Department of Neurosurgery, Yale University School of Medicine, New Haven, Connecticut 06520
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ABSTRACT |
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Gao, Xiao-Bing and
Anthony N. Van Den Pol.
GABA, Not Glutamate, a Primary Transmitter Driving Action
Potentials in Developing Hypothalamic Neurons.
J. Neurophysiol. 85: 425-434, 2001.
Neuronal
activity is critical for many aspects of brain development. It has
often been assumed that the primary excitatory transmitter driving this
activity is glutamate. In contrast, we report that during early
development, synaptic release of GABA, the primary inhibitory
neurotransmitter in the mature brain, is not only excitatory but in
addition plays a more robust role than glutamate in generating spike
activity in mouse hypothalamic neurons. Based on gramicidin perforated
whole cell and extracellular recording, which leave intracellular
Cl unperturbed in brain slices and cultures,
the GABAA receptor antagonist bicuculline induced
a dramatic decrease in spike frequency (83% decrease) in developing
neurons, three times greater than that generated by glutamate receptor
antagonists 2-amino-5-phosphono-pentanoic acid and
6-cyano-7-nitroquinoxalene-2,3-dione. Thus a number of factors related
to spike-dependent stabilization of neuronal connections, including
Hebbian mechanisms, that are generally applied to glutamate transmission may also participate in stabilization of GABA circuits.
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INTRODUCTION |
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Although synapses can
develop in the absence of neuronal activity, a large number of reports
have demonstrated the importance of neuronal activity, particularly
action potentials, during brain development. Such activity is critical
to define synaptogenesis and synaptic efficacy (Nakagami et al.
1997), regulate the expression of adhesion molecules
(Itoh et al. 1997
), define axon structure (Lin
and Constantine-Paton 1998
), prevent inappropriate connections (Jarecki and Keshishian 1995
), allow the segregation of
functionally related axons (Kalil 1990
), facilitate
development of inhibitory circuits (Seil and Drake-Baumann
1994
), and, in general, facilitate the correct wiring of the
brain and spinal cord (Kalil et al. 1986
; Nelson
et al. 1989
). The absence of normal neuronal activity during
development can lead to permanent dysfunction (Hubel and Wiesel
1998
). Although transmitters may produce depolarizing activity at the cell body, only if spike threshold is reached does the neuron
send the message down the axon to distant sites of synaptic termination; the action potential is critical for long distance signaling.
The assumption that has generally been held is that the
excitatory transmitter glutamate is the principal mediator
regulating neuronal activity and spike generation in the developing
brain as it is in the mature brain. A number of interesting experiments have demonstrated an important role for glutamate in refining synaptic
connections (Hofer and Constantine-Paton 1994;
Lin and Constantine-Paton 1998
). A large proportion of
synapses in the brain use GABA (Gribkoff et al. 1999
;
Kim and Dudek 1992
; Strecker et al. 1997
;
Tasker and Dudek 1993
), and half of all presynaptic hypothalamic boutons contain immunoreactive GABA (Decavel and van den Pol 1990
). GABA is the primary inhibitory transmitter in the mature brain. In the developing brain, GABA may exert
depolarizing actions due to an elevated Cl
reversal potential as reported in many brain regions including hypothalamus, spinal cord, olfactory bulb, cerebellum, and hippocampus in both cultured neurons and brain slices (Ben-Ari et al.
1989
; Chen et al. 1996
; Leinekugel et al.
1997
; LoTurco et al. 1995
; Obata
1974
; Owens et al. 1996
; Reichling et al.
1994
; Rohrbough and Spitzer 1996
; Serafni
et al. 1995
; Wu et al. 1992
). Furthermore during
development, GABA has been found to enhance neurite outgrowth, increase
synapse formation, alter cell division of neuronal precursors, modulate
neuron migration, and influence growth cone dynamics, suggesting a
developmental role for GABA neurotransmission (Barbin et al.
1993
; Behar et al. 1996
; LoTurco et al.
