γ-aminobutyric acid (GABA) is the major inhibitory neu-rotransmitter in the adult mammalian central nervous system. However, GABA depolarizes immature rat hip-pocampal neurons and increases intracellular Ca2+ ([Ca2+]i). Here we show, that GABA and the GABAA receptor agonist muscimol induce c-Fos immunoreactivity and increase BDNF mRNA expression in embryonic hip-pocampal neurons cultured for 5 days. In contrast, after 3 weeks in culture, GABA and muscimol failed to induce c-fos and BDNF expression. Fura-2 fluorescence microscopy revealed that muscimol produces a dihydropyridine-sensitive transient increase in [Ca2+]i, comparable to the effect of the non-NMDA receptor agonist kainic acid in neurons cultured for 5 days, but not in 3-week-old cultures. The increase in c-Fos immunoreactivity and BDNF mRNA levels by GABA were dependent upon the activation of voltage-gated Ca2+ channels, as shown using the L-type specific Ca2+ channel blocker nifedipine. The differential regulation of c-fos and BDNF expression by GABA and muscimol in developing and mature hippocampal neurons is due to a switch in the ability of GABAA receptors to activate voltage-gated Ca2+ channels. These observations support the hypothesis that GABA might have neurotrophic effects on embryonic or perinatal hippocampal neurons, which are mediated by BDNF.

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin gene family (Barde et al., 1982; Leibrock et al., 1989), which to date comprises BDNF, nerve growth factor (NGF), neurotrophin-3, neurotrophin-4/5 and neurotrophin-6 (Barde, 1990; Snider, 1994; Götz et al., 1994). In vitro and in vivo studies have established that BDNF plays an important role in controlling neuronal survival and differentiation in the peripheral nervous system (Barde, 1990; Davies, 1994; Ernfors et al., 1994; Jones et al., 1994). In the brain, BDNF mRNA is expressed in virtually all regions, the highest levels being found in the hippocampus and cerebral cortex (Hofer et al., 1990). However, the function of BDNF is less clear in the brain, although BDNF has been shown to promote the survival of cultured dopaminergic neurons from the substantia nigra (Hyman et al., 1991), cultured cerebellar granule cells (Lindholm et al., 1993) and cortical neurons (Gosh et al., 1994). Recently, it has been demonstrated that BDNF regulates neuropeptide expression in interneurons of various brain areas, including neocortex and hippocampus (Jones et al., 1994; Nawa et al., 1994; Croll et al., 1994), suggesting a function for BDNF during maturation of the GABAergic system and its maintenance.

GABA acts as the main inhibitory neurotransmitter at central synapses in adult mammals. However, it depolarizes various types of developing neurons including spinal (Wu et al., 1992; Reichling et al., 1994), cerebellar (Connor et al, 1987), cortical (Yuste and Katz, 1991) and hippocampal neurons (Mueller et al., 1984; Ben-Ari et al., 1989; Cherubini et al., 1990; Hosokawa et al., 1994). In the developing rat hippocampus, GABA is responsible for the expression of so-called ‘giant depolarizing potentials’ (GDPs) in immature CA3 pyramidal neurons (Ben-Ari et al., 1989). These bicuculline-sensitive potentials are expressed only during early postnatal life (Ben-Ari et al., 1989). As a consequence of its depolarizing action, stimulation by GABA leads to a rise in [Ca2+]i, as shown for developing cortical neurons using slice preparation (Yuste and Katz., 1991; Lin et al., 1994), and in cultured hippocampal neurons (Segal, 1993).

In neurons, the expression of several immediate early genes, such as the proto-oncogene c-fos, is regulated by neuronal activity (Morgan and Curran, 1986; Sheng and Greenberg, 1990). In PC12 cells, depolarization by high KCl concentra tions leads to an increase in c-fos expression, which is dependent on Ca2+ influx (Morgan and Curran, 1986). Moreover, both synaptic activation of NMDA receptors and seizures up-regulate c-fos expression in the rat hippocampus in vivo (Morgan et al., 1987; Cole et al., 1989). Recently, evidence has been provided that distinct Ca2+ signalling pathways might be employed for the up-regulation of c-fos, depending upon whether Ca2+ enters the cells via voltage-gated Ca2+ channels or NMDA receptors (Bading et al., 1993).

