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INTRODUCTION |
The amino acid neurotransmitter
-aminobutyric acid (GABA) inhibits neuronal activity in mature neurons. In developing neurons by contrast, GABA, acting at the GABAA receptor, can be excitatory. As an excitatory transmitter in the developing brain, GABA can depolarize the membrane potential and increase the frequency of action potentials (Chen et al. 1996
; Owens et al. 1996
). The mechanism for GABAA receptor-mediated excitation appears to depend on a Cl
reversal potential that is positive to the resting membrane potential in early development (Chen et al. 1996
). Thus in developing neurons, opening the GABA-gated Cl
channel generates a chloride exit from the cell and a resultant depolarization. Voltage-dependent Ca2+ channels are activated when the membrane potential depolarizes, leading to intracellular Ca2+ increases (Obrietan and van den Pol 1995
; Reichling et al. 1994
). Intracellular Ca2+ plays crucial roles in neurite extension and guidance (Kater and Mills 1991
; Mattson and Kater 1987
), as well as in filopodial extension (Rehder and Kater 1992
), gene expression (Vaccarino et al. 1992
), second messenger communication (Clampham 1995, review), and transmitter release (Dunlap et al. 1995
, review). GABA-mediated Ca2+ rises are found in the majority of developing neurons from all parts of the brain tested, including hippocampus, hypothalamus, cortex, striatum, olfactory bulb (Obrietan and van den Pol 1995
), and spinal cord (Reichling et al. 1994
).
Functional GABAA receptors are expressed at the earliest development time studied, embryonic day 15 in rats (Chen et al. 1995
; van den Pol et al. 1995
). GABA immunoreactivity is found in axonal growth cones (van den Pol 1997
) and GABA is released from axon terminals within three or four days of the final mitosis (Obrietan and van den Pol 1995
, 1996a
,b
). In addition to its actions at the ligand-gated GABAA receptor, GABA also acts on GABAB receptors that operate through G proteins (Misgeld et al. 1995
, review). GABAB receptors are expressed both presynaptically (Dutar and Nicoll 1988
; Howe et al. 1987
; Yoon and Rothman 1991
) and postsynaptically (Dutar and Nicoll 1989; Solis and Nicoll 1992
). Presynaptic GABAB receptors have been shown to decrease neurotransmitter release by increasing K+ conductance or decreasing Ca2+ conductance or through a mechanism independent of changes in membrane conductance (Misgeld et al. 1995
, review). Additionally, GABAB receptors have been shown to play a role as autoreceptors, providing a negative feedback control for synaptic GABA secretion (Anderson and Mitchell 1985
; Pittaluga et al. 1987
). GABAB receptors may show greater expression during development than in the adult, as detected with receptor autoradiography (Turgeon and Albin 1994
), yet studies have suggested that GABAB receptors, at least in postsynaptic sites, have relatively little action in some areas of the developing brain (Gaiarsa et al. 1995
). This raises the developmental question as to whether the GABAB receptor is functional during early development, and if so, what role it might play in modulating excitatory GABA actions and regulating cytosolic Ca2+ in developing neurons. Hypothalamic peptides may modulate the developmental actions of GABA (Obrietan and van den Pol 1996b
), but the modulation of GABA's excitatory actions has not previously been pursued relative to GABAB receptors.
To address this question we studied neurons cultured from the hypothalamus. The hypothalamus is a rich source of GABAergic neurons (Decavel and van den Pol 1990
; Tappaz et al. 1982
). Furthermore, GABA-mediated excitation in hypothalamic neurons occurs independently of a glutamate driving force (Obrietan and van den Pol 1995
, 1996b
). This is in contrast to the hippocampus, for instance, where GABA-mediated giant depolarizing potentials are aided by the synergistic actions of glutamate, acting at N-methyl-D-aspartate (NMDA) receptors (Ben-Ari et al. 1989
; Leinkugel et al. 1997
; McLean et al. 1995
). Computer-assisted digital imaging was used with the Ca2+-sensitive ratiometric dye fura-2 to test the hypothesis that GABAB receptors are functional at an early stage of development and that they act to inhibit the excitatory actions generated by GABAA receptors.
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METHODS |
Tissue culture
The mediobasal hypothalamus (extending from the mammillary bodies rostrally to the preoptic area and ventrally from the ventral surface of the hypothalamus to the top of the 3rd ventricle and laterally to the outside of the fornix) was removed from Sprague-Dawley rats at embryonic day 18 (E18). The tissue was placed in standard minimal essential culture media (GIBCO) and washed three times. The tissue was then enzymatically digested in Earl's balanced salt solution containing papain (10 units/ml) and L-cysteine (0.2 mg/ml) for 30 min. The tissue was pelleted and the protease solution was removed by aspiration. The tissue was triturated into a single-cell suspension in tissue culture medium [glutamate- and glutamine-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/ml penicillin/streptomycin and 6 gm/l glucose]. The suspended cells were plated onto poly-D-lysine (540,000 Da) coated glass coverslips (22 mm2). A high local neuronal density (~100,000 per cm2) was ensured by plating the neurons within a 7-mm glass cylinder placed on top of the coverslip. The glass cylinder was removed 60 min after plating. Cytosine arabinofuranoside (2 µM) was added to the tissue culture medium on the second day in culture to inhibit glial cell proliferation. Cytosine arabinofuranoside was removed on the fourth day in culture. Cell cultures were maintained at 37°C and 5% CO2 in a Napco 5410 incubator.