1995
; Obrietan and van den Pol 1998
).
The possibility that GABA-mediated depolarization under some
circumstances leads to action potentials has been suggested and demonstrated (Andersen et al. 1980; Ben-Ari et
al. 1989
; Gao et al. 1998
; Serafini et
al. 1995
). Although depolarizing events are often excitatory,
they can also be inhibitory. Related to GABA, such activity has been
called a conductance shunt type of inhibition, shunting inhibition, or
depolarizing inhibition (Alger and Nicoll 1979
;
Andersen et al. 1980
; Staley and Mody
1992
). In some cells, GABA-mediated depolarization may be
primarily inhibitory due to current shunt (Staley and Mody
1992
) or may depress glutamate actions by transient shunting
activity (Gao et al. 1998
).
Based on previous work showing that in some circumstances GABA is capable of evoking action potentials, we tested the hypothesis that GABA is responsible for most spike activity and that, in contrast, the excitatory actions of glutamate relating to spike generation are relatively weak during early development of hypothalamic neurons.
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METHODS |
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Brain slices and cultures
Coronal hypothalamic slices, 400 µm thick, were cut on a vibratome from the developing brains of P1-P4 mice or from an older group of P8-P10 mice. Briefly, mice were anesthetized with pentobarbital sodium (Nembutal; 80 mg/kg) and then decapitated. The brains were rapidly removed and immersed in cold (4°C) oxygenated bath solution [containing (in mM): 150 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose, pH 7.3 with NaOH]. After being trimmed to contain only hypothalamus, the slices were transferred to a recording chamber. Slices were constantly perfused with bath solution at 2 ml/min. Rodent use was approved by the university committee on animal use.
To generate cultures, neurons were dissociated from E15 to E18 mouse
embryo hypothalami and spinal cords and cultured as described previously (Gao et al. 1998).
Immunocytochemistry
To detect GABA immunoreactive cells, 4-5 days after culturing
neurons, some cultures (n = 6) were fixed with 3%
glutaraldehyde and after membrane permeabilization with 0.3% Triton
X-100 were immunostained with GABA antiserum made in rabbits. The
specificity of the antiserum has been described previously (van
den Pol 1997). After overnight incubation in the primary
antiserum diluted 1:5,000, cultures were washed and then stained with a
secondary antiserum of goat anti-rabbit immunoglobulin (Molecular
Probes) conjugated to Texas Red and diluted 1:150. Cultures were viewed
on an Olympus IX70 inverted microscope, and photomicrographs were taken
with a Spot-2 digital camera interfaced with a lab computer. Contrast was adjusted in Photoshop, and micrographs were printed on an Epson 900 digital printer.
Extracellular recording
Extracellular recordings were made with a glass electrode
(resistance = 1 M) with a DAM 50 differential amplifier (World Precision Instruments) in the area of the arcuate and ventromedial nuclei in the mediobasal hypothalamus from P1-P4 and P10 mice. The
band-pass was 10-3,000 Hz. All data were sampled at 500 Hz with an
Apple Macintosh computer using Axodata 1.2.2 (Axon Instruments).
Whole cell patch-clamp recording
Experiments were performed at room temperature on cultured
neurons after 4-7 days in vitro (DIV; young neurons) and after 20 DIV
(mature neurons). Recordings were made from the area of the arcuate and
ventromedial nuclei in hypothalamic slices. The recording chamber was
continuously perfused at a rate of 1.5-2 ml/min with a bath solution
containing (in mM): 150 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose, pH 7.3 with NaOH. The tip
resistance of recording pipettes was 4-6 M after being filled with
pipette solution containing (in mM): 145 KCl, 1 MgCl2, 10 HEPES, 1.1 EGTA, 2 Mg-ATP, and 0.5 Na2-GTP, pH 7.3 with KOH. Gramicidin-perforated
recording, which maintains a physiological Cl
concentration inside the recorded cell, was used in most experiments except where otherwise mentioned, as previously described (Gao et al. 1998
). The series resistance after 20-30 min gramicidin perforation was between 40 and 50 M
. Experiments were performed under current or voltage clamp.