Physiological and pathophysiological neuronal activity also increases the mRNA expression of two members of the neurotrophin gene family, NGF and BDNF, in hippocampal neurons in vitro and in vivo (Gall and Isackson, 1989; Zafra et al., 1990, 1991; Castrén et al., 1993; Lindefors et al., 1992; Berzaghi et al., 1993). Glutamatergic stimulation via non-NMDA and NMDA receptors as well as cholinergic activation via muscarinic receptors rapidly increases BDNF mRNA in the rat hippocampus (Zafra et al., 1990, 1991; Berzaghi et al., 1993). The increase in BDNF mRNA has been shown to be Ca2+ dependent and can be blocked by Ca2+/calmodulin inhibitors (Zafra et al., 1992). In contrast, intraperitoneal injection of the GABAA receptor agonist, muscimol reduces BDNF mRNA in adult rat hippocampal neurons (Zafra et al., 1991). On this basis, it has been suggested, that the interplay between excitatory and inhibitory activity determines the levels of BDNF expression (Zafra et al., 1991).

Given the depolarizing effect of GABA on developing hip-pocampal neurons, in the present study we have examined whether, in contrast to the adult situation, GABA is able to up-regulate the expression of c-fos and BDNF in developing neurons. We show that GABA and muscimol acting via GABAA receptors have a differential effect on BDNF and c-fos expression in developing and mature cultured hippocam-pal neurons. Stimulation of c-fos and BDNF expression by GABA or muscimol in developing hippocampal neurons could be attributed to the ability of GABAA receptors to activate voltage-gated Ca2+ channels, which is lost during maturation. These results provide a rational basis for the hypothesis that GABA might exert a neurotrophic effect on rat hippocampal neurons during embryonic and early postnatal development.

Cell culture

Hippocampal neurons were prepared from E17 rat embryos. Hip-pocampi were incubated for 20 minutes at 37°C in phosphate-buffered saline (PBS) without Ca2+ or Mg2+, containing 10 mM glucose, 1 mg ml−1 bovine serum albumin (Sigma, St. Louis, MO, USA), 1 μg ml− 1 DNase (Sigma) and 12 μg ml−1 papain (Sigma), dissociated with a plastic pipette and centrifuged (5 minutes at 1000 revolutions per minute). Cells were resuspended in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum (Gibco, Paisley, UK). 5×105 cells were plated in 35 mm dishes (Falcon, Becton Dickinson, Plymouth, UK) and after 3 hours the medium was changed to defined medium as described previously (Zafra et al., 1990).

Calcium imaging

For measurement of [Ca2+]i (Grynkiewicz et al., 1985) hippocampal neurons were cultured in coverglass chambers (Nunc, Wiesbaden, FRG). Cells were loaded for 40 minutes (37°C, 10% CO2) with 2 μM fura-2/AM (Calbiochem, Bad Soden, FRG, 1 mM stock dissolved in DMSO/10% pluronic F-127, Molecular Probes, Eugene, OR, USA), rinsed and incubated in fresh culture medium for 10 minutes prior to the measurement. During [Ca2+]i imaging, cells were kept in medium consisting of 142 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, 1 mM NaH2PO4, 5 mM Glucose, 25 mM Hepes and 0.1% bovine serum albumin, adjusted to pH 7.4. Cells were visualized with a Zeiss Fluar 40×/1.30 oil objective using an inverted microscope (Axiovert 100, Zeiss, Jena, FRG). Fluorescence was determined at the excitation wavelengths of 340 and 380 nm with an ICCD camera (C2400-87, Hamamatsu, Joko-cho, Japan) and images were processed with the Argus 50/CA fluorescence ratios. Corresponding [Ca2+]i was estimated with the equation [Ca2+]i = Kd (R-Rmin)/(Rmax-R) Sf2/Sb2, where Kd is the dissociation constant for fura-2/Ca2+ (224 nM), R the determined ratio, Rmin and Rmax the ratios at zero and saturating [Ca2+]i, respectively, and Sf2/Sb2 the ratio of excitation efficiencies for free and bound fura-2 at 380 nm (Grynkiewicz et al., 1985).

