GABAB Receptor-Mediated Regulation of Glutamate-Activated Calcium Transients in Hypothalamic and Cortical Neuron Development

Karl Obrietan and Anthony van den Pol

Department of Biological Science, Stanford University, Stanford, California 94305; and Department of Neurosurgery, Yale University School of Medicine, New Haven, Connecticut 06520


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Obrietan, Karl and Anthony van den Pol. GABAB Receptor-Mediated Regulation of Glutamate-Activated Calcium Transients in Hypothalamic and Cortical Neuron Development. J. Neurophysiol. 82: 94-102, 1999. In the mature nervous system excitatory neurotransmission mediated by glutamate is balanced by the inhibitory actions of GABA. However, during early development, GABA acting at the ligand-gated GABAA Cl- channel also exerts excitatory actions. This raises a question as to whether GABA can exert inhibitory activity during early development, possibly by a mechanism that involves activation of the G protein-coupled GABAB receptor. To address this question we used Ca2+ digital imaging to assess the modulatory role of GABAB receptor signaling in relation to the excitatory effects of glutamate during hypothalamic and cortical neuron development. Ca2+ transients mediated by synaptic glutamate release in neurons cultured from embryonic rat were dramatically depressed by the administration of the GABAB receptor agonist baclofen in a dose-dependent manner. The inhibitory effects of GABAB receptor activation persisted for the duration of baclofen administration (>10 min). Preincubation with the Gi protein inhibitor pertussis toxin resulted in a substantial decrease in the inhibitory actions of baclofen, confirming that a Gi-dependent mechanism mediated the effects of the GABAB receptor. Co-administration of the GABAB receptor antagonist 2-hydroxy-saclofen eliminated the inhibitory action of baclofen. Alone, GABAB antagonist application elicited a marked potentiation of Ca2+ transients mediated by glutamatergic neurotransmission, suggesting that tonic synaptic GABA release exerts an inhibitory tone on glutamate receptor-mediated Ca2+ transients via GABAB receptor activation. In the presence of TTX to block action potential-mediated neurotransmitter release, stimulation with exogenously applied glutamate triggered a robust postsynaptic Ca2+ rise that was dramatically depressed (>70% in cortical neurons, >40% in hypothalamic neurons) by baclofen. Together these data suggest both a pre- and postsynaptic component for the modulatory actions of the GABAB receptor. These results indicate a potentially important role for the GABAB receptor as a modulator of the excitatory actions of glutamate in developing neurons.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In neurons, tight regulation of intracellular Ca2+ is essential for normal cellular functions. Even slight changes in neuronal Ca2+ levels can influence a myriad of biochemical pathways involved in enzyme activation, gene expression, transmitter release, and cytoskeletal remodeling (Clapham 1995; De Camilli and Takei 1996; Ghosh et al. 1994). In addition, during early development changes to intracellular Ca2+ can alter growth cone motility and the rate of neurite outgrowth (Mattson and Kater 1987) as well as synapse formation (Nelson et al. 1990), neural migration (Komuro and Rakic 1996), and neural phenotype (Marty et al. 1996). One principal mechanism by which neuronal Ca2+ levels are regulated is through the interplay of excitatory and inhibitory neurotransmitter systems. In the CNS, the widespread amino acid neurotransmitters glutamate and GABA play a primary role in regulating synaptic neurotransmission and thereby cytosolic Ca2+ levels. The ability of glutamate to both elicit fast excitatory neurotransmission and increase cytosolic Ca2+ results from the activation of the ionotropic class of glutamate receptors (Nakanishi 1992) and subsequent activation of voltage-gated Ca2+ channels. In addition to the normal regulation of Ca2+, excitotoxicity can result from an extreme elevation in internal Ca2+ caused by excessive levels of glutamate or an increase in glutamate sensitivity (Choi 1992).

In the mature nervous system, GABA acts as the primary fast inhibitory neurotransmitter. The hyperpolarizing actions of GABA result from the activation of the ligand-gated GABAA receptor. For example, glutamate-evoked membrane depolarization and subsequent Ca2+ influx are decreased by GABAA receptor activation in mature neurons. In contrast to the mature nervous system, during early development GABAA receptors act to increase cytosolic Ca2+ levels. By activating Cl--dependent inward current at GABAA receptors, GABA can depolarize the membrane potential and increase the frequency of action potentials (Chen et al. 1996; Owens et al. 1996), thus resulting in membrane depolarization, activation of voltage-dependent Ca2+ channels, and intracellular Ca2+ increases (Obrietan and van den Pol 1995b; Reichling et al. 1994). These results raise the question of whether there is a Ca2+ inhibitory role for GABA independent of the GABAA receptor during development.