Immunocytochemistry
Cultures were washed with phosphate buffered saline (PBS), and then fixed with 3% glutaraldehyde. The next day, the cultures were washed five times with PBS with 0.3% triton X-100 to permeabilize the plasma membrane, immersed in 1% normal goat serum for 30 min and then incubated in GABA antiserum (1:5,000) overnight. Two GABA antisera were used, one from Incstar and another from Dr. Geffard, and both gave similar results. Both antisera are specific for GABA, and do not react with other amino acids (Decavel and van den Pol 1990
; van den Pol 1997
). Cultures were then washed five times in PBS and then stained with an avidin biotin peroxidase kit from Vector Labs. The peroxidase was revealed with diaminobenzidine and hydrogen peroxide as previously described (Decavel and van den Pol 1992
). Immunostained cultures were treated with 0.1% osmium tetroxide for 2 min to increase the staining intensity, dehydrated, and mounted with Permount on a glass slide.
Photomicrograph film of the stained cultures was scanned and digitized on a Kodak RFS 2035 digital slide scanner and printed with a Kodak 7700 dye sublimation printer.
Ca2+ digital imaging
Cells were loaded for 20 min in standard perfusion solution containing (in mM) 137 NaCl, 25 glucose, 10 N-2-hydroxyethylpiperazine-N
-2-ethanesulfonic acid (HEPES), 5 KCl, 1 MgCl2, 3CaCl2, pH 7.4 containing 5 µM fura-2 acetoxymethyl ester (fura-2).The cells were then washed to removed the excess fura-2 and allowed to sit for 20 min. The coverslip was then placed in a laminar-style perfusion chamber (Forscher et al. 1987
). This low volume (180 µL) perfusion chamber contained five ports that allowed for rapid (5 s) and uniform, unidirectional flow of perfusion solutions. Cells were imaged with an Olympus DApo ×40 objective with high UV light transmittance on a Nikon Diaphot 300 inverted microscope.
Data collection and all peripheral devices were controlled by a 486 computer with Fluor software (Universal Imaging). A Sutter filter wheel controlled by a Lambda 10 microprocessor was used to switch between the 340 and 380 nm excitation filters. Fluorescent light was passed through a 480-nm filter then directed at a silicon intensified target video camera (Hamamatsu, model 2400) located on the side port of the microscope. Excitation light from a 150 watt xenon lamp connected to an Optiquip power supply was attenuated by 90% with neutral density filters. Sixteen (500 ms) digital frames of data were collected every 4 s. Ca2+ calibrations were performed with Ca2+ standards and fura-2 acid as described by Grynkiewicz et al. (1985)
. Data analysis was performed on an Apple Quadra 840 with Igor Pro Software (WaveMetrics). In some experiments, a Grass SD9 Stimulator was used to initiate action potential-mediated transmitter release. A postsynaptic Ca2+ rise was elicited by passing 2.5 V/cm2, at 20 Hz, and a 2-ms duration through the chamber for 6 s. At this voltage, the Ca2+ rise from >95% of the neurons was blocked with tetrodotoxin (TTX, 1 µM).
To assess the modulatory effects of GABAB receptor activation on Ca2+ rises elicited by synaptic GABA release, the mean Ca2+ rise for the 15 s period immediately preceding GABAB receptor agonist administration was determined. This value was compared with the mean Ca2+ rise over a 15-s period 90 s after GABAB receptor agonist administration. The Ca2+ rise was determined by subtracting the Ca2+ level in the presence of the GABAA receptor antagonist bicuculline (20 µM) from the Ca2+ increase that resulted from its withdrawal during the time periods described above. Data are reported as the mean Ca2+ rise of all responsive neurons ± SE. Ca2+ rises elicited by either electrical stimulation or administration of GABAA receptor agonists to the bath solution were determined by subtracting the mean Ca2+ level for a 15-s period immediately preceding stimulation from the maximal evoked Ca2+ level.
Cytosine arabinofuranoside, GABA, muscimol and poly-D-lysine were acquired from Sigma, AP5, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), bicuculline, baclofen, 2-hydroxy-saclofen (2OH-SAC), 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 gift from Ciba-Geigy AG (Basel). GABA, muscimol, bicuculline, baclofen, and CGP 35348 were dissolved in physiological saline (HEPES buffer). AP5 was dissolved in mildly acidic HEPES-based buffer (pH 6.6). TTX was dissolved in 50% ethanol.2-hydroxy-saclofen and CNQX were dissolved in dimethyl sulfoxide (DMSO). At working concentrations, administration of vehicles did not alter physiological response characteristics.