c-Fos immunocytochemistry

Cultured hippocampal neurons were treated for 3 hours with 50 μM GABA, 25 μM muscimol or 25 μM kainic acid. After fixation for 15 minutes in 4% paraformaldehyde in PBS, cells were rinsed with PBS and incubated for 30 minutes in TBS containing 0.5% Triton X-100 and 3% normal goat serum (Serva, Heidelberg, FRG). Cells were then incubated overnight at 4°C with polyclonal antibodies against c-Fos (1:2000; sc-52, Santa Cruz Biotechnology, CA, USA). Subsequently, cells were rinsed and incubated for 1 hour with a solution of anti-rabbit biotinylated antibodies (1:100; Vector Labs, Burlingame, CA, USA) followed by incubation with an avidin-biotin-horseradish per-oxidase complex (1:100; Vector Labs) for 1 hour. Staining was developed with a TBS solution containing 0.05% diaminobenzidine (Sigma) and 0.01% hydrogen peroxide.

RNA analysis

Total RNA was extracted by the guanidinium thiocyanate method (Chomczynski and Sacchi, 1987), glyoxylated, fractionated on a 1% agarose gel and transferred to Hybond N filters (Amersham, Braun-schweig, FRG). Filters were hybridized in a solution consisting of 50% deionized formamide, 6× SSC, 5× Denhardts, 0.5% SDS, 50 mM NaH2PO4 (pH 7.0), 5 mM EDTA and 20 μg ml−1 salmon sperm DNA at 65.5°C to 32P-labelled mouse BDNF or β-actin cRNA probes. Filters were washed twice for 10 minutes in 2× SSC, 0.1% SDS at 65.5°C and for 15 minutes in 0.2× SSC, 0.1% SDS at 72°C. Subsequently, filters were exposed to an imaging plate (Fuji Type BAS-III S, Fuji, Japan) or X-ray film (Fuji) for 24 hours and signals were analysed with a scanning device (Fujix Bas 1000). Two BDNF transcripts were detected at 4.2 and 1.6 kb. Both transcripts were regulated in the same manner. For quantification, the levels of both BDNF transcripts were normalized to the amount of β-actin mRNA per lane. The levels of β-actin mRNA were not changed by any of the experimental procedures.

Drugs

γ-aminobutyric acid, muscimol, kainic acid and bicuculline were obtained from Sigma, nifedipine from Biomol (Hamburg, FRG).

Increase in [Ca2+]i in immature hippocampal neurons by GABA and muscimol

The effect of GABAergic stimulation on [Ca ]i in embyronic hippocampal neurons was studied by fura-2 fluorescence microscopy, 5 days after plating. The neurons had a basal [Ca2+]i of 51 nM ±24 (s.d.; n>15). As shown in Fig. 1, virtually all neurons responded to 1 μM GABA with a transient increase in [Ca2+]i, peaking at 197 nM ±119 (n=21). Decreasing the concentration of GABA down to 100 nM still induced a sig-nificant [Ca2+]i elevation (67 nM ±20; n=21), but the responsiveness varied markedly within a population of immature hip-pocampal neurons, many of them showing no change in [Ca2+]iat this dose (not shown). 100 μM bicuculline completely blocked the increase in [Ca2+]i by GABA providing evidence for the involvement of GABAA receptor activation (not shown). Moreover, the effect of GABA was mimicked by muscimol. The response to muscimol at the maximally effective concentration of 100 μM peaked at 867 nM ±105, comparable to that elicited by 100 μM kainic acid (621 nM ±278) (Fig. 2A). Pretreatment of the cells with 20 μM nifedip-ine completely abolished the effect of muscimol on [Ca2+]i suggesting that the increase was mainly due to Ca2+ influx through L-type Ca2+ channels (Fig. 2B). Neurons, cultured for 15 days, had a slightly higher basal [Ca2+]i (108 nM ±40) than the cells cultured for 5 days. Moreover, in the mature neurons oscillations in [Ca2+]i were observed, indicating spontaneous synaptic activity in these cells (not shown). At this stage of development, however, treatment with 100 μM muscimol had only a minor effect on [Ca2+]i (146 nM ±70), whereas 100 μM kainic acid evoked a very prominent response (582 nM ±345) in these neurons, which recovered much slower than in neurons cultured for 5 days, indicating an increase in glutamate responsiveness. After 3 weeks in culture, application of muscimol had still a modest effect on [Ca2+]i, raising [Ca2+]i from 134 nM ±80 to 223 nM ±107 (Fig. 3A). 100 μM bicuculline, however, induced a slow rise in [Ca2+]isuggesting that endogeneously released GABA, in fact, mediated inhibition (Fig. 3B). This indicates that the neurons had undergone a specific change in the Ca2+ response to GABAA receptor activation.