Another mechanism by which GABA may negatively affect cytosolic Ca2+ levels is through the activation of the GABAB subclass of receptors. GABAB receptors operate through G proteins (Bowery 1993; Misgeld et al. 1995) and are expressed both presynaptically (Dutar and Nicoll 1988a; Howe et al. 1987; Yoon and Rothman 1991) and postsynaptically (Solis and Nicoll 1992). At their presynaptic location, GABAB receptors have been shown to decrease neurotransmitter release by increasing K+ conductance or by decreasing Ca2+ conductance (Bowery 1993; Misgeld et al. 1995). Presynaptically, GABAB receptors may also play a role as autoreceptors, negatively regulating the synaptic release of GABA (Anderson and Mitchell 1985; Pittaluga et al. 1987). Activation of postsynaptic GABAB receptors has been shown to induce K+ currents, leading to membrane hyperpolarization (Alger and Nicoll 1982; Dutar and Nicoll 1988b) and to inhibit voltage-dependent Ca2+ channels (Lambert and Wilson 1996; Misgeld et al. 1995).

Although receptor autoradiography binding studies revealed that in some brain regions GABAB receptor expression is greater during development than in the adult (Turgeon and Albin 1994), the physiological effects of GABAB receptors on developmentally immature neurons were not thoroughly characterized. Given that GABA and glutamate are released from axon terminals within 4 days of final mitosis (van den Pol et al. 1995) and that both transmitters have the capacity to increase Ca2+ and act in synergy to increase action potentials (Gao et al. 1998), it is unclear what mechanisms are involved with attenuating runaway excitation generated by the depolarizing actions of GABA and glutamate during neural development. We tested the hypothesis that GABA acting at the GABAB receptor would decrease glutamate-dependent excitability, as detected by inhibition of glutamate-mediated Ca2+ elevations. Previously, we have shown that GABAB receptor activation regulates Ca2+ transients mediated by GABAA receptors during early development (Obrietan and van den Pol 1998).

The results presented here reveal a potent inhibitory action of GABAB receptors on glutamate-mediated Ca2+ transients. These results strongly suggest that GABAB receptors may play a substantial role in regulating glutamatergic neurotransmission in developing cortical and hypothalamic neurons.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue culture

The region of the mediobasal hypothalamus from the caudal preoptic area to the mammillary bodies was excised from Sprague-Dawley rats on embryonic day 18 (E18). The area of the fornix and mammillothalalmic tract was used as the general lateral boundary. The tissue was washed by resuspending it three times in culture medium (DMEM, Gibco) and then incubating it in a mild proteolytic solution (10 units/ml papain, 0.2 mg/ml L-cysteine in Earl's balanced salt solution). After 30 min, the digestion was stopped by pelleting the tissue and removing the protease solution. Tissue was then suspended in standard tissue culture medium (glutamate- and glutamine-free DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin/streptomycin and 6 gm/l glucose). Next a fire-polished pasteur pipette was used to triturate the tissue into a single cell suspension. The tissue was pelleted and washed three additional times. Cells were then plated (~100,000 cells/cm2) onto 22 mm2 glass coverslips coated with high molecular weight polylysine (MW 540 kD, Collaborative Research). To ensure high local neuronal density, cells were plated within a glass cylinder (7 mm diam) placed on the coverslip. The cylinder was removed 45 min after plating. Hypothalamic cells were cultured in a Napco incubator at 5% CO2 and 37°C and maintained in standard tissue culture medium. Two days after plating, cytosine arabinofuranoside (6 µM) was added to the media to inhibit cell proliferation. Media was changed twice a week. The identical culturing protocol as described for the hypothalamus was used to culture neurons from the cortex. To generate hyperexcitable hypothalamic neurons (Obrietan and van den Pol, 1995a) used in Fig. 6, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 µM) and D,L-2-amino 5 phosphono valerate (D,L-AP5) (100 µM) were added to the tissue culture medium 3 days after plating and maintained in the media throughout time in culture.

Calcium digital imaging

Cells were incubated in standard HEPES perfusion solution (137 mM NaCl, 25 mM glucose, 10 mM HEPES, 5 mM KCl, 1 mM MgCl2, 3 mM CaCl2, pH 7.4) containing 5 µM fura-2 acetoxymethyl ester (Molecular Probes) for 20 min at 37°C. To inhibit synaptic activity in chronically blocked cultures, ionotropic glutamate receptor inhibitors AP5 (100 µM) and CNQX (10 µM) or TTX (1 µM) were added to the incubation solution. The cells were washed two times after loading and then allowed to recover for 30 min. The coverslip was then loaded into an eight-port, 180 µl, microscope perfusion chamber (Forscher et al. 1987) and perfused at a constant rate. Solutions moved as a straight wave across the perfusion chamber, with total exchange of solution occurring in ~5 s. Neurons were identified by their phase-bright appearance and by their responsiveness to the application of N-methyl-D-aspartate (NMDA). Ca2+ recordings were made from the cell soma. All experiments were performed at room temperature. To enhance NMDA receptor activity during endogenous activity experiments, 2 µM glycine was added, and Mg2+ was removed from the standard HEPES perfusion solution.