In some experiments GABA or glutamate was applied to recorded neurons either through a micropipette placed 2-4 µm away from the recorded cell or by application through a large flow pipe aimed at the recorded cell. All data were sampled at 3-10 kHz and filtered at 1 kHz with an Apple Macintosh computer using AxoData 1.2.2 (Axon Instruments). 2-Amino-5-phosphono-pentanoic acid (AP5), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and bicuculline (BIC) were obtained from RBI (Research Biochemical International).
All data are reported as means ± SE. Because of the variation in the frequency of action potentials or spikes among different recorded neurons, all data were normalized to the percentage of control frequency before further statistical analysis. ANOVA was used to determine levels of statistical significance when three groups of data were compared, together with a post hoc Scheffe comparison; Student's t-tests were used for comparison of two groups of data.
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RESULTS |
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GABA immunocytochemistry
To detect the presence of GABAergic neurons in our hypothalamic
cultures, immunocytochemically labeled cells were examined. After 5 DIV, a time when many of our recordings described in the following text
were done, GABA immunoreactive cells and processes were common. Long,
thin immunoreactive axons extended from immunoreactive cells bodies and
boutons made contact with both GABA immunoreactive (Fig.
1A) and nonimmunoreactive
neurons. Astrocytes showed no GABA immunoreactivity, and a number of
cells with the appearance of neurons also lacked GABA immunostaining.
Cells and processes showing no GABA immunoreactivity were detected
using green fluorescent protein as a marker for cells in general (Fig.
1B), as described previously (Gao and van den Pol
2000). Approximately 35-40% of the neurons showed GABA
immunoreactivity after 5 DIV. Whether additional developing neurons in
culture synthesized GABA, but at levels too low to be detected with
immunostaining, remains to be determined. Control experiments with
preabsorption of the antiserum with GABA conjugated to a protein
carrier, or omission of the primary antiserum, resulted in an absence
of immunolabeling. These results with mouse cultures are parallel to
similar experiments done with GABA immunostaining of rat neurons
(van den Pol 1997
).
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GABA, not glutamate, drives action potentials in developing hypothalamic neurons
Initial experiments were done with conventional whole cell
recording in which the pipette solution contained 29 mM
[Cl]i to mimic the
intracellular chloride concentration previously reported in developing
rat hypothalamic neurons of similar age (Chen et al.
1996
). The resting membrane potential ranged from
31.6 to
57.5 mV with an average of
46.1 ± 2.0 mV (n = 12). During the application of the GABAA receptor
antagonist bicuculline (30 µM; BIC), the frequency of action
potentials decreased from 20 ± 6 to 2 ± 1/min (90%
reduction) and returned to 15 ± 6/min after BIC washout.
As the responses to GABA are dictated to a large degree by
intracellular Cl, to leave intracellular
Cl
undisturbed by the pipette solution, action
potentials were recorded with gramicidin-perforated whole cell
recording (Chen et al. 1996
; Gao et al.
1998
) under current clamp in 4-7 DIV hypothalamic neurons. Neurons had an average resting membrane potential of
44.6 ± 1.69 mV (range
38.5 to
52.8 mV, n = 9). Spike
frequency was based on a 1-min sample at the end of receptor antagonist
application or after washout. After a 10-min recording of the control
baseline, BIC (30 µM) was applied through a flow pipe. The frequency
of action potentials decreased from a control level of 13 ± 2 to 0.3 ± 0.2/min and returned to 18 ± 10/min after BIC
washout, indicating a decrease of 97.5 ± 1.5% in the frequency
of action potentials (n = 9) when
GABAA receptors were blocked (Fig.
2A). Combining the results of
gramicidin recordings with the conventional whole cell recordings, as
each gave similar results, BIC decreased the frequency of action
potentials by 93.1 ± 2.0% (n = 21), a very significant and substantial decrease (P < 0.01).