Fig. 1.

GABAA receptor stimulation induces a transient [Ca2+]i increase in immature hippocampal neurons. (A) Ratio image of neurons which were cultured for 5 days. (B) Ratio image of these cells stimulated for 20 seconds with 1 μM GABA. C. After 120 seconds [Ca2+]i had almost recovered to basal levels. The sequence shows an uniform response in all neurons. Colour scale from dark blue (50 nM) to red (400 nM).

Fig. 1.

GABAA receptor stimulation induces a transient [Ca2+]i increase in immature hippocampal neurons. (A) Ratio image of neurons which were cultured for 5 days. (B) Ratio image of these cells stimulated for 20 seconds with 1 μM GABA. C. After 120 seconds [Ca2+]i had almost recovered to basal levels. The sequence shows an uniform response in all neurons. Colour scale from dark blue (50 nM) to red (400 nM).

Fig. 2.

GABAA receptor stimulation activates voltage-gated Ca2+ channels in immature hippocampal neurons. (A) 100 μM muscimol and 100 μM kainic acid increase [Ca2+]i to a similar extent in neurons cultured for 5 days. (B) Pretreatment with 20 μM nifedipine (15 minutes prior to stimulation) completely blocked the Ca2+ increase induced by muscimol.

Fig. 2.

GABAA receptor stimulation activates voltage-gated Ca2+ channels in immature hippocampal neurons. (A) 100 μM muscimol and 100 μM kainic acid increase [Ca2+]i to a similar extent in neurons cultured for 5 days. (B) Pretreatment with 20 μM nifedipine (15 minutes prior to stimulation) completely blocked the Ca2+ increase induced by muscimol.

Fig. 3.

GABAA receptor activation produces only a minor rise in [Ca2+]i, whereas GABAA receptor blockade results in a high elevation of [Ca2+]i in neurons cultured for 3 weeks. (A) 100 μM muscimol did not induce an increase in [Ca2+]i, whereas 100 μM kainic acid produced a large response. (B) Treatment with 100 μM bicuculline, a GABAA receptor blocker led to a slow increase in [Ca2+]i.

Fig. 3.

GABAA receptor activation produces only a minor rise in [Ca2+]i, whereas GABAA receptor blockade results in a high elevation of [Ca2+]i in neurons cultured for 3 weeks. (A) 100 μM muscimol did not induce an increase in [Ca2+]i, whereas 100 μM kainic acid produced a large response. (B) Treatment with 100 μM bicuculline, a GABAA receptor blocker led to a slow increase in [Ca2+]i.

