Distinct patterns of expression and regulation of GABA receptors containing the delta subunit in cerebellar granule and hippocampal neurons

Neuronal plasticity is achieved by regulation of the expression of genes for neurotransmitter receptors such as the type A receptor (GABA(A)R) for gamma-aminobutyric acid. We now show that two different rat neuronal populations in culture manifest distinct patterns of GABA(A)R plasticity in response to identical stimuli. Whereas prolonged exposure to ethanol had no effect on expression of the delta subunit of GABA(A)Rs at the mRNA or protein level in cerebellar granule neurons, it increased the abundance of delta subunit mRNA and protein in hippocampal neurons. Subsequent ethanol withdrawal transiently down-regulated delta subunit expression in cerebellar granule neurons and gradually normalized that in hippocampal neurons. These effects of ethanol exposure and withdrawal were accompanied by corresponding functional changes in GABA(A)Rs. GABA(A)Rs containing the delta subunit were also distributed differentially in the cerebellar and hippocampal neurons. These findings reveal complex and distinct mechanisms of regulation of the expression of GABA(A)Rs that contain the delta subunit in different neuronal types.     

Antisense suppression of GABA(A) receptor beta subunit levels in cultured cerebellar granule neurons demonstrates their importance in receptor expression

GABA(A) receptors in the CNS are pentameric molecules composed of alpha, beta, gamma, delta, epsilon and theta subunits. Studies on transfected cells have shown that GABA(A) receptor beta subunit isoforms can direct alpha1 subunit localization within the cell. To examine the role of selected subunits in governing GABA(A) receptor expression in neurons, cultures of rat cerebellar granule cells were grown with antisense or sense oligodeoxynucleotides (ODNs) specific for the alpha 1, beta 2 or gamma 2 subunits. These subunits are all expressed in granule neurons where they are thought to contribute to an abundant receptor type. Following ODN treatment, subunit expression and distribution were examined by western blotting, immunocytochemistry and RT-PCR. Treatment of the cultures with the antisense, but not the corresponding sense, ODNs reduced the levels of the targeted subunit polypeptides. In addition, the beta 2 antisense ODN reduced the level of the alpha1 subunit polypeptide without altering the level of its mRNA. In contrast, treatment with the beta 2 subunit antisense ODN did not alter gamma 2 subunit polypeptide expression, distribution or mRNA level. These findings suggest that the alpha1 subunit requires a beta subunit for assembly into GABA(A) receptors in cerebellar granule neurons.     

Chronic blockade of N-methyl-D-aspartate receptors alters gamma-aminobutyric acid type A receptor peptide expression and function in the rat

Chronic in vivo or in vitro application of GABA(A) receptor agonists alters GABA(A) receptor peptide expression and function. Furthermore, chronic in vitro application of N-methyl-D-aspartate (NMDA) agonists and antagonists alters GABA(A) receptor function and mRNA expression. However, it is unknown if chronic in vivo blockade of NMDA receptors alters GABA(A) receptor function and peptide expression in brain. Male Sprague-Dawley rats were chronically administered the noncompetitive NMDA receptor antagonist MK-801 (0.40 mg/kg, twice daily) for 14 days. Chronic blockade of NMDA receptors significantly increased hippocampal GABA(A) receptor alpha4 and gamma2 subunit expression while significantly decreasing hippocampal GABA(A) receptor alpha2 and beta2/3 subunit expression. Hippocampal GABA(A) receptor alpha1 subunit peptide expression was not altered. In contrast, no significant alterations in GABA(A) receptor subunit expression were found in cerebral cortex. Chronic MK-801 administration also significantly decreased GABA(A) receptor-mediated hippocampal Cl- uptake, whereas no change was found in GABA(A) receptor-mediated cerebral cortical Cl- uptake. Finally, chronic MK-801 administration did not alter NMDA receptor NR1, NR2A, or NR2B subunit peptide expression in either the cerebral cortex or the hippocampus. These data demonstrate heterogeneous regulation of GABA(A) receptors by glutamatergic activity in rat hippocampus but not cerebral cortex, suggesting a new mechanism of GABA(A) receptor regulation in brain.     