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Gramicidin-perforated patch recordings were also made during the application of glutamate receptor antagonists CNQX and AP5 to hypothalamic neurons. The frequency of action potentials was decreased from 17 ± 3 to 10 ± 3/min and recovered after antagonist washout to 15 ± 4/min (Fig. 2A). Statistical analysis suggested that the decrease in the frequency of action potentials (34.6 ± 16.15%, n = 15) was significant (P < 0.01). The difference between the decrease caused by GABA and glutamate receptor antagonists was also significant (P < 0.01, ANOVA test). Thus these data are consistent with the concept that both GABA and glutamate contribute to spike generation during this developmental time period but that GABA played a greater role in triggering action potentials than glutamate did.
In older hypothalamic neurons (>20 DIV), gramicidin-perforated recordings were also performed (Fig. 2B). The frequency of action potentials dramatically decreased from 41 ± 26 to 5 ± 3/min and returned to 49 ± 18/min in the presence of CNQX and AP5, as expected. This decrease in the frequency of action potentials (75.4 ± 15.6%, n = 5; the percentage used here and elsewhere is based on the mean of percentages from each neuron, not on the spike frequency absolute value) was significant (P < 0.05). In contrast, the frequency of action potentials was not depressed but enhanced by treatment with BIC in the same neurons. Mean frequency increased from 40 ± 23 to 56 ± 26/min in BIC and returned to 47 ± 26/min after removal of BIC (P < 0.05; ANOVA). The difference between changes in the frequency of action potentials induced by block of GABA or glutamate receptors was significant (P < 0.01). Thus our data demonstrate that in mature hypothalamic neurons (>20 DIV) action potentials were mediated by glutamate receptors and GABA played an inhibitory role in spike generation, as expected.
GABA excitation is independent of glutamate receptors in hypothalamic neurons
Our data suggested that the generation of action potentials was
dependent on the activation of GABA receptors during development of
hypothalamic neurons. There are two possible mechanisms that may
contribute to this phenomenon. The first one may be due primarily to
inward current as Cl exits the neurons after
GABA stimulation, leading to the initiation of action potentials if the
depolarization induced by GABA receptor activation reaches the
threshold for action potentials (Gao et al. 1998
). The
second possibility is that the depolarization induced by GABA receptor
activation removes the voltage-dependent Mg2+
blockade of NMDA receptors and leads to NMDA receptor-dependent action
potentials, as reported in hippocampal neurons (Leinekugel et
al. 1997
). In this scenario, GABA acts synergistically with glutamate to evoke spikes. Since GABA and glutamate receptor
antagonists together block all synaptic currents in hypothalamic
cultures and slices from P1 mice, the direct synaptic contribution of
other neurotransmitters in the absence of GABA and glutamate is
probably negligible. We tested the effect of the NMDA receptor
antagonist AP5 on the frequency of action potentials in 4-7 DIV
hypothalamic neurons.
After 10 min recording of baseline spike activity, the NMDA
receptor antagonist AP5 was applied to the recording chamber. The
frequency of action potentials was not altered, changing from 20 ± 14 to 19 ± 13 spikes/min in the presence of AP5 and back to
22 ± 17/min after washout of AP5 (n = 6; Fig.
3). In contrast to the minor effect of
AP5, as indicated in the preceding text, BIC caused a substantial
decrease in the frequency of action potentials from 17 ± 3 to
1 ± 0.7/min (n = 21). These data suggest that
most action potentials in hypothalamic cells were dependent solely on
GABAergic transmission and were independent of the NMDA receptor activation found in hippocampal neurons (Leinekugel et al.
1997; Staley and Mody 1992
).