GABAA receptor activation induces c-Fos immunoreactivity and BDNF mRNA expression in immature hippocampal neurons

An increase in [Ca2+]i is known to activate a number of genes including the proto-oncogene c-fos (Morgan and Curran, 1986). Therefore we studied whether the [Ca2+]i rise induced by GABAA receptor stimulation in the immature hippocampal neurons would activate immediate early genes such as c-fos. 5 days after plating, neurons were treated for 3 hours with 50 μM GABA, 25 μM muscimol or 25 μM kainic acid. All of these treatments dramatically increased c-Fos immunoreactivity, when compared to control cultures (Fig. 4). 100 μM bicuculline blocked the induction of c-fos by GABA or muscimol (Fig. 5B). Moreover, when the neurons were pretreated with 20 μM nifedipine, GABA and muscimol failed to induce c-fos, showing that Ca2+ influx was indeed mediating the observed increase (Fig. 5C). In contrast to the effect of GABAA receptor activation on c-fos expression in immature neurons, there was no induction of c-Fos immunoreactivity by GABA or muscimol 3 weeks after plating (Fig. 6). Kainic acid, however, clearly induced c-fos expression (Fig. 6D).

Fig. 4.

Induction of c-Fos immunoreactivity by GABAA receptor activation in immature hippocampal neurons. Neurons were cultured for 5 days and treated for 3 hours as indicated. (A) Control. (B) 50 μM GABA. (C) 25 μM muscimol. (D) 25 μM kainic acid. An induction of c-Fos immunoreactivity is observed in response to both GABAA receptor agonists and kainic acid.

Fig. 4.

Induction of c-Fos immunoreactivity by GABAA receptor activation in immature hippocampal neurons. Neurons were cultured for 5 days and treated for 3 hours as indicated. (A) Control. (B) 50 μM GABA. (C) 25 μM muscimol. (D) 25 μM kainic acid. An induction of c-Fos immunoreactivity is observed in response to both GABAA receptor agonists and kainic acid.

Fig. 5.

The effect of GABAA receptor activation on c-Fos immunoreactivity is blocked by 100 μM bicuculline or 20 μM nifedipine. Neurons were cultured for 5 days. Bicuculline or nifedipine treatment started 15 minutes before the stimulation with GABA. (A) 50 μM GABA. (B) 100 μM bicuculline + 50 μM GABA. (C) 20 μM nifedipine + 50 μM GABA.

Fig. 5.

The effect of GABAA receptor activation on c-Fos immunoreactivity is blocked by 100 μM bicuculline or 20 μM nifedipine. Neurons were cultured for 5 days. Bicuculline or nifedipine treatment started 15 minutes before the stimulation with GABA. (A) 50 μM GABA. (B) 100 μM bicuculline + 50 μM GABA. (C) 20 μM nifedipine + 50 μM GABA.

Fig. 6.

GABAA receptor activation fails to induce c-Fos immunoreactivity in mature hippocampal neurons. Neurons were cultured for 3 weeks. (A)Control. (B) 50 μM GABA (C) 25 μM muscimol. (D) 25 μM kainic acid.

Fig. 6.

GABAA receptor activation fails to induce c-Fos immunoreactivity in mature hippocampal neurons. Neurons were cultured for 3 weeks. (A)Control. (B) 50 μM GABA (C) 25 μM muscimol. (D) 25 μM kainic acid.

In addition to c-fos, GABAA receptor activation might activate other genes such as BDNF, which is expressed in embryonic hippocampal neurons. Stimulation of hippocampal neurons 5 days after plating with 50 μM GABA for 3 hours, resulted in an approximate 3-fold increase in the steady state levels of BDNF mRNA (Fig. 7A,D). The induction of BDNF mRNA by GABA was blocked by bicuculline and mimicked by muscimol (Fig. 7D) providing evidence that the effect was specifically mediated via GABAA receptors. A very marked induction was already seen with a concentration of 1 μM muscimol and was maximal at 25 μM (Fig. 7B). BDNF mRNA expression increased within 1 hour, reached its maximum after 3 hours and remained above basal levels for more than 12 hours (Fig. 7C). BDNF mRNA expression was also up-regulated by 25 μM kainic acid (Fig. 7D). As was observed for c-fos, the increase in BDNF mRNA by GABA and muscimol in these neurons was completely abrogated by 20 μM nifedipine, demonstrating that Ca2+ influx mediated the effect of GABAA receptor activation on BDNF expression (Fig. 7D). As shown in Fig. 8, after 2 weeks in culture, muscimol failed to induce BDNF mRNA expression. In contrast, kainic acid clearly up-regulated BDNF mRNA levels. Moreover, treatment with 100 μM bicuculline dramatically increased BDNF mRNA levels in these cultures, in contrast to immature neurons, suggesting that endogenously released GABA acting via GABAA receptors had a suppres-sive effect upon BDNF mRNA at this stage (Fig. 8).