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RESULTS |
Immunocytochemistry
To demonstrate that GABA was present in cultured cells during the developmental stage that was used for digital imaging, cultures were immunostained with GABA antiserum (Fig. 1). After four days in vitro strong GABA immunoreactivity was found in about 25% of the neurons and their associated neurites. Other neurons showed no immunoreactivity. Underlying astrocytes showed no immunostaining. Control experiments where the primary antiserum was not used or in which solid phase adsorption with GABA conjugated with glutaraldehyde to a carrier protein not part of the immunogen, were negative.

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| FIG. 1.
A culture of hypothalamic cells after 4 days in vitro was immunostained with -aminobutyric acid (GABA) antiserum. Some neurons (large arrows) and their neurites (small arrows) show strong GABA immunoreactivity. Other cells (curved arrows) show no immunoreactivity, but can be faintly seen because of the differential interference contrast condenser used. Scale bar: 30 µm.
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Calcium digital imaging
GABAB RECEPTOR ACTIVATION REDUCES Ca2+ RISES EVOKED BY SYNAPTIC ACTIVATION OF GABAA RECEPTORS.
In developing cultures from five to seven days in vitro (DIV), Ca2+ transients were generated by synaptic release of GABA. This is demonstrated in Fig. 2. When GABAA receptors were blocked with the antagonist bicuculline (20 µM), the mean cytosolic Ca2+ level was lowered and Ca2+ transients were lost. When baclofen (10 µM), a GABAB agonist (Bowery et al. 1980
; Hill and Bowery 1981
), was bath applied, there was a rapid decrease in both the mean cellular Ca2+ level and a loss of Ca2+ transients. This and the other imaging experiments described below were all done in the presence of the glutamate receptor antagonists AP5 (100 µM) and CNQX (10 µM) to prevent synaptic glutamate-mediated intracellular Ca2+ rises. In the presence of both the glutamate receptor antagonists and GABAA receptor antagonists, a baseline cytosolic Ca2+ level was maintained and transients were not found. In many cells (Fig. 2, top) the effect of baclofen was dramatically large, depressing GABA-mediated Ca2+ rises to the same baseline Ca2+ level as that observed on bicuculline administration. In other cells (Fig. 2, bottom) baclofen caused a substantial reduction in the GABA-mediated Ca2+ rise, but to a lesser extent than that induced by bicuculline. After baclofen washout, the neurons returned to the same level of Ca2+ activity as found before baclofen introduction.

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| FIG. 2.
GABAB receptor activation depresses Ca2+ rises mediated by synaptic release of GABA. Withdrawal of GABAA receptor antagonist bicuculline (BIC, 20 µM) from the perfusion solution elicited a rapid and sustained Ca2+ rise. Administration of GABAB receptor agonist baclofen (BAC, 10 µM) caused a large depression in cytosolic Ca2+ levels. Ca2+ levels recovered to prebaclofen levels on withdrawal of baclofen from perfusion solution. Calibrated Ca2+ values are shown on Y-axis; minutes (MIN) are on X-axis. To ensure that postsynaptic ionotropic glutamate receptor activity did not contribute to Ca2+ rises, all perfusion solutions contained glutamate receptor antagonists D-2-amino-5-phosphonovalerate (AP5, 100 µM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 µM). These 2 neurons were representative of majority of neurons assayed.
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The effect of different concentrations of baclofen on GABA-mediated Ca2+ rises were examined, as shown in two typical neurons (Fig. 3). In total, 188 neurons were tested with multiple concentrations of baclofen (10 nM-10 µM). With the use of analysis of variance (ANOVA), we found no difference in the level of the Ca2+ rise immediately preceeding each of the baclofen administrations (F = 2.0287, P > 0.1) indicating a full recovery. Ten nanomolar showed no effect and 100 nM baclofen showed a slight depression that was not statistically different from controls. Concentrations
1 µM elicited a substantial reduction in Ca2+ levels. The mean Ca2+ depression trigger by 1, 5, and 10 µM baclofen was statistically different (P < 0.0001, 2-tailed t-test) from controls. Additional tests with 100 µM baclofen showed no increased inhibition over that found with 10 uM baclofen. The relative efficiencies of different concentrations of baclofen are shown in the bar graph in Fig. 3B.

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| FIG. 3.
Baclofen inhibition of GABA-mediated Ca2+ rises was dose dependent. A: 2 representative neurons show that increasing concentration of baclofen (from 100 nM to 10 µM) evoked increasing levels of Ca2+ inhibition. At 10 µM, Ca2+ depression level was almost equivalent to that observed for bicuculline administration. These experiments were performed in the constant presence of AP5 (100 µM) and CNQX (10 µM) to block ionotropic glutamate receptor actions. B: dose-dependent effects of baclofen. Control (normalized to 100%) represents the mean Ca2+ increase from basal, bicuculline blocked Ca2+ levels. * Statistically significant (P < 0.0001, 2-tailed t-test) mean Ca2+ depression compared with control Ca2+ rise. N refers to total number of neurons assayed. Error bars = SE.
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| FIG. 4.
