Steroid requirements for regulation of the α4 subunit of the GABA_A receptor in an in vitro model

Abstract

The α4 subunit of the GABA_A receptor (GABAR) has relatively low expression in the CNS, but is increased in vivo following 48 h administration of the GABA-modulatory steroid 3α-OH-5α[β]-pregnan-20-one (THP or [allo]pregnanolone) to female rats. The purpose of the following study was to determine the optimal conditions for steroid-induced upregulation of α4 expression in an in vitro model. To this end, we used the IMR-32 cell, a neuroblastoma cell line, which normally expresses α4 mRNA at low levels. In undifferentiated IMR-32 cells, 48 h administration of THP increased α4 expression when ambient THP levels were reduced by the 5α-reductase blocker 4MA, suggesting that the background steroid milieu affects steroid regulation of this subunit. Following neuronal differentiation in serum-free medium, 48 h THP treatment significantly increased α4 expression two-fold following application of nerve growth factor (NGF) suggesting that development of neuronal processes facilitates this effect of the steroid. In the absence of NGF treatment, combined administration of 17β-estradiol (E₂) plus THP also increased α4 expression to a similar extent as THP following NGF treatment. In addition, E₂ alone effectively increased α4 expression to maximal levels following NGF treatment. In contrast, neuronal differentiation in the absence of serum deprivation did not increase α4 levels. These results suggest that both THP and E₂ can increase expression of the GABAR α4 subunit, but that this effect is dependent upon the background steroid milieu as well as the degree of neuronal development. These findings demonstrate optimal conditions for steroid-induced upregulation of the α4 subunit in an in vitro system.

The GABA_A receptor (GABAR) is a pentameric structure which mediates most fast inhibition in the brain, and is typically composed of 2α, 2β and 1γ subunit [3]. Although the α1-α3 subunits are most abundantly expressed in the CNS [38], the α4 subunit is capable of rapid plasticity. Indeed, there are numerous reports suggesting that chronic in vivo administration of positive GABA modulators, such as benzodiazepines and ethanol increase expression of this subunit in CNS [4, 13, 21]. In addition, the GABA-modulatory [22] metabolite of progesterone, THP (3α-OH-5α[β]-pregnan-20-one or [allo]pregnanolone), when administered to female rats at physiological doses, also increases α4 expression in hippocampus after 48-72 h [11, 34].

However, despite the in vivo evidence that chronic THP treatment can upregulate expression of the α4 protein, there is no direct evidence for this using in vitro techniques. In contrast, effects of other GABA-modulators on α4 expression have been established in vitro [7, 31]. In one recent study, 7 day administration of the parent compound progesterone was seen to increase α4 mRNA levels in differentiated NT2-N cells [27]. However, a second study showed that 5 day application of THP reduced α4 mRNA levels in developing P19 cells [10]. The experimental conditions of these two studies were different than the in vivo protocols, in that developing cells were used, mRNA levels rather than protein levels were assessed and, in the second study, α4 levels were assessed beyond the 48-72 h timepoint which produces maximal effects in vivo [11]. In addition, cells were grown in serum-containing medium [15], where the presence of steroids such as progesterone and THP may have influenced the ability of THP to alter α4 expression. Thus, the present study was conducted to examine effects of 48 h THP exposure on expression of α4 protein under varying conditions of neuronal differentiation and background steroid levels. To this end, we used 4MA (N,N-diethyl-4-methyl-3-oxo-4-aza-5α-androstane-17β-carboxamide), a blocker of THP formation [36], as well as serum-free conditions to compare with effects of 48 h THP treatment on α4 expression in developing or differentiated IMR-32 cells. This is a well-established neuroblastoma cell line previously shown to express GABAR subunits, including α4 and θ, which yield functional receptors [32]. In addition, the currents recorded from these cells are responsive to neurosteroid potentiation [32], further suggesting it as an appropriate model.