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GABA reversal potential is positive to spike threshold
Previous data suggested that GABA-mediated depolarization was
based on a relatively high intracellular chloride concentration in the
early period of development; when Cl channels
were opened by GABA, Cl
exited the cell
(Chen et al. 1996
). GABA-mediated depolarization could
be inhibitory if it acted to shunt current evoked by another excitatory
transmitter or it would be excitatory if it could trigger action
potentials. A critical mechanistic question is whether the
depolarization induced by GABA would reach or exceed the threshold for
the generation of an action potential.
The threshold for the generation of action potentials was measured in
42 hypothalamic neurons (4-7 DIV) recorded with gramicidin perforated
patches and ranged from 48 to
21 mV with an average of
34 ± 1 mV. The reversal potential of GABA was determined by applying GABA
(10 µM) and recording inward or outward currents at different holding
potentials. EGABA was
31 ± 5 mV
(n = 6) in 4-7 DIV hypothalamic neurons. With an
extracellular Cl
level of 157 mM, based on the
Nernst equation, the estimated concentration of
Cl
in the cytoplasm of developing mouse
hypothalamic neurons is 45 mM, substantially greater than the 8 mM
Cl
reported in mature neurons of rat
(Chen et al. 1996
). Thus the mean GABA reversal
potential was positive to the threshold for spike generation.
To demonstrate that GABA, even at relatively low concentrations, can
evoke action potentials under conditions when intracellular Cl is unperturbed, we used
gramicidin-perforated whole cell recording. Several concentrations of
GABA were applied to the recorded neuron (n = 21)
during current clamp. As shown in Fig.
4A, the application of GABA
induced action potentials even at very low concentrations, including 2, 5, and 10 µM. After the first action potential, there was a plateau
of depolarization whose period was dependent on the length and
concentration of GABA application. With 10 µM GABA, spikes were found
at both the beginning and end of GABA stimulation; during the phase
when the membrane potential recovered and returned to a more negative
resting membrane potential, firing of one spike or trains of action
potentials were observed (Fig. 4A). One micromolar GABA
evoked a subthreshold depolarization but no action potential.
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In another set of experiments, GABA was applied to the recorded neuron from a micropipette by a brief pressure pulse at a frequency of 1 Hz (Fig. 4D). Each GABA application evoked an action potential. Thus our data here demonstrate that GABA-mediated depolarization could induce action potentials that were phase locked to GABA presence.
To compare the relative ability of GABA and glutamate to evoke action potentials, we used a pair of flow pipes to deliver equimolar concentrations of GABA or glutamate (2 µ or 10 µM each). Although the different GABA and glutamate ionotropic receptors may have different affinities for their respective ligands, similar concentrations of transmitters were used on the working assumption that roughly equivalent amounts may be released, and to test the relative sensitivities to GABA and glutamate in developing hypothalamic neurons. In young cells, GABA evoked action potentials at these relatively low concentrations in all cells (n = 10) tested with 2 µM (n = 5) and 10 µM (n = 5). In contrast, at 2 µM, glutamate had little effect on membrane potential and did not evoke spikes (Fig. 4B). At 10 µM, glutamate depolarized all five neurons but evoked a spike in only one neuron (Fig. 4C). In all cases, glutamate evoked a depolarization of smaller amplitude than that evoked by the same concentration of GABA. These data with evoked responses are consistent with our earlier data showing that synaptic release of GABA was similarly more powerful than glutamate in evoking action potentials.
To examine the ionic mechanism of GABA-evoked spikes,
Na+ and Ca2+ channel
blockers were used. Cd2+ (100 µM), a
wide-spectrum Ca2+ channel blocker, did not block
GABA evoked spikes. In contrast, tetrodotoxin (1 µM) completely
blocked GABA evoked action potentials (n = 6; not
shown). In the presence of TTX, GABA still evoked a depolarization due
to Cl efflux, but no spike. These data suggest
that GABA evokes a voltage-dependent Na+ spike,
and that a Ca2+ component is not critical.
GABA drives action potentials in spinal cord neurons
In early development, depolarizing actions of GABA have been
reported in spinal cord neurons (Reichling et al. 1994).