Baclofen acts at the GABAB receptor to inhibit GABAA receptor-mediated Ca2+ rises. A: in 2 representative neurons, administration of baclofen (1 µM) resulted in a large Ca2+ depression. However, when the GABAB receptor antagonist 2-hydroxy saclofen (2OH-SAC) (200 µM) was added during baclofen administration, baclofen-mediated Ca2+ depression was largely blocked. Application of bicuculline (20 µM) at the end of the experiment caused a rapid Ca2+ depression and cessation of large Ca2+ transients. B: in these 2 representative neurons coadministration of GABAB receptor antagonist CGP-35348 (200 µM) blocked the actions of baclofen (5 µM). These experiments were performed in the constant presence of AP5 (100 µM) and CNQX (10 µM).
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To ensure that the effects of baclofen were mediated at 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 coadministered with baclofen (1 µM). 2-Hydroxy-saclofen attenuated the baclofen-mediated depression of GABA-mediated Ca2+ transients (Fig. 4A). The mean Ca2+ rise on bicuculline withdrawal was 61 ± 7 (SE) nM. After baclofen administration, the Ca2+ rise was reduced to 9 ± 2 (SE) nM. In the presence of the antagonist 2-hydroxy-saclofen, the mean Ca2+ rise of 60 ± 5 nM from a bicuculline-defined baseline was decreased to 34 ± 3 nM. On average, 2-hydroxy-saclofen coadministration resulted in a more than threefold reduction in baclofen-mediated Ca2+ depression (n = 44). Figure 4B shows that a second specific GABAB receptor antagonist, CGP-35348 (200 µM), also inhibited baclofen (5 µM)-mediated Ca2+ depression. Alone, baclofen triggered a depression of the GABAA-mediated Ca2+ rise from 57 ± 5 nM to 23 ± 4 nM. In the presence of CGP-35348 baclofen did not significantly reduce the cytosolic Ca2+ level; the control Ca2+ rise immediately preceding baclofen/CGP-35348 administration was 62 ± 5 nM and after baclofen/CGP-35348 administration, the Ca2+ rise was 62 ± 4 nM, n = 15. These data support the conclusion that the agonist baclofen was acting specifically on GABAB receptors.
POSTSYNAPTIC AND PRESYNAPTIC ACTIONS OF GABAB RECEPTORS.
Next, we assessed the cellular location at which GABAB receptors exert inhibitory actions on Ca2+ rises. To test for a postsynaptic (i.e., cell body) modulatory role of GABAB receptors we blocked action potential-mediated release of GABA with tetrodotoxin (1 µM) and bath-applied the GABAA agonist muscimol (3 µM) to generate a Ca2+ rise. In a considerable number of neurons the coadministration of baclofen reduced the postsynaptic Ca2+ rise elicited by muscimol (>20% inhibition observed in 66 of 100 neurons) (Fig. 5A, top). In other neurons in the same culture, baclofen had relatively little effect (Fig. 5A, bottom). The mean Ca2+ rise elicited by muscimol was 177 ± 11 nM. After the addition of baclofen the muscimol-evoked Ca2+ rise decreased to 127 ± 8 nM, representing a statistically significant (P < 0.0001, 2-tailed t-test) reduction in the GABAA receptor-mediated Ca2+ rise of 28%, n = 100. For control purposes we also examined older neurons after 16 DIV. In the older neurons, GABA did not generate Ca2+ rises, as expected (Fig. 5B) (Obrietan and van den Pol 1995
). In experiments where we combined GABA immunostaining with Ca2+ digital imaging, we found that both cells immunoreactive for GABA and others not immunoreactive for GABA showed Ca2+ rises in response to GABA (not shown). In general, these results show that young hypothalamic neurons express postsynaptic GABAB receptors capable of depressing Ca2+ rises elicited by GABAA receptor activation.

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| FIG. 5.
Postsynaptic GABAB receptor activation depresses GABAA receptor-evoked Ca2+ rises. Hypothalamic neurons were stimulated ( ) for 15 s with GABAA receptor-specific agonist muscimol (3 µM) in both the absence and presence of baclofen (10 µM). A: repeated administration of muscimol elicited rapid and reproducible Ca2+ rises. In the top neuron, administration of baclofen caused a large reduction in the postsynaptic muscimol-evoked Ca2+ rise. On baclofen withdrawal, the magnitude of evoked Ca2+ rises recovered to a level similar to that observed before baclofen administration. In the bottom neuron, administration of baclofen had little effect on muscimol-evoked Ca2+ rises. The voltage-activated sodium channel blocker tetrodotoxin (TTX) (1 µM) was used to block action potential-mediated transmitter release. This experiment was performed with embryonic day 18 (E18) neurons cultured for 6 days in vitro (DIV). B: GABA-mediated Ca2+ rises were only observed during early development. Administration of GABA to more mature neurons (E18: 16 DIV) did not trigger a Ca2+ rise.