Image collection was performed with a ×40 Olympus objective with high 340/380 nm transmittance on a Nikon Diaphot 300 inverted microscope. Fluorescent light was passed back through the objective, through a 420 nm cutoff filter, and directed at a Hamamatsu 2400 silicon-intensified target video camera. Recordings at 340 and 380 nm excitation were controlled by a 486 PC with Fluor software (Universal Imaging). Switching between the two excitation filters was performed with a Sutter filter wheel connected to a Lambda 10 microprocessor; 90% neutral density filter attenuation of light from a 150-W xenon lamp allowed for recordings of >90 min without detectable phototoxicity. Sixteen video frames (500 ms) were collected every 5 s and averaged after background subtraction. The equation [Ca2+]i = Kd(R - Rmin)/(Rmax - R) was employed to determine free Ca2+ concentrations from fura-2 ratiometric fluorescent Ca2+ values. R is defined as the ratio of the two fluorescence intensities, Rmin is the ratio in the absence of Ca2+, and Rmax is the ratio in a saturating concentration of Ca2+. The Kd for binding of Ca2+ to fura-2 was taken to be 224 nM (Grynkiewicz et al. 1985). Data analysis was performed with Igor Pro software (WaveMetrics, Lake Oswego, OR).

For assays that evoked a Ca2+ response by exogenous administration of glutamate, the peak Ca2+ rise (either in the presence or absence of baclofen) for individual neurons was determined by subtracting the mean basal Ca2+ level for a 15 s period just before stimulation from the peak glutamate-evoked Ca2+ rise. The mean Ca2+ rise triggered by synaptic glutamate release after withdrawal of receptor antagonists (or TTX) was determined for a 15-s period immediately preceding the administration of GABAB agonists and/or antagonists. This value was compared with the mean Ca2+ rise over a 15 s period 30 s after addition of the GABAB receptor antagonist or 90 s after addition of GABAB agonists. Data for both experimental protocols are reported as a mean (pooled) Ca2+ rise of all responsive neurons ± SE. A responsive neuron was defined as a neuron that showed a >= 20 nM Ca2+ rise to either exogenous glutamate application or synaptic glutamate release.

Reagents

Cytosine arabinofuranoside, glutamate, and poly-D-lysine were acquired from Sigma; AP5, CNQX, bicuculline, R(+)-baclofen, 2-hydroxy-saclofen, NMDA, and TTX from Research Biochemicals International; papain from Worthington Biochem; DMEM from Gibco; and fura-2 acetoxymethyl ester from Molecular Probes. CGP 35348 was a generous gift of Ciba-Geigy AG (Basel).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The removal of the ionotropic glutamate receptor antagonists AP5 (100 µM) and CNQX (10 µM) from the perfusion solution initiated an increase in basal Ca2+ levels and a series of complex Ca2+ transients in both hypothalamic and cortical neurons cultured for 7 days from E18 rats (Fig. 1, A and B). Previous work has shown that by 7 days in vitro neurons cultured under these conditions form synaptic connections and secrete neurotransmitter (van den Pol et al. 1998). That the Ca2+ transients shown in Fig. 1A were initiated by the withdrawal of ionotropic glutamate receptor antagonists indicates the synaptic release of glutamate. The mean Ca2+ rise elicited by AP5 and CNQX withdrawal was 123 ± 8 nM in hypothalamic and 156 ± 11 nM in cortical neurons. GABAB receptor activation mediated by the administration of the specific agonist R(+)-baclofen (baclofen: 10 µM) depressed the mean Ca2+ rise to 3 ± 5 nM, representing a decrease in the glutamate-mediated rise of ~95% in hypothalamic neurons (n = 38; Fig. 1A). In cortical neurons baclofen (10 µM) decreased the Ca2+ rise to 17 ± 5 nM (n = 43; Fig. 1B). Large Ca2+ transients were again found immediately after baclofen washout. Ca2+ levels in 82 of 119 synaptically active hypothalamic neurons were depressed >80% by baclofen. The effects of baclofen were dose dependent in hypothalamic neurons (Fig. 1E) as well as in cortical neurons (data not shown). The inhibitory effects of baclofen were observed from the earliest [6 days in vitro (DIV)] to the latest (32 DIV) time point tested.



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Fig. 1. GABAB receptor-mediated depression of cytosolic Ca2+ levels. A: withdrawal of glutamate receptor antagonists D,L-2-amino-5-phosphonovalerate (AP5) (100 µM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 µM) from the perfusion solution initiated spontaneous Ca2+ transients in synaptically activated neurons cultured for 7 days in vitro (7 DIV). Baclofen (Bac) administration (10 µM) reversibly depressed glutamate-mediated Ca2+ transients. Arrow in the second trace indicates that the level of Ca2+ depression exceeded that observed under conditions of glutamate receptor blockade. The 2 neurons were recorded at the same time and show similar response characteristics. B: baclofen depressed Ca2+ transients in cortical neurons cultured for 7 days. C: in the presence of TTX (1 µM) baclofen depressed basal Ca2+ levels in hypothalamic neurons. D: no effect of baclofen on basal Ca2+ levels was observed in cortical neurons in the presence of TTX. E: dose-response representation of baclofen-mediated depression of Ca2+ transient triggered by synaptic glutamate release in hypothalamic neurons. F: comparison of the basal Ca2+ levels in the presence of TTX (open bars) to the levels in the presence of TTX and baclofen. **P < 0.0001. Error bars denote SE. Hypo, hypothalamus. In A-D the bar along the x-axis shows time in minutes (min), and the vertical bar to the left of each trace is the calibrated cytosolic Ca2+ value for each neuron.