Brain-derived neurotrophic factor (BDNF)-induced synthesis of early growth response factor 3 (Egr3) controls the levels of type A GABA receptor alpha 4 subunits in hippocampal neurons

Altered function of gamma-aminobutyric acid type A receptors (GABA(A)Rs) in dentate granule cells of the hippocampus has been associated with temporal lobe epilepsy (TLE) in humans and in animal models of TLE. Such altered receptor function (including increased inhibition by zinc and lack of modulation by benzodiazepines) is related, in part, to changes in the mRNA levels of certain GABA(A)R subunits, including alpha4, and may play a role in epileptogenesis. The majority of GABA(A)Rs that contain alpha4 subunits are extra-synaptic due to lack of the gamma2 subunit and presence of delta. However, it has been hypothesized that seizure activity may result in expression of synaptic receptors with altered properties driven by an increased pool of alpha4 subunits. Results of our previous work suggests that signaling via protein kinase C (PKC) and early growth response factor 3 (Egr3) is the plasticity trigger for aberrant alpha4 subunit gene (GABRA4) expression after status epilepticus. We now report that brain derived neurotrophic factor (BDNF) is the endogenous signal that induces Egr3 expression via a PKC/MAPK-dependent pathway. Taken together with the fact that blockade of tyrosine kinase (Trk) neurotrophin receptors reduces basal GABRA4 promoter activity by 50%, our findings support a role for BDNF as the mediator of Egr3-induced GABRA4 regulation in developing neurons and epilepsy and, moreover, suggest that BDNF may alter inhibitory processing in the brain by regulating the balance between phasic and tonic inhibition.     

Role of the GABA(A)beta2, GABA(A)alpha6, GABA(A)alpha1 and GABA(A)gamma2 receptor subunit genes cluster in drug responses and the development of alcohol dependence

gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter of the central nervous system and it acts at the GABA(A) and GABA(B) receptors. A possible role for the GABA(A) receptors in alcohol action has been derived from in vitro cell models, animal studies and human research. GABA(A) subunit mRNA expression in cell models has suggested that the long form of the gamma2 subunit is essential for ethanol enhanced potentiation of GABA(A) receptors, by phosphorylation of a serine contained within the extra eight amino acids. Several animal studies have demonstrated that alterations in drug and alcohol responses may be caused by amino-acid differences at the GABA(A)alpha6 and GABA(A)gamma2 subunits. An Arg(100)/Glu(100) change at the GABA(A)alpha6 subunit conferring altered binding efficacy of the benzodiazepine inverse agonist Ro 15-4513, was found between the AT (alcohol tolerance) and ANT (alcohol non-tolerance) rats. Several loci related to alcohol withdrawal on mouse chromosome 11 which corresponds to the region containing four GABA(A) subunit (beta2, alpha6, alpha1 and gamma2) genes on human chromosome 5q33-34, were also identified. Gene knockout studies of the role of GABA(A)alpha6 and GABA(A)gamma2 subunit genes in mice have demonstrated an essential role in the modulation of other GABA(A) subunit expression and the efficacy of benzodiazepine binding. Absence of the GABA(A)gamma2 subunit gene has more severe effects with many of the mice dying shortly after birth. Disappointingly few studies have examined the effects of response to alcohol in these gene knockout mice. Human genetic association studies have suggested that the GABA(A)beta2, alpha6, alpha1 and gamma2 subunit genes have a role in the development of alcohol dependence, although their contributions may vary between ethnic group and phenotype. In summary, in vitro cell, animal and human genetic association studies have suggested that the GABA(A)beta2, alpha6, alpha1 and gamma2 subunit genes have an important role in alcohol related phenotypes (300 words).     

Flumazenil selectively prevents the increase in alpha(4)-subunit gene expression and an associated change in GABA(A) receptor function induced by ethanol withdrawal

The actions of ethanol on gamma-aminobutyric acid type A (GABA(A)) receptors are still highly controversial issues but it appears that some of its pharmacological effects may depend on receptor subunit composition. Prolonged ethanol exposure produces tolerance and dependence and its withdrawal alters GABA(A) receptor subunit gene expression and function. Whereas benzodiazepines are clinically effective in ameliorating ethanol withdrawal symptoms, work in our laboratory showed that benzodiazepines also prevent, in vitro, some of the ethanol withdrawal-induced molecular and functional changes of the GABA(A) receptors. In the present work, we investigated the effects, on such changes, of the benzodiazepine receptor antagonist flumazenil that can positively modulate alpha(4)-containing receptors. We here report that flumazenil prevented both the ethanol withdrawal-induced up-regulation of the alpha(4)-subunit and the increase in its own modulatory action. In contrast, flumazenil did not inhibit ethanol withdrawal-induced decrease in alpha(1)- and delta-subunit expression as well as the corresponding decrease in the modulatory action on GABA(A) receptor function of both the alpha(1)-selective ligand zaleplon and the delta-containing receptor preferentially acting steroid allopregnanolone. These observations are the first molecular and functional evidence that show a selective inhibition by flumazenil of the up-regulation of alpha(4)-subunit expression elicited by ethanol withdrawal.     