In 4-7 DIV spinal cord neurons, gramicidin-perforated whole cell
recording was performed under current clamp to test the relative
contribution of GABA and glutamate in the induction of action
potentials (Fig. 5). Similar to what was
found in young hypothalamic neurons, the application of BIC depressed
the generation of action potentials in developing spinal cord neurons.
The frequency of action potentials was decreased from 48 ± 27 to
3 ± 1/min in the presence of BIC (statistically significant,
P < 0.05) and reversed to 37 ± 26/min after
washout of BIC. The decrement in the frequency (71.1 ± 12.3%, n = 5 neurons) was very significant (P < 0.01, ANOVA). Application of CNQX and AP5 also caused a reversible
reduction in the frequency of action potentials from 42 ± 24 to
23 ± 11/min (n = 4; P > 0.05, ANOVA test). BIC caused a substantially greater reduction in spike frequency than AP5/CNQX (P < 0.05), suggesting that
GABA contributed more in the induction of action potentials than
glutamate did in young spinal cord neurons, similar to our findings in
hypothalamic neurons.
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Electrophysiology in brain slices
The experiments in the preceding text used cultured neurons to
show the critical role GABA plays in generating spikes during neuronal
development under conditions of random synaptogenesis. To test the
hypothesis that synaptic GABA activity plays the same critical role in
brain slices, we used whole cell recording and extracellular recording
in acute slices. Whole cell recordings were made from mediobasal
hypothalamic slices with pipettes containing 29 mM
Cl, a conservative estimate of intracellular
Cl
in developing hypothalamic neurons of this
age. The baseline frequency of spikes was greater in slices than in
cultures. BIC caused a 73.4 ± 15.1% reduction in the frequency
of these action potentials, dropping from 36 ± 8 to 13 ± 8/min in the presence of BIC, a statistically significant decrease
(n = 5, P < 0.05).
Extracellular recording is useful in that it does not disturb intracellular ion concentrations, critical for the assessment of GABA's excitatory actions. An extracellular recording pipette was placed in the arcuate nucleus area of hypothalamic slices and controlled through a motorized micromanipulator. When spike firing was detected, the movement of the recording pipette was stopped. Two developmental ages of hypothalamic slices were studied. One set of slices (P1 group) was obtained from postnatal day 1 to postnatal day 4 mice and the other from P10 mice, which represent different periods of neuronal development. To determine the spike frequency, a 10-min control period was recorded before treatment. The antagonists of GABA and glutamate receptors were given for 10 min, and the spike frequency within the last minute was used as the result of treatment. After 10 min of washout, the last minute of recorded spike frequency was used as the recovery period. In most cases, only a single cell was recorded in each slice. In hypothalamic slices from P1 mice, the frequency of spikes ranged from 0.3 to 13 Hz, with a mean of 2.4 ± 1.4 Hz (n = 12). The amplitude of recorded spikes ranged from 117 to 650 µV with a mean of 294 ± 50 µV. After a 10-min recording of the baseline, BIC (30 µM) was bath-applied to the slices. The frequency of spikes was dramatically decreased from 2.4 ± 1.4 to 0.3 ± 0.1 Hz with the minimal decrease from baseline of 1.3 to 0.9 Hz and the maximal decrease was from a pre-BIC baseline of 13.4 to 0 Hz after BIC treatment (Fig. 6A). In the presence of BIC, the mean percent spike frequency decrease per neuron was significantly decreased to only 27.9 ± 9.8% of the control (P < 0.05, n = 9, range from 0 to 73.6% of control; Fig. 6C). In contrast, when the glutamate receptor antagonists CNQX (10 µM) and AP5 (100 µM) were applied to hypothalamic slices, the frequency of spikes showed only a modest decrease, from 2.4 ± 1.1 to 1.7 ± 0.7 Hz with 88.6 ± 24.2% of control spikes left (n = 7, P > 0.05; Fig. 6, A and C).
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Parallel experiments were used to examine older slices from P10 mice.