In some hypothalamic neurons baclofen depressed cytosolic Ca2+ to a concentration lower than the concentration in the presence of AP5 and CNQX. An example of this is shown in Fig. 1A (bottom neuron: arrow). To asses whether this effect resulted from a capacity of the GABAB receptor to alter Ca2+ homeostasis independent of glutamate transmission, baclofen was administered in the presence of TTX (1 µM; Fig. 1C). TTX blocks action potential-mediated neurotransmitter release. Under this condition, baclofen administration resulted in a small but significant (P < 0.0001, two tailed t-test) 12% decrease in basal Ca2+ levels in hypothalamic neurons (Fig. 1F, n = 189). Similar results were observed when baclofen was administered in the presence of AP5 and CNQX (data not shown). No significant alteration in basal Ca2+ levels was observed in cortical neurons (Fig. 1, D and F; n = 98).

Early in hypothalamic development GABA acts as an excitatory neurotransmitter, leading to membrane depolarization (Chen et al. 1996) and triggering cytosolic Ca2+ rises (Obrietan and van den Pol 1995b). Given this, the data presented in Fig. 1 may have resulted from a modulation of excitatory GABAergic stimulation that in turn would affect glutamatergic transmission. To address this possibility, 6 DIV neurons released from glutamate receptor blockade were exposed to the GABAA receptor antagonist bicuculline (20 µM; Fig. 2). Under these conditions, administration of baclofen (10 µM) resulted in a dramatic decrease in glutamate-dependent Ca2+ transients (Fig. 2). In the presence of bicuculline, the mean Ca2+ rise after AP5/CNQX withdrawal was 101 ± 6 nM. Baclofen treatment depressed the mean Ca2+ rise to 21 ± 6 nM, representing an ~80% decrease in the glutamate-mediated Ca2+ rise (n = 44). These results reveal that in the absence of excitatory GABAA receptor activity the GABAB receptor can modulate glutamate neurotransmission.



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Fig. 2. GABAB receptor-mediated depression of glutamate-dependent Ca2+ elevations are not dependent on GABAA receptor activity. Six DIV hypothalamic neurons showed marked increases in intracellular Ca2+ after withdrawal of AP5 and CNQX. Elevated Ca2+ levels were maintained in the presence of the GABAA antagonist bicuculline (20 µM). Under this condition baclofen (Bac:10 µM) administration reversibly depressed glutamate-mediated Ca2+ transients.

Given the potent short-term Ca2+ modulatory effects of GABAB receptor activation, we tested whether this effect would persist for an extended time period. Figure 3A shows that spontaneous glutamate-mediated Ca2+ transients could be totally inhibited for >10 min by baclofen (100 µM). The modulatory effects of baclofen did not significantly desensitize in any of the neurons assayed (n = 47). Ca2+ transients resumed after baclofen washout. Administration of AP5 (100 µM) and CNQX (10 µM) at the end of the experiment returned Ca2+ to basal levels that were slightly higher than the Ca2+ level observed in the presence of baclofen.



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Fig. 3. A: extended administration of baclofen (100 µM) depressed Ca2+ transients mediated by synaptic glutamate release in hypothalamic neurons. After withdrawal of baclofen, Ca2+ levels immediately resumed prebaclofen levels. Pretreatment with the Gi protein inhibitor pertussis toxin (200 ng/ml: 16 h) attenuated baclofen-mediated depression of Ca2+ transients triggered by synaptic transmitter release in hypothalamic (B) and cortical (C) neurons cultured for 10 days in vitro. D: bar graph shows the mean Ca2+ rise under control conditions and the Ca2+ rise after baclofen administration in pertussis toxin-treated neurons. Error bars denote SE.

One possible mechanism by which GABAB receptors could affect glutamate-mediated Ca2+ transients is through the activation of Gi-coupled proteins. To test this cellular signaling mechanism, hypothalamic and cortical neurons were pretreated with the Gi inhibitor pertussis toxin (200 ng/ml, 16 h). Pertussis toxin pretreatment largely blocked the inhibitory effects of baclofen (10 µM) in both hypothalamic (Fig. 3B) and cortical neurons (Fig. 3C). Figure 3D shows the mean Ca2+ rise in the absence (open bars) and in the presence (solid bars) of baclofen in pertussis toxin-treated neurons. Compared with the inhibitory effects of baclofen (10 µM) described in Fig. 1, pertussis toxin pretreated reduced the efficacy of baclofen by more than eightfold for hypothalamic and by more than twofold for cortical neurons. These data suggest that a principal mechanism by which GABAB receptor activation alters glutamate responsiveness is via activation of Gi.