gamma-Aminobutyric acid(A) receptor subunit expression predicts functional changes in hippocampal dentate granule cells during postnatal development

Profound alterations in the function of GABA occur over the course of postnatal development. Changes in GABA(A) receptor expression are thought to contribute to these differences in GABAergic function, but how subunit changes correlate with receptor function in individual developing neurons has not been defined precisely. In the current study, we correlate expression of 14 different GABA(A) receptor subunit mRNAs with changes in the pharmacological properties of the receptor in individual hippocampal dentate granule cells over the course of postnatal development in rat. We demonstrate significant developmental differences in GABA(A) receptor subunit mRNA expression, including greater than two-fold lower expression of alpha1-, alpha4- and gamma2-subunit mRNAs and 10-fold higher expression of alpha5-mRNA in immature compared with adult neurons. These differences correlate both with regional changes in subunit protein level and with alterations in GABA(A) receptor function in immature dentate granule cells, including two-fold higher blockade by zinc and three-fold lower augmentation by type-I benzodiazepine site modulators. Further, we find an inverse correlation between changes in GABA(A) receptor zinc sensitivity and abundance of vesicular zinc in dentate gyrus during postnatal development. These findings suggest that developmental differences in subunit expression contribute to alterations in GABA(A) receptor function during postnatal development.     

Identification of the sites for CaMK-II-dependent phosphorylation of GABA(A) receptors

Phosphorylation can affect both the function and trafficking of GABA(A) receptors with significant consequences for neuronal excitability. Serine/threonine kinases can phosphorylate the intracellular loops between M3-4 of GABA(A) receptor beta and gamma subunits thereby modulating receptor function in heterologous expression systems and in neurons (1, 2). Specifically, CaMK-II has been demonstrated to phosphorylate the M3-4 loop of GABA(A) receptor subunits expressed as GST fusion proteins (3, 4). It also increases the amplitude of GABA(A) receptor-mediated currents in a number of neuronal cell types (5-7). To identify which substrate sites CaMK-II might phosphorylate and the consequent functional effects, we expressed recombinant GABA(A) receptors in NG108-15 cells, which have previously been shown to support CaMK-II modulation of GABA(A) receptors containing the beta3 subunit (8). We now demonstrate that CaMK-II mediates its effects on alpha1beta3 receptors via phosphorylation of Ser(383) within the M3-4 domain of the beta subunit. Ablation of beta3 subunit phosphorylation sites for CaMK-II revealed that for alphabetagamma receptors, CaMK-II has a residual effect on GABA currents that is not mediated by previously identified sites of CaMK-II phosphorylation. This residual effect is abolished by mutation of tyrosine phosphorylation sites, Tyr(365) and Tyr(367), on the gamma2S subunit, and by the tyrosine kinase inhibitor genistein. These results suggested that CaMK-II is capable of directly phosphorylating GABA(A) receptors and activating endogenous tyrosine kinases to phosphorylate the gamma2 subunit in NG108-15 cells. These findings were confirmed in a neuronal environment by expressing recombinant GABA(A) receptors in cerebellar granule neurons.     

GABAA receptor phosphorylation and functional modulation in cortical neurons by a protein kinase C-dependent pathway

GABA(A) receptors are critical mediators of fast synaptic inhibition in the brain, and the predominant receptor subtype in the central nervous system is believed to be a pentamer composed of alpha, beta, and gamma subunits. Previous studies on recombinant receptors have shown that protein kinase C (PKC) and PKA directly phosphorylate intracellular serine residues within the receptor beta subunit and modulate receptor function. However, the relevance of this regulation for neuronal receptors remains poorly characterized. To address this critical issue, we have studied phosphorylation and functional modulation of GABA(A) receptors in cultured cortical neurons. Here we show that the neuronal beta3 subunit is basally phosphorylated on serine residues by a PKC-dependent pathway. PKC inhibitors abolish basal phosphorylation, increasing receptor activity, whereas activators of PKC enhance beta3 phosphorylation with a concomitant decrease in receptor activity. PKA activators were shown to increase the phosphorylation of the beta3 subunit only in the presence of PKC inhibitors. We also show that the main sites of phosphorylation within the neuronal beta3 subunit are likely to include Ser-408 and Ser-409, residues that are important for the functional modulation of beta3-containing recombinant receptors. Furthermore, PKC activation did not change the total number of GABA(A) receptors in the plasma membrane, suggesting that the effects of PKC activation are on the gating or conductance of the channel. Together, these results illustrate that cell-signaling pathways that activate PKC may have profound effects on the efficacy of synaptic inhibition by directly modulating GABA(A) receptor function.     