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To determine whether or not GABAB receptors were functionally expressed at a very early time point, before synapse formation, we studied the modulatory action of GABAB receptors stimulation in neurons cultured at low density from embryonic day 15 neurons cultured for only 3 DIV. Figure 6A shows that GABAB receptor activation with baclofen (10 µM) depressed the Ca2+ rise evoked by bath applied muscimol (3 µM) in the cell body. Muscimol elicited a mean Ca2+ rise of 86 ± 15 nM. Coactivation of the GABAB receptor decreased this Ca2+ rise to 31 ± 6 nM. Many of the cells that showed a GABAB mediated inhibition of muscimol-induced Ca2+ rises were isolated from other cells and when examined with phase contrast, no neuritic connections were seen between these isolated cells at this stage of in vitro development. Thus GABAB receptor activation can trigger a statistically significant (P < 0.01, 2-tailed t-test) depression of muscimol-evoked Ca2+ rises in neurons before they have established synaptic contact.

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| FIG. 6.
Muscimol-evoked Ca2+ rises were depressed by baclofen in E15 neurons after 3 days in the culture. A: in the constant presence of TTX (500 nM), muscimol (3 µM) triggered a Ca2+ rise in the cell soma that was depressed by baclofen (10 µM) administration. On baclofen washout, Ca2+ responsiveness to muscimol returned to a level approximately equivalent to that observed before baclofen administration. B: muscimol-evoked Ca2+ rises were also observed in neurites. Baclofen (10 µM) coadministration reduced the neurite Ca2+ rise triggered by muscimol (3 µM).
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GABAB receptor stimulation also depressed muscimol-evoked Ca2+ rises in developing neurites (Fig. 6B). Muscimol evoked a mean Ca2+ rise of 188 ± 46 nM, coadministration of baclofen reduced the Ca2+ rise to 116 ± 33 nM,n = 9. These findings suggest that functional GABAB receptors are expressed very early in hypothalamic neurogenesis and that their activity can depress Ca2+ in both perikarya and neurites.
Next, experiments were performed in cultures that were synaptically coupled, but that had little evidence of Ca2+ transients to determine whether or not presynaptic GABAB receptors also contribute to the inhibition of GABA excitation. Electrical stimulation of the presynaptic neurons (2.5 V/cm2) generated a Ca2+ rise evoked by synaptic GABA release (Fig. 7A). As the experiments were done in the presence of glutamate receptor antagonists (AP5 and CNQX), synaptically released glutamate was not responsible for the Ca2+ rise. Tetrodotoxin (1 µM) blocked the Ca2+ rise (Fig. 7, A and B), indicating that the rise was dependent on action potential release of transmitter and was not the result of a direct electrical depolarization of the recorded cells. Bicuculline (20 µM) efficiently blocked the response to electrical stimulation, indicating that the Ca2+ rise resulted from activation of GABAA receptors. Baclofen (10 µM) reduced the GABA-mediated Ca2+ rise by over 25% in all neurons assayed (n = 85). Additionally, some neurons (30 of 85) showed an almost complete block (>90%) of the Ca2+ rise. The mean electrically evoked, GABA-mediated, Ca2+ rise in the absence of baclofen was 217 ± 13 nM. After baclofen was added the mean Ca2+ rise was 30 ± 5 nM, representing a statistically significant (P < 0.0001, 2-tailed t-test) mean Ca2+ rise decrease of 86% (n = 85). These experiments were performed at 4 DIV. Figure 7B shows that the inhibitory actions of baclofen (5 µM) were attenuated by administration of the GABAB receptor antagonist CGP-35348 (200 µM). The mean electrically evoked, GABA-mediated, Ca2+ rise in the absence of baclofen was 100 ± 7 nM. The mean Ca2+ rise in the presence of baclofen was 27 ± 3 nM; whereas, in the presence of both baclofen and CGP-35348, the mean Ca2+ rise was 64 ± 5 nM (n = 49). These experiments were performed at 3 DIV. These data provide additional evidence that activation of the GABAB receptor leads to a decrease in GABAA receptor-mediated Ca2+ rises and that GABAB receptor antagonists reduce this baclofen-mediated inhibition.

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| FIG. 7.
Electrical stimulation (E.S.)-evoked Ca2+ rises are depressed by baclofen. Application of electrical stimulation ( ) (2.5 V/cm2, at 20 Hz, and a 2-ms duration) through the chamber for 6 s triggered reproducible Ca2+ rises. A: administration of baclofen (10 µM) to perfusion solution resulted in a dramatic depression of evoked Ca2+ rise. Ca2+ responsiveness recovered on baclofen withdrawal. Administration of the GABAA receptor antagonist bicuculline (20 µM) also depressed electrically induced Ca2+ rises. Addition of sodium channel blocker TTX (1 µM) blocked the Ca2+ rise, suggesting that electrically induced Ca2+ rises were the result of action potential-mediated neurotransmitter release. B: administration of GABAB receptor antagonist CGP-35348 (200 µM) blocked the inhibitory effects of baclofen (5 µM). After removal of CGP-35348 from the perfusion solution, inhibitory actions of baclofen recovered.