Fig. 7.

GABAA receptor activation up-regulates BDNF mRNA expression in immature hippocampal neurons. Neurons were cultured for 5 days. (A) Northern blot analysis for BDNF mRNA expression under control condition (C) and stimulation for 3 hours with 50 μM GABA (G) or 25 μM kainic acid (K). (B) Regulation of BDNF mRNA expression by muscimol. BDNF induction was maximal at a concentration of 25 μM. (C) Time course of the effect of 25 μM muscimol on BDNF induction. BDNF mRNA levels increased within 1 hour and showed maximal up-regulation after 3 hours. (D) Quantitative analysis of BDNF mRNA expression. Steady state levels of BDNF mRNA are shown as a percentage of the control ± s. e. m. (n=3). 50 μM GABA, 25 μM muscimol or 25 μM kainic acid increased BDNF mRNA levels. Pretreatment for 15 minutes with 100 μM bicuculline blocked the BDNF induction by GABA. Bicuculline alone had no effect. Pretreatment for 15 minutes with 20 μM nifedipine blocked the increase in BDNF mRNA by muscimol. None of these treatments induced a change in the expression of β-actin mRNA, which was therefore used to normalize the BDNF mRNA levels.

Fig. 7.

GABAA receptor activation up-regulates BDNF mRNA expression in immature hippocampal neurons. Neurons were cultured for 5 days. (A) Northern blot analysis for BDNF mRNA expression under control condition (C) and stimulation for 3 hours with 50 μM GABA (G) or 25 μM kainic acid (K). (B) Regulation of BDNF mRNA expression by muscimol. BDNF induction was maximal at a concentration of 25 μM. (C) Time course of the effect of 25 μM muscimol on BDNF induction. BDNF mRNA levels increased within 1 hour and showed maximal up-regulation after 3 hours. (D) Quantitative analysis of BDNF mRNA expression. Steady state levels of BDNF mRNA are shown as a percentage of the control ± s. e. m. (n=3). 50 μM GABA, 25 μM muscimol or 25 μM kainic acid increased BDNF mRNA levels. Pretreatment for 15 minutes with 100 μM bicuculline blocked the BDNF induction by GABA. Bicuculline alone had no effect. Pretreatment for 15 minutes with 20 μM nifedipine blocked the increase in BDNF mRNA by muscimol. None of these treatments induced a change in the expression of β-actin mRNA, which was therefore used to normalize the BDNF mRNA levels.

Fig. 8.

GABAA receptor activation suppresses BDNF mRNA expression in mature hippocampal neurons. Neurons were cultured for 15 days. Steady state levels of BDNF mRNA are shown as a percentage of the control ± s. e. m. (n=3). In contrast to 5 days old cells, 25 μM muscimol did not increase BDNF mRNA expression. 100 μM bicuculline, however, greatly up-regulated BDNF mRNA steady state levels. BDNF mRNA levels were normalized to the amount of β-actin mRNA.

Fig. 8.

GABAA receptor activation suppresses BDNF mRNA expression in mature hippocampal neurons. Neurons were cultured for 15 days. Steady state levels of BDNF mRNA are shown as a percentage of the control ± s. e. m. (n=3). In contrast to 5 days old cells, 25 μM muscimol did not increase BDNF mRNA expression. 100 μM bicuculline, however, greatly up-regulated BDNF mRNA steady state levels. BDNF mRNA levels were normalized to the amount of β-actin mRNA.