Modulation of GABA(A) receptor function by neuroactive steroids: evidence for heterogeneity of steroid sensitivity of recombinant GABA(A) receptor isoforms

Neuroactive steroids are potent, selective allosteric modulators of gamma-aminobutyric acid type A (GABA(A)) receptor function in the central nervous system, and may serve as endogenous anxiolytic and analgesic agents. In order to study the influence of subunit subtypes of the GABA(A) receptor on modulation of receptor function by neuroactive steroids, we expressed human recombinant GABA(A) receptors in Xenopus oocytes. GABA-activated membrane current, and the modulatory effects of the endogenous neurosteroid 5alpha-pregnan-3alpha-ol-20-one (allopregnanolone) and the synthetic steroid anesthetic 5alpha-pregnan-3alpha-ol-11,20-dione (alphaxalone) were measured using two-electrode voltage-clamp recording techniques. Allopregnanolone had similar effects to potentiate GABA-activated membrane current in the alpha1beta1gamma2L and alpha1beta2gamma2L receptor isoforms. In contrast, alphaxalone was much more effective as a positive allosteric modulator on the alpha1beta1gamma2L receptor isoform. In the absence of the gamma2L subunit subtype, allopregnanolone had much greater efficacy, but its potency was decreased. Allopregnanolone was much more effective on the alpha1beta1 receptor isoform compared with the alpha1beta2 receptor isoform. The potency for alphaxalone to potentiate the GABA response was not altered in the absence of the gamma2L subunit subtype, although its efficacy was greatly enhanced. Both allopregnanolone and alphaxalone produced nonparallel leftward shifts in the GABA concentration-response relationship in the absence of the gamma2L subunit, decreasing the EC50 concentration of GABA and increasing the maximal response. Only alphaxalone increased the maximal GABA response when the gamma2L subunit subtype was present. The 3beta-pregnane isomers epipregnanolone and isopregnanolone both inhibited the ability of allopregnanolone and alphaxalone to potentiate GABA(A) receptor function. However, the degree of block produced by the 3beta-pregnane steroid isomers was dependent on the type of receptor isoform studied and the neuroactive steroid tested. Isopregnanolone, the 3beta-isomer of allopregnanolone, was significantly more effective as a blocker of potentiation caused by allopregnanolone compared with alphaxalone in all receptor isoforms tested. Epipregnanolone had a greater efficacy as a blocker at the alpha1beta2gamma2L receptor isoform compared with the alpha1beta1gamma2L receptor isoform, and also produced a greater degree of block of potentiation caused by allopregnanolone compared with alphaxalone. Our results support the hypothesis that the heteromeric assembly of different GABA(A) receptor isoforms containing different subunit subtypes results in multiple steroid recognition sites on GABA(A) receptors, which in turn produces distinctly different modulatory interactions between neuroactive steroids acting at the GABA(A) receptor. The alpha and gamma subunit subtypes may have the greatest influence on allopregnanolone modulation of GABA(A) receptor function, whereas the beta and gamma subunit subtypes appear to be most important for the modulatory effects of alphaxalone.     

Neuroactive steroids and GABAA receptor plasticity in the brain of the WAG/Rij rat, a model of absence epilepsy

