Department of Biological Science, Stanford University, Stanford, California 94305; and Department of Neurosurgery, Yale University School of Medicine, New Haven, Connecticut 06520
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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METHODS |
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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).
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RESULTS |
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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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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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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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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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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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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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DISCUSSION |
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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
- and
-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.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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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REFERENCES |
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