The effects of GABA and glutamate receptor antagonists on spike firing
frequency were examined (Fig. 6B). In the presence of
glutamate receptor antagonists CNQX and AP5, the spike frequency was
depressed by 37.3 ± 13.4% (n = 4;
P < 0.05; Fig. 6D). In contrast, spike
frequency showed no decrease (99.0 ± 18.7% of control) in the
presence of GABA receptor antagonists (P > 0.5, n = 3; Fig. 6D). We have previously shown
that addition of BIC to hypothalamic neurons older than 10 days evoked
a strong increase in spike frequency (van den Pol et al.
1998).
Thus the data from hypothalamic slices are consistent with the data from cultures, suggesting that GABA contributed more to spike firing than glutamate in young (P1-P4) brain slices and that this pattern was reversed by postnatal day 10 at which time glutamate served as the primary driving force behind spikes.
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DISCUSSION |
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We find that in developing brain slices and cultures most action potentials are due not to transmitter-independent spikes nor to glutamate activated excitation but rather are due to GABA release from developing axons. The GABAA receptor antagonist bicuculline blocks most impulse activity. In contrast, blocking ionotropic glutamate receptors had considerably less effect on spike frequency in early development. We found similar actions of GABA in hypothalamus and spinal cord neurons. In later development, glutamate assumed its well-known role as the primary excitatory transmitter, and GABA took on its inhibitory role in reducing spike generation.
The mechanism for the GABA-mediated spikes appears to be due to the
synaptically mediated depolarizing actions of
Cl efflux from the cell, activating
voltage-gated Na+ channels. If sufficient GABA is
released by local axon terminals to summate enough to drive the
depolarization to spike threshold, an action potential occurs. If a low
level of GABA is constantly present, this would not be sufficient to
drive spikes and may instead serve a shunting function. GABA-mediated
depolarization might lead to shunting inhibition as well characterized
by Staley and Mody (1995)
. The core of this shunt
concept was that the reversal potential for GABA was positive to the
resting membrane potential but negative to the spike threshold
potential and that GABA transmission occurred simultaneously with
glutamate transmission. In our study, the reversal potential of
GABA-mediated responses (EGABA) was measured with
undisturbed intracellular chloride concentration by using
gramicidin-perforated whole cell recording. EGABA
was positive to the threshold for action potential in developing
hypothalamic neurons, allowing GABA to depolarize the membrane
potential to the point that an action potential was generated as shown
with both synaptic release and flow pipe administration of GABA.
Previous reports have shown that bicarbonate ion can also pass through the GABA-gated anion channel (Kaila et al. 1993
). We
found that GABA-mediated activation of spikes has no bicarbonate
dependence and occurs even in HEPES buffer in the relative absence of bicarbonate.
Intracellular Cl was about four to five times
greater in developing than in mature neurons, suggesting an active pump
or transporter is moving Cl
into the developing
cell, perhaps a cation/Cl
cotransporter moving
Cl
inward, or that a Cl
transporter that is outward in mature neurons may have a reversed polarity in developing neurons. In addition, many outward
Cl
transporters are relatively inactive during
early development (Clayton et al. 1998
; Lu et al.
1999
; Luhmann and Prince 1991
; Rivera et
al. 1999
; Staley et al. 1996
). One mechanism
that may explain GABA's stronger role than glutamate in development is that GABA receptors develop earlier than glutamate receptors in the
hypothalamus (Chen et al. 1995
; van den Pol et
al. 1995
) and spinal cord (Walton et al. 1993
),
and, as we demonstrate here, GABA application can evoke spikes at a
developmental stage when an equimolar glutamate concentration does not
even depolarize neurons. GABA depolarizing actions begin early in
development as GABA is released from advancing axon growth cones even
before synaptic contact is established (Gao and van den Pol
2000
). That GABA evolves from an excitatory to inhibitory
transmitter during neuronal development in cultures, as it does in the
brain, suggests that the negative shift in ECl
may either be an intrinsic property of developing neurons or that the
necessary factors generating the developmental shift are available in vitro.