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Figure 8A compares the mean modulatory effects of baclofen on muscimol-evoked Ca2+ rises (purely postsynaptic GABAB receptor effect) and on electrically induced Ca2+ rises (a mix of both presynaptic and postsynaptic GABAB receptor effects). Scatter plots compare the relative modulatory action of baclofen on muscimol- (Fig. 8B) or electrically (Fig. 8C) evoked Ca2+ rises. Each neuron is shown as a single point that is a representation of the first Ca2+ rise in the presence of baclofen divided by the control Ca2+ rise immediately preceding baclofen administration. Data are displayed as a percentage. Zero percent on the Y-axis signifies that the peak rise in the presence of baclofen was equivalent to the control Ca2+ rise (no modulation). Values below 0% represent depression; values above 0% represent response potentiation. When the data from the two experiments are plotted on the same axis, two populations with minimal overlap are observed. These results suggest that functional GABAB receptors are expressed both presynaptically and postsynaptically. Further, presynaptic GABAB receptors exert a greater inhibitory action on synaptic GABA release than on postsynaptic Ca2+ rises triggered by GABAA receptor activity.

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| FIG. 8.
A: bar graph comparison of ability of GABAB receptor activation to depress muscimol- or electrically evoked Ca2+ rises. White bars: mean Ca2+ rises elicited by muscimol. Coadministration of baclofen significantly (P < 0.05) depressed the muscimol-evoked Ca2+ rise. This modulatory effect was purely postsynaptic. Black bars: mean Ca2+ rises elicited by electrical stimulation (E.S.). Coadministration of baclofen, bicuculline, or tetrodotoxin (TTX) significantly reduced the evoked Ca2+ rise. These agents depressed the electrically induced Ca2+ rises through both presynaptic and postsynaptic mechanisms. B and C: scatter plot analysis of data in A. Each circle is a percentage comparison for a single neuron of the peak Ca2+ rise elicited in presence of baclofen to the control peak Ca2+ rise immediately preceding baclofen administration. Levels of Ca2+ depression elicited by baclofen using these 2 different methods of Ca2+ stimulation show little overlap.
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ONGOING SYNAPTIC ACTIVATION OF GABAB RECEPTORS INHIBITS GABA-MEDIATED Ca2+ RISES.
To determine whether or not GABAB receptors normally exert an inhibitory tone on GABAA-mediated Ca2+ rises, the GABAB antagonist 2-hydroxy-saclofen (200 µM) was added to synaptically active cultures. In 27 of 100 neurons tested, the addition of 2-hydroxy-saclofen caused an increase in mean cytosolic Ca2+ and an increase in the amplitude of GABA mediated Ca2+ transients (Fig. 9). In the 27 responsive neurons, 2-hydroxy-saclofen caused a 27 ± 5 nM mean Ca2+ rise. Addition of bicuculline (20 µM) blocked the Ca2+ transients (not shown), verifying they were the result of GABA release. This result suggests that tonic GABAB receptor activation may depress Ca2+ rises elicited by GABAA receptor activation.

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| FIG. 9.
Tonic GABAB receptor activation decreases Ca2+ rises elicited by spontaneous synaptic GABA release. These 2 representative neurons showed large Ca2+ transients and an elevated Ca2+ level because of GABA release in the absence of bicuculline. Administration of the GABAB receptor antagonist 2-hydroxy-saclofen (2OH-SAC) (200 µM) during GABA-mediated Ca2+ rises triggered increased mean Ca2+ levels and larger Ca2+ transients. Ca2+ levels recovered after 2-hydroxy-saclofen wash out. Vertical dashed lines of 2OH-SAC show periods during which antagonist was added and washed out. This experiment was performed in constant presence of AP5 (100 µM) and CNQX (10 µM).
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DISCUSSION |
The findings in this study indicate that early in neural development, the Ca2+ elevating actions of GABA, acting on the GABAA receptor, may be regulated by GABAB receptor coactivation. GABAB receptors are expressed at both presynaptic and postsynaptic locations in developing hypothalamic neurons. The observation that tonic GABAB receptor activation decreases GABA-mediated Ca2+ rises suggests that GABAB receptor autoinhibition may play an important role in regulating GABA's ability to raise cytosolic Ca2+ levels. As GABA-mediated excitation was found in the majority of neurons tested from a number of brain regions (Obrietan and van den Pol 1995
), GABAB-mediated inhibition may play a widespread, critical role in modulating GABA-regulated neuronal excitability during early development.
Presynaptic and postsynaptic GABAB actions
In the present study we demonstrate that the bath application of the GABAA receptor agonist muscimol elicited Ca2+ rises that were depressed by coadministration of baclofen. That this modulatory effect of baclofen was observed in the constant presence of TTX (to block action potential-mediated transmitter release) suggests that postsynaptic GABAB receptors decreased Ca2+ entry through the plasma membrane. Postsynaptic GABAB receptors have been observed in some brain regions, including the spinal cord (Beattie et al. 1989
), cortex (Karlsson and Olpe 1989
), and hippocampus (Yoon and Rothman 1991
), but appear to be absent in other brain regions such as the striatum (Nisenbaum et al. 1993
). A postsynaptic physiological effect often resulting from GABAB receptor stimulation is an increase in K+ conductance (Misgeld et al. 1995
). Increased K+ conductance decreases neural excitability as a result of membrane hyperpolarization. Our observations that postsynaptic GABAB receptor activation decreases Ca2+ influx mediated by GABAA receptor activation fits well with this finding. GABAA-elicited Ca2+ rises result from Cl
efflux (Chen et al. 1996
) leading to membrane depolarization-mediated activation of voltage-dependent Ca2+ channels. Hyperpolarization as a result of increased GABAB-mediated K+ channel conductance would lead to a decrease in the relative level of GABAA-mediated membrane depolarization. In addition, GABABreceptor activation could inhibit Ca2+ conductance at voltage activated Ca2+ channels.
