cl Ϫ ] in is elevated, E GABA is depolarizing, and GABA consequently is excitatory. Depolarizing GABAergic synaptic responses may be recapitulated in various neuropathological conditions, including epilepsy. In the present study, rat hippocampal dentate granule cells were recorded using gramicidin perforated patch techniques at varying times (1-60 d) after an epileptogenic injury, pilocarpine-induced status epilepticus (STEP). In normal, non-epileptic animals, these strongly inhibited dentate granule cells act as a gate, regulating hippocampal excitation, controlling seizure initiation and/or propagation. For 2 weeks after STEP, we found that E GABA was positively shifted in granule cells. This shift in E GABA altered synaptic integration, increased granule cell excitability, and resulted in compromised "gate" function of the dentate gyrus. E GABA recovered to control values at longer latencies post-STEP (2-8 weeks), when animals had developed epilepsy. During this period of shifted E GABA , expression of the Cl Ϫ extruding K ϩ /Cl Ϫ cotransporter, KCC2 was decreased. Application of the KCC2 blocker, furosemide, to control neurons mimicked E GABA shifts evident in granule cells post-STEP. Furthermore, post-STEP and furosemide effects interacted occlusively, both on E GABA in granule cells, and on gatekeeper function of the dentate gyrus. This suggests a shared mechanism, reduced KCC2 function. These findings demonstrate that decreased expression of KCC2 persists for weeks after an epileptogenic injury, reducing inhibitory efficacy and enhancing dentate granule cell excitability. This pathophysiological process may constitute a significant mechanism linking injury to the subsequent development of epilepsy.

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.

Increasing evidence suggests that postnatal events, such as handling or maternal separation, can produce long-term changes in brain function. These are often expressed as changes in the profile of endocrine or behavioral responses to stress. Changes in ␥-aminobutyric acid type A receptors (GABARs), which mediate the majority of fast synaptic inhibition in adult brain, have been proposed as one potential mediator of these behavioral effects. In the current article, we use a combination of single-cell electrophysiology and antisense mRNA amplification to demonstrate permanent molecular and functional differences in GABARs within hippocampal dentate granule neurons after as few as two episodes of neonatal handling with brief maternal separation. Adult animals that as pups experienced handling with maternal separation maintained a more immature GABAR phenotype and exhibited increased activity in response to swim stress. These findings demonstrate the exquisite sensitivity of the developing GABAergic system to even subtle environmental manipulations and provide an unique molecular mechanism by which postnatal handling with maternal separation may alter stress-related behavior.development ͉ glucocorticoid ͉ dentate granule neurons ͉ patch clamping ͉ single-cell antisense mRNA amplification T he developing nervous system can be exquisitely sensitive to even minor perturbations in the environment. It has been recognized for decades that handling of neonatal rats could produce profound effects on later neuroendocrine and behavioral responses to stress (1, 2). Repetitive brief handling in neonatal rats has been shown to result in permanent alterations in hippocampal glucocorticoid (GC) receptors, decreased GC responses to stress in adulthood, and a relative protection against age-related hippocampal neuronal death and cognitive impairments (3). Adult animals handled as pups also demonstrate decreased expression of fear-related behaviors under stressful conditions (4-6). The molecular mechanisms underlying handling-induced behavioral and cognitive changes are not fully understood. ␥-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in mammalian brain and regulates both endocrine and behavioral responses to stress (7-9). Benzodiazepines (BZs) and other drugs that potentiate GABA currents can be potent anxiolytics. Rats exposed to early-life handling have been found to have altered BZ receptor levels in several brain regions including brainstem nuclei, amygdala, and frontal cortex (5, 6), but handling effects on GABA type A receptor (GABAR) subunit expression and function in hippocampal neurons have not, to our knowledge, previously been reported.GABARs are chloride ion channel-associated ligand-gated heteromeric receptors composed of five subunits, which are modulated by BZs, barbiturates, zinc, and neurosteroids. Many genetically distinct subunit subtypes, including ␣1-6, ␤1-4, ␥1-3, ␦, , , , and 1-3, have been identified (10, 11). GABARs can be assembled in different subunit combinations, resulting in a st...