Human neuroblastoma IMR-32 cells (ATCC) were maintained in EMEM (ATCC) supplemented with 2 mM L-glutamine and 10% FBS, and were incubated in humidified 5% CO₂ at 37°C. After growing for 3-4 days, cells were differentiated into neurons with one of two different protocols: i. serum deprivation or ii. 5-bromo-2-deoxyuridine (BrdU)-induced. For i, we used DMEM/F-12 (ATCC), supplemented with 0.2% BSA for 2-5 days, with or without 20 ng/ml human NGF-β [29]. For ii, we used 10 μM BrdU (Fluka) added to DMEM/F-12 with 5% FBS for 10-11 days. All culture media contained 100 I.U./ml penicillin and 100 μg/ml streptomycin.

In some cases 4MA (N,N-diethyl-4-methyl-3-oxo-4-aza-5α-androstane-17β-carboxamide, 5α-reductase blocker, 500 nM), THP (100 nM, P8150-000, Steraloids, Newport, RI) and/or E₂ (600 pM, Steraloids) were added to the cells for 48 h (vehicle, 0.001% DMSO).

Levels of the α4 subunit were assessed using standard Western blot procedures as described previously [36] following isolation of cell membranes and determination of protein content. In all cases, two or three protein loading amounts were tested per sample to ensure density readings in the linear range. Cell membranes electrophoresed onto 8% or 10% Tris-Glycine or NuPage Bis-Tris gels were transferred to nitrocellulose membranes (Invitrogen). The α4 subunit was detected with a rabbit antibody [36] against the C-terminal end as a 67 kDa band and visualized by enhanced chemoluminesence (ECL) (Pierce). The optical density of the immunoreactive bands was analyzed using a Microtek scanner and One-DScan Gel Analysis Software. Results were normalized to a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control protein from the same samples (36 kDa band).

GABA-activated current was recorded using whole cell patch clamp procedures at -50 mV at room temperature (20-25°C) in a bath solution containing (in mM): NaCl 140, KCl 5, CaCl₂ 1, MgCl₂ 1, HEPES 10, and D-glucose 24, pH 7.4, 320 mOsm/kg H₂O. The pipette solution contained (in mM): N-methyl-D-glutamine chloride 120, Cs₄BAPTA 5, Mg-ATP 5. The ATP regeneration system, Tris phosphocreatinine (20 mM) and creatine kinase (50 U/ml), was added to minimize GABA rundown. Currents were filtered at 3 kHz and digitally sampled at 10 kHz using the pClamp 8.2 software package (Axon Instruments). Drug delivery was accomplished via a solenoid-activated rapid superfusion system positioned within 50 μm of the cell, as previously described [36]. Peak GABA-gated current was calculated during THP application as the percentage GABA potentiation relative to the current gated by 10 μM GABA (EC₂₀). All chemicals were obtained from Sigma or Calbiochem unless otherwise indicated.

Data are expressed as the mean ± S.E.M. Differences between groups were assessed using standard ANOVA and Tukey (>2 groups) post hoc procedures. Significance was determined when p < 0.05.

We initially tested the effect of 48 h THP administration on GABAR α4 expression in undifferentiated IMR-32 cells. Cells were treated once per day with 100 nM THP in some cases following blockade of endogenous THP in the medium using the 5α-reductase blocker 4MA (500 nM). In fact, 48 h THP treatment was effective in significantly (P<0.05) increasing α4 expression by 2-3-fold only when basal levels of the steroid in serum-containing medium were reduced with 4MA (Fig. 1). In contrast, 48 h treatment with THP or 4MA applied alone had no effect on α4 expression.

Fig. 1.

48 h THP administration alters expression of the α4 subunit of the GABA_A receptor in undifferentiated IMR-32 cells. A, Representive Western blot of α4 and GAPDH immunoreactivity. Con, control. B, Statistical analysis of the data is represented as the integrated optical density of the immunoreactive bands relative to the GAPDH level and is expressed as a ratio of the control condition (mean ± S.E.M.). (ANOVA, ^* p < 0.05 vs. Con, n = 8).

Because this result suggests that the background steroid milieu is a factor in α4 expression, we chose the serum deprivation method with or without addition of NGF to differentiate IMR-32 cells into a neuronal phenotype. After 2-5 days in serum-free medium, IMR-32 cells grew large neuronal processes, which were increased following NGF administration (20 ng/ml), as previously described [29].