The postsynaptic modulatory effect of baclofen in the presence of TTX was observed at the earliest time point tested, embryonic day 15 (E15) neurons cultured for 3 days. E15 is a early stage of hypothalamic development, before the final mitosis of most neurons and neural differentiation is still primitive (Altman and Bayer 1978
). In accordance with our observations of functional GABAB receptors at early stages of neuron development, several ligand binding studies have shown the expression of GABAB receptors at early stages of CNS development (Snead 1994
; Turgeon and Albin 1994
). In contrast to our detection of postsynaptic GABAB actions in hypothalamic neurons even at the earliest age studied, relatively little postsynaptic physiological activity was found in the developing hippocampus (Gaiarsa et al. 1995
).
In our study, GABAB receptor activation exerted a much greater depression of Ca2+ rises elicited by synaptic GABA release than of postsynaptic muscimol-evoked Ca2+ rises (in the presence of TTX). This is consistent with the robust expression of presynaptic GABAB receptors. These large GABAB-mediated decreases were observed for Ca2+ rises triggered by electrically evoked synaptic GABA release or for Ca2+ rises elicited by spontaneous synaptic GABA release. A number of studies have demonstrated that GABAB receptors are expressed by axon terminals where they act to reduce transmitter release (Dutar and Nicoll 1988
; Fukuda et al. 1993
; Howe et al. 1987
; Isaacson et al. 1993
; Yoon and Rothman 1991
). Reduction of transmitter release may depend on a mechanism involving inhibition of 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
). Additionally, GABAB receptors may also hyperpolarize the membrane potential via K+ current enhancement (Gerber and Gahwiler 1994
). The GABAB receptor, working via an inhibitory G protein-coupled mechanism has also been shown to decrease transmitter release through a mechanism independent of changes in Ca2+ or K+ channel conductance (Jarolimek and Misgeld 1992
; Scanziani et al. 1992
).
GABAB in early development
A critical point here is that both pre- and postsynaptic responses to GABAB receptors are found early in the embryonic development of hypothalamic neurons. Whereas developing CA3 hippocampal neurons show a robust presynaptic effect of GABAB receptor activation, relatively little postsynaptic effect is found (Gaiarsa et al. 1995
). In contrast, our data demonstrate that the GABAB receptor is functional both presynaptically and postsynaptically at early stages of neuronal development, at a time when GABA can generate a greater Ca2+ rise than glutamate (Obrietan and van den Pol 1995
). Many neurons including isolated neurons in low-density cultures showed responses to GABAB activation before substantial synapse formation (3 DIV), and two-thirds of the hypothalamic neurons kept in vitro for 5 days showed GABAB-mediated postsynaptic actions.
GABAB responses are also found presynaptically at the earliest stage when robust GABA-mediated synaptic activity can be detected with digital imaging (5 DIV). All neurons tested with electrical stimulation driving presynaptic GABA release showed a GABAB-mediated Ca2+ depression, suggesting that most neurons in these cultures either expressed GABAB receptors, or were postsynaptic to neurons that did express them. Additionally, GABAB receptor antagonists generated a Ca2+ rise in synaptically active neurons. The fact that all ionotropic glutamatergic activity was blocked by the constant presence of AP5 and CNQX (glutamate is the only other synaptically released transmitter we have found to elicit Ca2+ rises in young hypothalamic neurons), suggests that tonic GABAB receptor activity sets an inhibitory tone on GABA's ability to raise cytosolic Ca2+ levels. The tonic actions of GABA on the GABAB receptor may be working at both presynaptic and postsynaptic sites to both decrease vesicle release and the Ca2+ responsiveness to GABA.
Our general observation is consistent with the finding that tonic GABAB receptor activation in the neonatal rat hippocampus reduces excitatory GABAergic activity (McLean et al. 1996
). However, in contrast to the hippocampus where GABA's giant depolarizing potentials are driven synergistically by GABA and NMDA-type glutamate receptor actions (Ben-Ari et al. 1989
; Leinkugel et al. 1997
; McLean et al. 1995
), hypothalamic neurons in our study act completely independent of glutamate. In developing hypothalamic neurons, Ca2+ rises (Obrietan and van den Pol 1995
) and excitatory postsynaptic potentials (EPSPs) (Chen et al. 1996
) are mediated by excitatory axons driven solely by GABA and found even in the presence of glutamate receptor blockers. In addition to the excitatory actions of GABA during development that we focus on here, we recently found that GABA may exert excitatory actions after neuronal trauma (van den Pol et al. 1996
). Similar to the mechanism in developing neurons, this action is based on a positive shift in the Cl
reversal potential (van den Pol et al. 1996
) and is not dependent on bicarbonate actions (Kaila 1994
). GABAB receptor activation in traumatized neurons would probably have a similar effect to that described here for developing neurons
that is, a reduction in GABA's excitatory activity. Excitatory actions of GABA may not be restricted to developing neurons, but have been suggested in slices that contain the adult suprachiasmatic nucleus (Wagner et al. 1997
), suggesting that GABAB receptors could play an inhibitory role with neurons involved in circadian rhythms or phase shifts of these rhythms.