The role of neuroactive steroids and GABA(A) receptors in the generation of spontaneous spike-and-wave discharges (SWDs) was investigated in the WAG/Rij rat model of absence epilepsy. The plasma, cerebrocortical, and thalamic concentrations of the progesterone metabolite 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH PROG) were increased in the WAG/Rij rat at 2 months of age compared with those in control (Wistar) rats. In contrast, the brain and peripheral levels of 3alpha,5alpha-tetrahydrodeoxycorticosterone (3alpha,5alpha-TH DOC) did not differ between the two rat strains at this age. At 6 months of age, when absence epilepsy worsens in WAG/Rij rats, the plasma concentration of 3alpha,5alpha-TH PROG remained high whereas that of 3alpha,5alpha-TH DOC had increased, the cerebrocortical levels of both 3alpha,5alpha-TH PROG and 3alpha,5alpha-TH DOC had increased, and the thalamic concentrations of these metabolites had decreased. At 6 months of age the expression of the alpha(4) and delta subunits of the GABA(A) receptor in relay nuclei was increased. Finally, chronic stress induced by social isolation elicited a reduction in the amount of 3alpha,5alpha-TH PROG in the thalamus of 2-month-old WAG/Rij rats that was associated with a reduction in the number and overall duration of SWDs at 6 months of age. Absence epilepsy in the WAG/Rij rat is thus associated with changes in the abundance of neuroactive steroids and in the expression of specific GABA(A) receptor subunits in the thalamus, a brain area key to the pathophysiology of this condition.     

Targeted disruption of the GABA(A) receptor delta subunit gene leads to an up-regulation of gamma 2 subunit-containing receptors in cerebellar granule cells

GABA(A) receptors are chloride channels composed of five subunits. Cerebellar granule cells express abundantly six subunits belonging to four subunit classes. These are assembled into a number of distinct receptors, but the regulation of their relative proportions is yet unknown. Here, we studied the composition of cerebellar GABA(A) receptors after targeted disruption of the delta subunit gene. In membranes and extracts of delta-/- cerebellum, [(3)H]muscimol binding was not significantly changed, whereas [(3)H]Ro15-4513 binding was increased by 52% due to an increase in diazepam-insensitive binding. Immunocytochemical and Western blot analysis revealed no change in alpha(6) subunits but an increased expression of gamma(2) subunits in delta-/- cerebellum. Immunoaffinity chromatography of cerebellar extracts indicated there was an increased coassembly of alpha(6) and gamma(2) subunits and that 24% of all receptors in delta-/- cerebellum did not contain a gamma subunit. Because 97% of delta subunits are coassembled with alpha(6) subunits in the cerebellum of wild-type mice, these results indicated that, in delta-/- mice, alpha(6)betagamma(2) and alphabeta receptors replaced delta subunit-containing receptors. The availability of the delta subunit, thus, influences the level of expression or the extent of assembly of the gamma(2) subunit, although these two subunits do not occur in the same receptor.     

Role of the PLC-related, catalytically inactive protein p130 in GABA(A) receptor function

The protein p130 was isolated from rat brain as an inositol 1,4,5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-delta1 but lacking PLC activity. We show that p130 plays an important role in signaling by the type A receptor for gamma-aminobutyric acid (GABA). Yeast twohybrid screening identified GABARAP (GABA(A) receptor-associated protein), which is proposed to contribute to the sorting, targeting or clustering of GABA(A) receptors, as a protein that interacts with p130. Furthermore, p130 competitively inhibited the binding of the gamma2 subunit of the GABA(A) receptor to GABARAP in vitro. Electrophysiological analysis revealed that the modulation of GABA-induced Cl- current by Zn2+ or diazepam, both of which act at GABA(A) receptors containing gamma subunits, is impaired in hippocampal neurons of p130 knockout mice. Moreover, behavioral analysis revealed that motor coordination was impaired and the intraperitoneal injection of diazepam induced markedly reduced sedative and antianxiety effects in the mutant mice. These results indicate that p130 is essential for the function of GABA(A) receptors, especially in response to the agents acting on a gamma2 subunit.     

Effects of treatment with GABA(A) receptor subunit antisense oligodeoxynucleotides on GABA-stimulated 36Cl- influx in the rat cerebral cortex