The aim of this study was to investigate the effect of GABAA receptor activation on gene expression in immature rat hip-pocampal neurons. Since it was shown that GABA depolarizes immature hippocampal neurons (Ben-Ari et al., 1989; Cherubini et al., 1990; Fiszman et al., 1990) leading to Ca2+ influx (Segal, 1993), we have examined here the effect of GABA on the expression of two genes, c-fos and BDNF, which are regulated in an activity-dependent manner in hippocampal neurons (Morgan and Curran, 1986; Zafra et al., 1990). The main finding of this study was the observation that immature hippocampal neurons respond to GABAA receptor activation by a transient Ca2+ influx, which up-regulates c-Fos immunore-activity and BDNF mRNA expression. The causal link between the Ca2+ influx and the increased c-fos and BDNF expression was demonstrated by the finding that treatment with nifedipine, which blocks L-type voltage-gated Ca2+ channels, completely abolished the transient [Ca2+]i rise and also abrogated both the increase in c-Fos immunoreactivity and the up-regulation of BDNF mRNA. In the course of the maturation of these neurons, however, Ca2+ transients and also the increase in c-Fos immunoreactivity and BDNF mRNA expression in response to GABAA receptor activation disappear. Moreover, as shown by bicuculline, endogenously released GABA sup-pressed BDNF mRNA expression at this stage. Muscimol, however, failed to reduce BDNF mRNA expression below the basal level, in contrast to observations made in vivo (Zafra et al., 1991). However, BDNF expression might be already in a maximally repressed state under control conditions due to the endogenous GABAergic activity in mature hippocampal cultures. Therefore, GABAA receptor activation differentially regulates the expression of c-fos and BDNF in immature and mature neurons, due to a switch in the ability of GABA to activate Ca2+ channels during development.

In adult neurons GABAA receptor activation leads to hyper-polarization (Mody et al., 1994), thereby reducing Ca2+ currents. During development, however GABA depolarizes various types of neurons such as spinal (Wu et al., 1992; Reichling et al., 1994), cerebellar (Connor et al., 1987), cortical (Yuste and Katz, 1991) and hippocampal neurons (Mueller et al., 1984; Ben-Ari et al., 1989; Cherubini et al., 1990; Hosokawa et al., 1994) leading to the activation of voltage-gated Ca2+ channels. Upon maturation of these neurons, however, GABA looses the ability to depolarize these cells and Ca2+ transients are no longer observed (Ben-Ari et al., 1989; Lin et al., 1994; Wang et al., 1994). These effects are clearly GABAA receptor mediated as they are mimicked by the GABAA receptor agonist muscimol, but not by the GABAB receptor agonist baclofen (Lin et al., 1994; Reichling et al., 1994). Since muscimol precisely mimicked and bicuculline completely blocked the increase in c-Fos immunoreactivity and BDNF mRNA expression induced by GABA in immature hippocampal neurons, we assume, that these effects of GABA are mediated by GABAA but not by GABAB receptors. The GABAA receptor complex controls Cl conductance, leading to a Cl-dependent inward current after channel opening (Mody et al., 1994). It has been suggested that a difference in the Cl gradient due to a reversed Cl membrane transport might be responsible for the depolarizing action by GABA in immature neurons (Misgeld et al., 1986; Cherubini et al., 1991; Reichling et al., 1994). Interestingly, the subunit composition of GABAA receptors in developing and in adult brain seems to differ, which suggests that the embryonic and early postnatal GABAA receptor complexes may have different properties compared to adult ones (Poulter et al., 1992; Fritschy et al., 1994).

Activation of GABAA receptors may be of functional impor-tance during neuronal maturation and differentiation. Embryonic hippocampal neurons express GABAA receptors as early as day E15 (Poulter et al., 1992), as shown by in situ hybridization for various GABAA receptor subunits. Moreover, these subunits form functional receptors, since E17 embryonic hippocampal neurons respond to muscimol at low nanomolar concentrations (Fiszman et al., 1990). In the early postnatal hippocampus, glutamatergic synaptic transmission seems to be low (Hosokawa et al., 1994). Excitatory potentials at this stage might instead be mediated via GABAergic activity. Ben-Ari and colleagues previously described the occurrence of so-called ‘giant depolarizing potentials’ (GDPs) in the CA3 subfield of the rat hippocampus in vitro (Ben-Ari et al., 1989; Cherubini et al., 1990; Hosokawa et al., 1994). These GDPs are likely to be mediated by GABAA receptor activation as they are blocked by bicuculline, and the corresponding currents reverse at the same potential as produced by exogenously applied GABA. (Ben-Ari et al., 1989). These potentials can be observed only until the postnatal day 7, after which the first inhibitory potentials appear, indicative of the maturation of the GABAergic system (Ben-Ari et al., 1989).