We initially verified that GABA application generated bicuculline-sensitive currents following NGF treatment of IMR-32 cells differentiated in serum-free medium (Fig. 2A), as has been reported in undifferentiated IMR-32 cells [32]. Further, 100 nM THP application significantly (P<0.05) potentiated this GABA(EC₂₀)-gated current by 102 ± 12 % in NGF-treated differentiated IMR-32 cells (n=13), but was without effect in the absence of GABA application (Fig. 2B), suggesting that this steroid is a potent positive GABA-modulator as described in other systems [22, 32].

Fig. 2.

NGF-differentiated IMR-32 cells express functional GABA_A receptors which are steroid-sensitive. A, GABA-gated whole-cell current was completely blocked by bicuculline. Representative of results from 6 cells. Horizontal bars, drug application. B, 100 nM THP potentiated 10 μM GABA-induced current by 102 % (mean), but did not elicit a current without GABA application. Representative of results from 13 cells.

Under conditions of serum deprivation, combined administration of THP plus E₂ significantly increased α4 expression by 2-3-fold (Fig. 3E,F), while neither steroid alone was effective in this regard. However, following treatment with NGF, all steroid treatment regimens produced significant increases in α4 expression: THP; E₂; and THP plus E₂ (P<0.05). In contrast, THP was ineffective in upregulating α4 expression following neuronal differentiation in serum-containing medium (Fig. 3C,D). In addition, neuronal differentiation by serum deprivation alone (Fig. 3A, B) did not alter α4 expression compared to the undifferentiated state of the cells. These data suggest that neuronal differentiation by serum deprivation plus NGF administration is the optimal condition for steroid-induced increases in α4 expression in IMR-32 cells by THP as well as by E₂.

Fig. 3.

48 h steroid treatment increases α4 expression in differentiated IMR-32 cells: An effect dependent upon nerve growth factor. Representative Western blot (A) and averaged data (B) of α4 and GAPDH immunoreactivity in differentiated (Diff) and undifferentiated (Undiff) IMR32 cells (10 μg loading protein). There are no significant differences in α4 expression between these two stages of cells (Mean ± SEM, p > 0.05, n = 4). C, BrdU-differentiated IMR-32 cells in serum-containing medium. THP did not increase α4 expression. D, Averaged data, P>0.05, n-4. E, In contrast, E₂ + THP treatment increased α4 expression in differentiated IMR-32 cells cultured without NGF (left), while 48 h treatment with THP, E₂ or E₂ + THP all increased α4 expression in cells differentiated with 100 nM NGF (right). F, Averaged optical densities (mean ± S.E.M), normalized to GAPDH level and expressed relative to control. (ANOVA, ^* p < 0.05 vs. control. n = 6; ^** p < 0.05 vs. NGF. n = 5).

The results from this study suggest that both the background steroid milieu as well as the developmental stage of the IMR-32 cell influence the ability of THP to increase expression of the α4 subunit of the GABAR. In both differentiated and undifferentiated IMR-32 cells, THP produced significant effects on α4 expression only with low background steroid levels. These results are consistent with findings from both in vitro and behavioral studies showing that THP exerts more significant effects when endogenous THP levels are lowest across the estrous cycle [6]. Unpublished results from this laboratory also demonstrate that THP administration to female mice does not alter α4 expression when administered during the nocturnal surge in CNS THP levels. These findings imply that the critical factor regulating α4 expression is not the absolute level of steroid, but rather the change in steroid level which triggers α4 expression, as has been suggested by other groups [18].

In differentiated neuroblastoma cells grown in serum-free medium to lower the ambient steroid levels, THP produced significant effects on α4 expression only following NGF treatment. The increased development of neuronal processes produced by NGF [29] may be responsible for this effect, as α4 expression in native receptors has been localized to dendritic processes rather than the perisomatic region [25]. Alternatively, expression of factors involved in transcription or trafficking may also play a role. In fact, NGF is known to increase several cell signaling cascades [14], including Map Kinase, PI 3-kinase and phospholipase C-γ, as well as a diverse array of G-proteins, which are known to regulate receptor trafficking in neurons, while early growth factor (Egr) is a key element in activating the α4 promoter [30]. However, our data as well as other studies [27] have not observed steroid-induced upregulation of the α4 subunit after 48 h treatment of differentiated neurons, suggesting that this event requires both differentiation as well as a serum-free environment.