GABA(A) receptor function was studied in cerebral cortical vesicles prepared from rats after intracerebroventricular microinjections of antisense oligodeoxynucleotides (aODNs) for alpha1, gamma2, beta1, beta2 subunits. GABA(A) receptor alpha1 subunit aODNs decreased alpha1 subunit mRNA by 59+/-10%. Specific [3H]GABA binding was decreased by alpha1 or beta2 subunit aODNs (to 63+/-3% and 64+/-9%, respectively) but not changed by gamma2 subunit aODNs (94+/-5%). Specific [3H]flunitrazepam binding was increased by alpha1 or beta2 subunit aODNs (122+/-8% and 126+/-11%, respectively) and decreased by gamma2 subunit aODNs (50+/-13%). The "knockdown" of specific subunits of the GABA(A )receptor significantly influenced GABA-stimulated 36Cl- influx. Injection of alpha1 subunit aODNs decreased basal 36Cl- influx and the GABA Emax; enhanced GABA modulation by diazepam; and decreased antagonism of GABA activity by bicuculline. Injection of gamma2 subunit aODNs increased the GABA Emax; reversed the modulatory efficacy of diazepam from enhancement to inhibition of GABA-stimulation; and reduced the antagonist effect of bicuculline. Injection of beta2 subunit aODNs reduced the effect of diazepam whereas treatment with beta1 subunit aODNs had no effect on the drugs studied. Conclusions from our studies are: (1) alpha1 subunits promote, beta2 subunits maintain, and gamma2 subunits suppress GABA stimulation of 36Cl- influx; (2) alpha1 subunits suppress, whereas beta2, and gamma2 subunits promote allosteric modulation by benzodiazepines; (3) diazepam can act as an agonist or inverse agonist depending on the relative composition of the receptor subunits: and (4) the mixed competitive/non-competitive effects of bicuculline result from activity at alpha1 and gamma2 subunits and the lack of activity at beta1 and beta2 subunits.     

Social isolation-induced increase in alpha and delta subunit gene expression is associated with a greater efficacy of ethanol on steroidogenesis and GABA receptor function

Previously we have demonstrated that social isolation of rats reduces both the cerebrocortical and plasma concentrations of 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH PROG), and potentiates the positive effects of acute ethanol administration on the concentrations of this neurosteroid. We now show that the ethanol-induced increase in 3alpha,5alpha-TH PROG is more pronounced in the brain than in the plasma of isolated rats. The ability of ethanol to inhibit isoniazid-induced convulsions is greater in isolated rats than in group-housed animals and this effect is prevented by treatment with finasteride. Social isolation modified the effects of ethanol on the amounts of steroidogenic regulatory protein mRNA and protein in the brain. Moreover, ethanol increased the amplitude of GABA(A) receptor-mediated miniature inhibitory postsynaptic currents recorded from CA1 pyramidal neurones with greater potency in hippocampal slices prepared from socially isolated rats than in those from group-housed rats, an effect inhibited by finasteride. The amounts of the alpha(4) and delta subunits of the GABA(A) receptor in the hippocampus were increased in isolated rats as were GABA(A) receptor-mediated tonic inhibitory currents in granule cells of the dentate gyrus. These results suggest that social isolation results in changes in GABA(A) receptor expression in the brain, and in an enhancement of the stimulatory effect of ethanol on brain steroidogenesis, GABA(A) receptor function and associated behaviour.     

Withdrawal properties of a neuroactive steroid: implications for GABA(A) receptor gene regulation in the brain and anxiety behavior

Early work in the field established that the 5 alpha-reduced metabolite of progesterone 3 alpha-OH-5 alpha-pregnan-20-one (allopregnanolone or 3 alpha,5 alpha-THP) is a potent positive modulator of the GABA(A) receptor (GABAR), the receptor mediating the effects of the primary inhibitory transmitter in the brain. This steroid acts in a manner similar to sedative drugs, such as the barbiturates, both in terms of potentiating GABA-induced inhibition in vitro and in behavioral assays, by reducing anxiety and seizure susceptibility. Because sedative compounds exhibit withdrawal properties that result in behavioral hyperexcitability, our laboratory has more recently investigated the effect of prolonged application and rapid removal (i.e. 'withdrawal') of this steroid, administered in vivo to female rats. Withdrawal from 3 alpha,5 alpha-THP produces a state of increased anxiety and lowered seizure threshold, similar to withdrawal from other GABA-modulatory drugs such as the benzodiazepines and alcohol. Hormone withdrawal also produced increases in the alpha 4-containing GABAR, an effect correlated with insensitivity of the GABAR to modulation by the benzodiazepine class of tranquilizers, as would normally occur under control conditions. In addition, changes in intrinsic channel properties, including a marked acceleration in the decay rate was also observed as a result of declining levels of 3 alpha,5 alpha-THP. Such a change would result in less inhibitory total current, and the resulting increase in neuronal excitability could then underlie the observed behavioral excitability following hormone withdrawal. These results suggest that actions of this steroid on a traditional transmitter receptor in the brain lead to alterations in GABAR subunit composition that result in changes in the intrinsic channel properties of the receptor and behavioral excitability. These results may have implications for endogenous fluctuations in this hormone which may accompany premenstrual dysphoric disorder.