GABAB receptors may affect glutamate-dependent signaling by altering transmitter release and/or by altering the postsynaptic responsiveness to glutamate. To address the latter possibility, postsynaptic Ca2+ responses evoked by exogenous glutamate administration were compared with Ca2+ responses evoked by glutamate in the presence of baclofen. All perfusion solutions contained TTX (1 µM) to block presynaptic transmitter release. Administration of glutamate (Fig. 4, A and B, arrows: 10 µM) triggered rapid and reproducible Ca2+ rises in both hypothalamic and cortical neurons. Co-administration of baclofen (10 µM) dramatically reduced peak Ca2+ transients evoked by glutamate. By comparing sequential glutamate application (the second evoked Ca2+ rise to the first evoked Ca2+ rise in the presence of baclofen), we found that of the 317 neurons assayed baclofen reduced the glutamate-dependent Ca2+ increase by >20% in 90% of hypothalamic neurons and by >20% in 98% of cortical neurons. In some hypothalamic (Fig. 4A, top neuron) and cortical (Fig. 4B) neurons baclofen almost totally abolished glutamate responsiveness. GABAB receptor activation resulted in a >90% inhibition of glutamate-evoked Ca2+ rises in 9% of hypothalamic neurons and in 54% of cortical neurons. Figure 4C shows significant (P < 0.001 two-tailed t-test) decreases in the peak glutamate-evoked Ca2+ transient mediated by GABAB activation in hypothalamic and cortical neurons. These data reveal a potent postsynaptic modulatory role for the GABAB receptor in both hypothalamic and cortical neurons.



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Fig. 4. Postsynaptic GABAB receptor stimulation reduces glutamate-evoked Ca2+ rises in neurons cultured for 9 days in vitro. A: glutamate (10 µM) administration (arrows) elicited rapid and reproducible Ca2+ rises that were depressed by administration of the GABAB receptor agonist baclofen (10 µM, solid line) in hypothalamic neurons. B: postsynaptic GABAB receptor stimulation potently depressed evoked Ca2+ rises mediated by glutamate administration in cortical neurons. After removal of baclofen from the perfusion solution glutamate responsiveness increased to prebaclofen levels in both hypothalamic and cortical neurons. The voltage-activated sodium channel blocker TTX (1 µM) was used to inhibit action potential-mediated transmitter release. C: bar graph representation showing the mean glutamate-evoked Ca2+ rise in the absence (open bars) and in the presence (solid bars) of baclofen for hypothalamic and cortical neurons. Error bars denote SE.

To assess whether baclofen depression of Ca2+ transients evoked by synaptic glutamate release could result entirely from the postsynaptic actions of GABAB receptors or if there was a presynaptic component, we compared baclofen modulation of Ca2+ rises mediated by synaptic glutamate release with baclofen modulation of Ca2+ rises triggered by exogenous glutamate application in the presence of TTX. By comparing modulation under these two protocols it should be possible to subtract the purely postsynaptic site of action (exogenous application) from the pre- and postsynaptic site of action (revealed by synaptic glutamate release protocol). To control for a varied Ca2+ responsiveness under these two protocols, only neurons exhibiting a rise of 150-300 nM were used. For hypothalamic neurons, the Ca2+ rise to synaptically released glutamate was 207 ± 21 nM; in the presence of baclofen the Ca2+ rise was 20 ± 11 nM n = 31. For exogenously evoked glutamate response, the Ca2+ rise was 239 ± 10 nM. In the presence of baclofen the Ca2+ rise was 83 ± 15 nM (n = 27). The significantly (P < 0.005, two-tailed t-test) greater level of baclofen depression of synaptic glutamate-dependent Ca2+ rises compared with bath-applied glutamate suggests a modulatory role for presynaptic GABAB receptors. In cortical neurons, the Ca2+ rise to synaptically release glutamate was 210 ± 10 nM; in the presence of baclofen the Ca2+ rise was 25 ± 4 nM (n = 42). The exogenously evoked Ca2+ rise was 240 ± 7 nM. In the presence of baclofen the Ca2+ rise was 57 ± 10 (n = 42). For cortical neurons, the difference in the modulatory actions of baclofen between these two protocols was statistically significant (P < 0.005, two-tailed t-test). Consistent with electrophysiological recordings (see DISCUSSION), these results suggest that functional GABAB receptors are expressed presynaptically and augment the postsynaptic actions of GABAB receptors.