Interestingly, 48 h E₂ exposure was also effective in increasing α4 expression in NGF-treated cells. It has been shown that long-term exposure to NGF enhances E₂ binding in neuronal differentiating PC12 cells. Reciprocally, E₂ up-regulates TrkA mRNA and transiently down-regulates p75 mRNA [23], which are NGF receptors, suggesting that E₂ may increase the efficiency of NGF binding in IMR-32 which express TrkA but not p75 [12]. Another recent study has shown that E₂ can increase α4 expression in NT2-N cells after a 7-day exposure [27]. The difference in timecourse may be due to the fact that serum-free medium was used in the present study to reduce ambient steroid levels, which would be expected to increase steroid sensitivity. In addition, differences in characteristics of cell lines and maturity of neurons [24, 32] might also play a role.

Our findings also suggest that THP and E₂ produce additive effects, consistent with the idea that these steroids act via distinct mechanisms. THP is a positive modulator of the GABAR [22], while E₂ has no direct effect at GABAR [22], but can initiate a variety of cell signaling mechanisms known to affect α4 transcription and receptor trafficking [16], such as PKC [28], which can also increase activation of the α4 promoter [30]. Our earlier in vivo work also suggested that E₂ could enhance the ability of progesterone, the parent compound of THP, to increase α4 expression [34]. The recently characterized α4 promoter [20] has multiple start sites and lacks a conventional TATA box, suggesting that it is regulated by a variety of conditions, consistent with the findings from the present study.

The effect of E₂ may also be related to indirect effects on neuronal development. Recent studies suggest that E₂ can increase brain-derived neurotrophic factor in the CNS [33, 39], leading to dendritic plasticity and neuronal development [33]. In addition, it has been reported that 48 h E₂ exposure prolongs and enhances Ca transients induced by GABAR activation or voltage-gated channels in developing neurons [26]. Calcium influx has been identified as a critical factor in GABAR regulation in response to prolonged GABA exposure [19], and may contribute to the observed effect of E₂ in the present study.

The results from the present study are consistent with our previous in vivo findings, which demonstrated that 48 h exposure of female rats to THP increases expression of the α4 subunit in CA1 hippocampus [11, 34]. The timecourse of steroid exposure required for α4 expression in fact was in the narrow range of 48-72 h, where longer exposures returned α4 expression back to control levels [11], although withdrawal from the steroid was also effective in increasing α4 expression [36]. In fact, one study which used a longer exposure time reported a decrease in α4 mRNA expression, although this reversed 24 h after withdrawal [10]. Other studies have reported increases in α4 expression in other CNS areas, such as the periaqueductal grey, after steroid withdrawal [9]. Study of the withdrawal effects of the steroid require a 21 day exposure period [36] which is not possible with the current model, and awaits further study. It is entirely possible that different mechanisms mediate the effects of chronic steroid administration and steroid withdrawal on α4 expression, however.

α4-containing GABAR have unique kinetic properties compared to the more abundantly expressed α1-, α2-, and α3-containing GABAR [8, 17]. They deactivate more quickly [35], with a faster τ-fast, and have a lower open probability as well as a significantly shorter mean open time [1]. These characteristics would tend to decrease the total charge transfer, and reduce the inhibition produced by current gated through these receptors. Indeed, increases in α4 expression are seen in models of temporal lobe epilepsy [2], as well as during the withdrawal hyperexcitability and increased anxiety that follows chronic administration of GABA-modulatory compounds [4, 7, 13, 21, 36]. In fact, down-regulation of α4 expression with in vivo antisense treatment prevents increases in seizure susceptibility following neurosteroid withdrawal [36], thus providing evidence that these faster deactivating receptors may mediate hyperexcitability states. Thus, regulation of the expression of the α4 subunit has significant consequences for the behavioral state of the animal.

In addition, α4-containing GABARs are insensitive to modulation by benzodiazepines [38]. These characteristics suggest that alterations in α4 expression may be relevant for premenstrual dysphoric disorder, when increased anxiety [5] and decreased benzodiazepine responsiveness [37] have been reported shortly after the onset of the luteal phase, following increases in endogenous, circulating levels of THP. The results from this study may suggest conditions which underlie changes in α4 expression relevant for changes in mood and behavior across the menstrual cycle.