Changing GABA's ability to increase cytosolic Ca2+ levels during the early stages of development may be important for several reasons. GABA has been shown to trigger motility of embryonic cortical neurons through an increase in intracellular Ca2+ (Behar et al. 1996
). Likewise, DNA synthesis in cortical neural progenitor cells can be blocked by inhibiting GABAA receptor activity (LoTurco et al. 1995
).
Previously, we have shown that GABA can increase Ca2+ levels in neurites and growth cones (Obrietan and van den Pol 1996a
), raising the possibility that GABA working at the GABAA receptor may alter growth cone motility and the rate of neurite extension as a result of Ca2+ increases. In growing neurites, we observed modulatory actions of baclofen on Ca2+ rises elicited by GABAA receptor stimulation, suggesting that GABAB receptors may play a role in the formation of synaptic connections. Administration of the GABAA receptor antagonist bicuculline decreased neurite length (Barbin et al. 1993
) and GABA-mediated Ca2+ influx in growth cones has been shown to increase GAP43 and MARCKS protein phosphorylation (Fukura et al. 1996
). As GABAB receptor agonists would reduce GABA actions, this might also be expected to modulate neurite and growth cone outgrowth.
The general phenomena and underlying mechanisms observed in culture with Ca2+ digital imaging offer a window on developmental actions of transmitters that cannot be readily examined in vivo. GABA shows depolarizing actions and raises cytosolic Ca2+ in developing neurons in both culture and in slices (LoTurco et al. 1995
; Obrietan and van den Pol 1995
; Owens et al. 1996
). Additionally, neuromodulators such as neuropeptide Y have similar physiological effects on excitatory GABA activity in the developing slice as that observed in cultured neurons (Obrietan and van den Pol 1996b
). There are some advantages to studying cultured neurons, particularly related to clear digital imaging, the simultaneous recording of synaptically coupled neurons, and the ability to rapidly introduce drugs and wash them away. During developmental aging, neurons in slices become more difficult to load with calcium sensitive dyes, in large part because of the increasing astrocyte processes surrounding neurons. Responses to drug application in brain slices are slowed by a factor of 10 to 100 because of the slowed diffusion into a slice and recovery times after drug washout can be even longer. A limitation of culture is that developmental times in culture may not be identical with those found in vivo and the specific synaptic connections generated in vivo are probably not generated in culture. Nonetheless, the findings in the present paper provide strong support that functional GABAB receptors are expressed very early in neuronal development and that GABAB receptors can reduce the excitatory actions of GABAA receptors at both pre- and postsynaptic locations in developing hypothalamic neurons.
Functional significance for synapse formation
Our study demonstrates that both GABAA and GABAB receptors are present and functionally active in early neuronal development. During this early stage of development, activation of the GABAA receptor usually leads to depolarization and the resultant opening of plasmalemmal Ca2+ channels that raise intracellular Ca2+ (Obrietan and van den Pol 1995
). In contrast, activation of the GABAB receptor tends to reduce the GABA-mediated elevations in Ca2+, at both presynaptic and postsynaptic sites of action. Thus GABA would generate two opposing actions, one at the GABAA receptor that initially depolarizes the cell, raising Ca2+, and a slightly later effect at the GABAB receptor that would reduce the Ca2+ rise.
This could have important ramifications during periods of synaptogenesis, relating to the idea that use strengthens synaptic connections and that with disuse, the synapse may become weaker (Hebb 1949
). Hypothetically, if two developing GABAergic axons terminate on a single postsynaptic neuron, the one that releases GABA first, or in the greatest amount, may stimulate the postsynaptic cell to generate a Ca2+ rise at the GABAA receptor and this may strengthen the synapse. At the same time, GABA released from the first axon could diffuse to the second adjacent axon terminal and would tend to reduce GABA release from the second axon by activation of axonal GABAB receptors. Whereas the first axon might also have GABAB receptors, these would not be stimulated until after the initial excitation, giving the first axon a competitive advantage over the second in establishing stable synaptic contact. Heterosynaptic actions of GABA acting at presynaptic GABAB receptors have been reported in mature brain slices (Isaacson et al. 1993
). Thus, the more active developing axon would both strengthen its own synaptic connection and reduce the synaptic strength of any adjacent developing axon bearing GABAB receptors.
In conclusion, these findings suggest a developmentally important interaction between the excitatory actions of GABAA and the substantial inhibitory actions of GABAB receptors.