To ensure that the effects of baclofen were specific to the GABAB receptor, 2-hydroxy-saclofen (200 µM), a GABAB receptor antagonist (Curtis et al. 1988; Harrison et al. 1990; Lambert et al. 1989), was co-administered with baclofen (10 µM). Figure 5, A and B, shows that the inhibitory effects of baclofen were attenuated by 2-hydroxy-saclofen. The mean Ca2+ rise triggered by AP5 and CNQX withdrawal from hypothalamic neurons was 127 ± 9 nM. Baclofen reduced this Ca2+ rise to 5 ± 6 nM. In combination with 2-hydroxy-saclofen the mean Ca2+ decrease mediated by baclofen was 76 ± 10 nM (n = 83).



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Fig. 5. Synaptic GABAB receptor activation decreases Ca2+ rises elicited by spontaneous synaptic glutamate release in hypothalamic neurons after 12 days in vitro. A: withdrawal of AP5 and CNQX triggered a large Ca2+ rise that was depressed by baclofen (Bac: 10 µM) administration. When the GABAB receptor antagonist 2-hydroxy saclofen (OH-SAC) (200 µM) was administered with baclofen, the inhibitory effects of baclofen were largely blocked. AP5 and CNQX administration at the end of the experiment returned Ca2+ to basal levels. B: as with the neuron in A, baclofen-mediated Ca2+ depression was blocked with 2-hydroxy saclofen. In addition, administration of 2-hydroxy saclofen alone resulted in potentiation of the Ca2+ elevation mediated by synaptic glutamate release. The withdrawal of AP5 and CNQX was not shown for this neuron. C: repeated Ca2+ rises were mediated by 2-hydroxy saclofen (200 µM) administration. D: in the presence of TTX (1 µM) to block synaptic transmitter release, 2-hydroxy saclofen (200 µM) did not alter basal Ca2+. N-Methyl-D-aspartate (100 µM) was added at the end of the experiment to show that cells were neurons responsive to glutamate receptor stimulation. E: administration of the GABAB receptor antagonist CGP 35348 (400 µM) potentiated Ca2+ elevations mediated by synaptic glutamate release. F: bar graph depicting the modulatory effects of baclofen (10 µM), 2-hydroxy saclofen (200 µM), baclofen (10 µM) + 2-hydroxy saclofen (200 µM), or CGP 35348 (CGP: 400 µM) on Ca2+ rises mediated by synaptic glutamate release. Error bars denote SE.

Next we assessed whether the synaptic release of GABA could depress glutamatergic Ca2+ rises via activation of the GABAB receptor. When administered alone, 2-hydroxy-saclofen dramatically potentiated the glutamate-regulated Ca2+ level (Fig. 5B). This effect is also shown for repeated 2-hydroxy-saclofen (200 µM) administrations in Fig. 5C. In this neuron, the Ca2+ transients mediated by glutamatergic neurotransmission are potentiated by approximately threefold by blockade of GABAB receptor activation. On average, 2-hydroxy-saclofen increased glutamate-mediated Ca2+ transients by 54 ± 10 nM (n = 87). Similar results were observed with another GABAB receptor antagonist, CGP 35348 (400 µM; Fig. 5E). A graphic representation of these results is shown in Fig. 5F. Figure 5D shows that 2-hydroxy-saclofen (200 µM) did not significantly alter basal Ca2+ levels when spontaneous glutamatergic neurotransmission was blocked with TTX (1 µM; n = 79). Administration of NMDA (100 µM) at the end of the experiment was used to show that the neurons were healthy and responsive to glutamate receptor stimulation. These results reveal that synaptically released GABA acts at GABAB receptors to depress Ca2+ transients triggered by synaptic glutamate release.

For normal cellular functions, intracellular Ca2+ must be maintained within a relatively narrow concentration range. If cytosolic Ca2+ levels exceed a critical threshold, cell death will result. In the CNS, excessive glutamatergic stimulation can lead to cell death as a result of triggering massive Ca2+ influx (Choi 1992). Previously, we have shown that hypothalamic neurons raised in the constant presence of AP5 and CNQX become hyperexcitable, thus leading to toxic levels of intracellular Ca2+ after glutamate receptor antagonist withdrawal (Obrietan and van den Pol 1995a). To asses the modulatory role of GABAB receptor activation under this condition of hyperexcitability, hypothalamic neurons were cultured for 29 days in the constant presence of AP5 and CNQX. Withdrawal of short-term TTX (1 µM) pretreatment elicited a Ca2+ rise (Fig. 6A). Enhancing NMDA receptor responsiveness by the withdrawal of Mg2+ and addition of glycine (2 µM) to the perfusion solution increased the Ca2+ activity further. Additionally, blockade of the inhibitory influence of tonic GABAA receptor activation by administration of the antagonist bicuculline (20 µM) triggered a massive potentiation of glutamatergic Ca2+ rises. Under this condition Ca2+ levels exceeded 1,500 nM in the representative neuron shown in Fig. 6. We have previously shown that this level of cytosolic Ca2+ is potentially toxic to hypothalamic neurons over an extended period (Obrietan and van den Pol 1995a). Under this condition, the administration of baclofen (100 µM) resulted in a large decrease in the glutamate-mediated Ca2+ rise. After withdrawal of baclofen, Ca2+ levels immediately rose to prebaclofen levels. Of the neurons showing >500 nM Ca2+ rise after bicuculline administration, 35% had a >50% reduction in Ca2+ levels, whereas 70% of neurons exhibited >30% inhibition after baclofen administration (n = 58). Single-cell response profiles are displayed in a histogram format (Fig. 6B). These results reveal that even under extreme, potentially toxic levels of glutamate neurotransmission GABAB receptors have the potent capacity to reduce cytotoxic Ca2+ levels.