In a number of seminal papers focusing on hippocampal neurons in vitro
and in slices, GABA was shown to play a critical role in generating
giant depolarizing potentials (Ben-Ari et al. 1989; Cherubini et al. 1990
, 1991
; Leinekugel et al.
1997
). This is based on a mechanism where GABA relieved the
NMDA receptor of its Mg2+ block, resulting in
glutamate- mediated giant depolarizing potentials. Blockade of the NMDA
receptor eliminated the ability of GABA to elicit giant depolarizing
potentials (Leinekugel et al. 1997
). Thus GABA and
glutamate have equivalent and synergistic roles during this stage of
hippocampal development. Our preliminary results with hippocampal cells
are consistent with these previous published observations. But only in
hippocampal cells did glutamate play a substantial role in spike
generation in developing cells; this was not true in neurons of the two
CNS regions examined in the present study, hypothalamus and spinal
cord. In hypothalamic neurons in vitro and in slices, the blockade of
glutamate transmission with CNQX and AP5 did not depress the majority
of the action potentials, and by itself the NMDA receptor antagonist
AP5 had little effect on the frequency of action potentials. Together
these results suggest that hippocampal cells in this context may not
necessarily be representative of other brain regions. One mechanism
that may account for this difference is the high level of expression of glutamate receptors, particularly the NMDA receptor, in the developing hippocampus (Tremblay et al. 1988
). Another factor that
may account for the strong role of GABA in the hypothalamic neurons
compared with the hippocampal pyramidal neurons is that
hypothalamic neurons are smaller and have smaller dendritic arbors and
a higher input resistance (van den Pol et al. 1990
),
which would increase the probability that low levels of GABA would
evoke spikes. Thus GABA plays a bigger role than glutamate in driving
long distance signaling via action potentials in early hypothalamic development.
Another critical factor is the GABA reversal potential. In developing
hippocampal cells, EGABA was suggested to be
about 51 mV (Cherubini et al. 1990
), whereas in
hypothalamic neurons of similar age in the present study
EGABA was
31 mV. GABA would generate greater
depolarizing actions in the presence of the more positive
EGABA.
A number of interesting studies have demonstrated that the excitatory
actions of glutamate can play a profound role in modulating the
formation of connections in the developing brain (Hofer and Constantine-Paton 1994). Enhancement of synaptic stabilization through Hebbian mechanisms has focused on glutamatergic synapses, and
the role of GABA has been relegated to some degree to the modulation of
the glutamate synapse (Ben-Ari et al. 1997
). In hippocampal neurons, GABA-mediated long-term depression was dependent on both GABA and NMDA actions (Caillard et al. 1999
).
The concept of a Hebbian synapse has been applied primarily to
glutamate synapses, leaving the other half of the synapses in the
brain, the GABA synapses, as a mechanistic mystery in terms of
activity-dependent synapse stabilization. As our data indicate that
axonal release of GABA routinely evokes spikes in neurons from some
regions of the developing brain, perhaps the concept of a Hebbian
synapse can apply to GABA synapses independent of glutamate activity.
In conclusion GABA appears to play a more powerful role than glutamate in generating spikes during early development in some brain regions. Thus many aspects of neuronal development in these regions that can be modulated by activity including synaptic stabilization, axonal pathfinding or pruning, growth cone orientation, and elaboration of functional efficacious circuitry may be regulated by GABA in early neuronal development, and by glutamate later in development.
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ACKNOWLEDGMENTS |
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Grant support was provided by National Institute of Neurological Disorders and Stroke Grants NS-10174, NS-31573, and NS-34887 and by the National Science Foundation.
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FOOTNOTES |
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Address for reprint requests: A. N. van den Pol, Dept. of Neurosurgery, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520 (E-mail: Anthony.vandenpol{at}Yale.Edu).
Received 7 July 2000; accepted in final form 12 September 2000.
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REFERENCES |
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