BDNF mRNA is highly expressed in the adult rat hippocampus. Moreover, the level of expression is developmen-tally regulated (Maisonpierre et al., 1990) and depends upon cholinergic and glutamatergic afferent inputs, such as those arising from the medial septum or the entorhinal cortex (Lindefors et al., 1992; Berzaghi et al., 1993). Systemic injection of kainic acid or intraventricular injection of NMDA rapidly up-regulate BDNF mRNA expression in vivo (Zafra et al., 1990; Berzaghi et al., 1993). In the developing hippocam-pus, however, kainic acid does not increase BDNF expression before postnatal day 15, whereas NMDA readily up-regulates BDNF mRNA synthesis at earlier ages (Dugich-Djordjevic et al., 1992; Berzaghi et al., 1993). This study demonstrates that in hippocampal neurons, GABAA receptor activation up-or down-regulates BDNF mRNA expression, depending upon the developmental stage of the neurons. It can be speculated, therefore, that GABAergic activity might enhance BDNF mRNA expression at early stages and thus contribute to the low level of BDNF mRNA during early development (Maison-pierre et al., 1990). With the establishment of mature GABAergic synapses mediating inhibition, GABA, however, starts to down-regulate BDNF mRNA levels as has been demonstrated in the adult (Zafra et al., 1991).

It has been suggested that GABA exerts a neurotrophic effect on embryonic neurons (Cherubini et al., 1991; Barbin et al., 1993). For example, GABA influences the expression of GABA receptors in rat cerebellar granule neurons (Meier et al., 1987). Moreover, GABA stimulates cytokinesis in early spinal neurons (Behar et al., 1993). Interestingly, it has been reported that blocking the endogeneous GABAergic activity in cultured hippocampal neurons by bicuculline leads to reduction of neurite arborization (Barbin et al., 1993). As shown here, GABA regulates the expression of c-fos, which is known to enhance transcription of different genes (Sheng and Greenberg, 1990) and to mediate the increase in Ca2+ currents in PC12 cells by NGF (Cavalié et al., 1994). The regulation of BDNF expression by GABA, might directly regulate neuronal differ-entiation and survival.

Hippocampal neurons express the BDNF receptor TrkB and respond to BDNF in vitro and in vivo (Berninger et al., 1993; Ip et al., 1993; Marsh et al., 1993; Ernfors et al, 1994; Jones et al., 1994). In cultured hippocampal neurons BDNF induces TrkB autophosphorylation on tyrosine residues, thereby eliciting downstream signalling events like MAP kinase activation and [Ca2+]i increase (Berninger et al., 1993; Marsh et al., 1993). Moreover, BDNF regulates neuropeptide and NT-3 mRNA expression in hippocampal neurons in vivo (Croll et al., 1994; Nawa et al., 1994; Lindholm et al., 1995). Although the hippocampal formation seems to be largely intact in mice deficient for the BDNF gene, the expression of calbindin, par-valbumin and neuropeptide Y in GABAergic interneurons is reduced, suggesting that these interneurons might be affected (Jones et al., 1994). Therefore, BDNF may be involved in the maturation of the GABAergic system in the hippocampus. These results, together with our observation that GABA up-regulates BDNF in embryonic hippocampal neurons, suggest an important interaction between BDNF and the neurotransmitter GABA in early brain development. BDNF induced by GABA might stabilize synaptic contacts, promote differentiation and, in conjunction to other growth factors, support neuronal survival.

We are grateful to Jens Richter for skillful technical assistance and Dr Jonathan Cooper for linguistic revision of the manuscript.

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