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Fig. 6. Potentially toxic increases in cytosolic Ca2+ are depressed by GABAB receptor stimulation. A: representative neuron showing that TTX (1 µM) withdrawal results in a Ca2+ rise that is enhanced by the withdrawal of Mg2+ and the addition of glycine (2 µM) to the perfusion solution. Removal of GABAA receptor activity by administration of bicuculline (20 µM) resulted in a marked potentiation of glutamate mediated Ca2+ rises. Baclofen (Bac: 100 µM) reduced this Ca2+ rise substantially. B: histogram representation of the inhibitory effects of baclofen on glutamate-dependent Ca2+ rises of >500 nM. Each bar represents a single neuron. Values along the x-axis show the mean Ca2+ elevation immediately before baclofen administration. The y-axis shows the percentage inhibition of the Ca2+ rise resulting from baclofen. Percentage decreases varied from ~5-90%.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The results presented here are the first to characterize a potent Ca2+ regulatory role for the GABAB receptor in hypothalamic and in cortical glutamatergic neurons during development. Stimulation of GABAB receptors depressed Ca2+ transients mediated by the synaptic release of glutamate and by postsynaptic stimulation of glutamate receptors. Additionally, the finding that GABAB receptors are tonically activated and thus lead to a decrease in Ca2+ transients mediated by synaptic glutamate release suggests an important regulatory role for the GABAB receptor during development.

We observed potent, dose-dependent GABAB receptor-mediated depression of Ca2+ transients resulting from the synaptic release of glutamate in hypothalamic neurons. This Ca2+ rise was the result of glutamate release because administration of the glutamate receptor antagonists AP5 and CNQX to the perfusion solution blocked the Ca2+ transients. Relative to other glutamate neuromodulators that we characterized (neuropeptide Y, adenosine, and dopamine), we found that the GABAB receptor was very effective at depressing glutamate-mediated Ca2+ transients (Obrietan et al. 1995; van den Pol et al. 1996a,b). For example, adenosine receptor activation could, at saturating agonist concentration, only decrease a maximum of 78% of the Ca2+ rise resulting from synaptic glutamate release, whereas the Ca2+ rise resulting from synaptic glutamate release could be completely blocked by baclofen. Possible reasons for this may include a greater number of GABAB receptors per neuron or tighter coupling to second-messenger signaling pathways

The inhibitory effects of baclofen persisted for the duration of its presence. We did not observe recovery of glutamatergic Ca2+ transients suggestive of GABAB receptor desensitization, even during extended treatments (>10 min). However, immediately after withdrawal of baclofen, glutamatergic transients were re-initiated. This potent inhibition of glutamatergic Ca2+ transients has not been previously observed. However, electrophysiological studies have shown that decreased membrane conductance mediated by baclofen was not attenuated during extended treatments lasting for ~10 min (Bon and Galvan 1996; Fujikawa et al. 1997).

Stimulating GABAB receptors with baclofen resulted in a potent depression of Ca2+ rises evoked by exogenous administration of glutamate to cortical or hypothalamic neurons. Because the presynaptic release of glutamate was blocked with TTX, the observed inhibitory effect of GABAB receptor activation in those experiments reflected a postsynaptic site of action. Postsynaptic GABAB receptors were previously observed in a number of brain regions, including the hippocampus (Yoon and Rothman 1991) and spinal cord (Beattie et al. 1989), but appear to be absent in other brain regions such as the striatum (Nisenbaum et al. 1993). Our observed postsynaptic effect of baclofen in cortical neurons is in agreement with work showing postsynaptic expression of GABAB receptors in the cortex (Karlsson and Olpe 1989). Besides our previous work (Obrietan and van den Pol 1998) and the data presented here, little has been done to characterize postsynaptic actions of the GABAB receptor in the hypothalamus. The postsynaptic physiological effect most often resulting from GABAB receptor stimulation is an increase in K+ conductance (Loose et al. 1991; Misgeld et al. 1995), although this may be absent from some hypothalamic regions, such as the suprachiasmatic nucleus (Chen and van den Pol 1998). Membrane hyperpolarization resulting from increased K+ conductance would decrease neural excitability.

The depression of postsynaptic glutamate responses by baclofen could be explained by the ability of the GABAB receptor to modulate second-messenger systems. For example, the GABAB receptor has been shown to couple to decreased cAMP production (Desrues et al. 1995; Hashimoto and Kuriyama 1997). We observed that GABAB receptor-mediated inhibition was significantly decreased by pretreatment with pertussis toxin, suggesting that GABAB receptors in cortical and hypothalamic neurons are coupled to heteromeric Gi proteins. Decreased adenylyl cyclase activity resulting from Gi release from the beta - and gamma -regulatory subunits would negatively regulate cAMP-dependent protein kinase activity and thereby potentially alter the phosphorylation state of glutamate receptors. Along these lines cAMP-dependent protein kinases were found to modulate kainate receptors (Keller et al. 1992; Raymond et al. 1993) and NMDA receptor conductance (Cerne et al. 1993). Other Ca2+ regulatory proteins that could be altered by GABAB receptors-activated signaling pathways may include voltage-activated Ca2+ channels, Ca2+ transporters, and Ca2+ pumps. GABAB receptor signaling may activate several other physiological signaling mechanisms to regulate postsynaptic Ca2+ responsiveness. For example, GABAB facilitation of postsynaptic membrane hyperpolarization mediated by increased K+ conductance would strengthen the Mg2+ block of NMDA receptors and thus decrease Ca2+ responsiveness. Likewise, a direct inhibition of voltage-dependent Ca2+ channels would decrease glutamate-evoked Ca2+ rises. For example, in hippocampal interneurons, somatic Ca2+ influx mediated by N-type voltage-activated Ca2+ channels has been shown to be inhibited by GABAB receptors (Lambert and Wilson 1996). The varied level of GABAB-mediated inhibition of glutamate-evoked Ca2+ rises observed in hypothalamic cultures may result from the varied expression of Ca2+ channel subtypes found in hypothalamic neurons (Zeilhofer et al. 1991) or to differences in glutamate receptor expression (van den Pol et al., 1994) or phosphorylation state of the glutamate receptors.

Given that the level of baclofen-mediated depression of postsynaptic glutamate-evoked Ca2+ rises (in the presence of TTX) was not as great as the level of depression resulting from synaptically released glutamate, our results suggests the functional expression of presynaptic GABAB receptors. Along these lines, several studies have shown that GABAB receptors are expressed in presynaptic nerve terminals where they act to reduce transmitter release from hypothalamic (Chen and van den Pol, 1998) and nonhypothalamic (Fukuda et al. 1993; Howe et al. 1987; Isaacson et al. 1993) neurons. There may be several presynaptic mechanisms initiated by GABAB receptors that result in decreased transmitter release. For example, GABAB receptor activation has been shown to reduce transmitter release by hyperpolarizing the membrane potential via elevated K+ currents (Gerber and Gahwiler 1994) and by inhibiting several different types of voltage-activated Ca2+ channels (Doze et al. 1995; Matsushima et al. 1993; Mintz and Bean 1993; Shibuya and Douglas 1993; Wall and Dale 1994).

Changes in cytosolic Ca2+ levels affect a variety of processes essential for nervous system development. The ability of glutamate to increase cytosolic Ca2+ levels during development (van den Pol et al. 1995) indicates that it plays an important role defining CNS cytoarchitechture. Given the widespread expression of glutamate-secreting cells in the CNS, one would expect that glutamate-mediated Ca2+ rises should be negatively regulated by other transmitter systems. One candidate inhibitory transmitter is GABA. This possibility is supported by the high percentage of GABAergic cells in the CNS, the high levels of GABA immunoreactivity found in axonal growth cones (van den Pol 1997), and the observation that GABA is released from axon terminals within 3 or 4 days of the final mitosis (Obrietan and van den Pol 1995b; van den Pol et al. 1995, 1998). Given that during early development GABA, acting at the GABAA receptor, is primarily an excitatory neurotransmitter (Chen et al. 1996; Owens et al. 1996), we turned our focus to the GABAB receptor. The work presented here reveals a prominent inhibitory action on the excitatory effects of glutamate. Coupled with our previous report (Obrietan and van den Pol 1998) showing that GABAB receptors also reduce the excitatory actions of GABA acting at the GABAA receptor these results are consistent with the idea that the GABAB receptor may play an important modulatory role in reducing transmitter-regulated Ca2+ transients in developing neurons.

Under some conditions, GABA- and glutamate-containing axons may compete for postsynaptic sites during early synaptogenesis. On the basis of Hebbian models of synaptic strengthening, the ability of GABA to reduce glutamate actions in developing neurons at both pre- and postsynaptic sites hypothetically may provide a competitive advantage for the depolarizing actions of GABA at the GABAA receptor.


    ACKNOWLEDGMENTS

We thank Dr. T. Meier for kind assistance with some of the neuronal culturing.

This work was supported by National Institute of Neurological Disorders and Stroke Grants NS-34887 and NS- 31573. This work was also supported by the National Science Foundation and the Air Force Office of Scientific Research.


    FOOTNOTES

Address for reprint requests: A. N. van den Pol, Dept. of Neurosurgery, Yale University Medical School, 333 Cedar St., New Haven, CT 06520-8082.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 17 June 1998; accepted in final form 1 December 1998.


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