GABA Itself Promotes the Developmental Switch of Neuronal GABAergic Responses from Excitation to Inhibition
GABA is the main inhibitory neurotransmitter in the adult brain. Early in development, however, GABAergic synaptic transmission is excitatory and can exert widespread trophic effects. During the postnatal period, GABAergic responses undergo a switch from being excitatory to inhibitory. Here, we show that the switch is delayed by chronic blockade of GABA(A) receptors, and accelerated by increased GABA(A) receptor activation. In contrast, blockade of glutamatergic transmission or action potentials has no effect. Furthermore, GABAergic activity modulated the mRNA levels of KCC2, a K(+)-Cl(-) cotransporter whose expression correlates with the switch. Finally, we report that GABA can alter the properties of depolarization-induced Ca(2+) influx. Thus, GABA acts as a self-limiting trophic factor during neural development.
Cell, Vol. 105, 521–532, May 18, 2001, Copyright 2001 by Cell Press
GABA Itself Promotes the Developmental Switch
of Neuronal GABAergic Responses
from Excitation to Inhibition
voltage at which GABAergic currents change their direc-
tion). Recent work has shown that the developmental
transformation of GABAergic synaptic transmission
from depolarizing to hyperpolarizing is due to a shift in
toward a more hyperpolarized potential, which is
Alejandro F. Schinder,
Scott T. Wong,
and Mu-ming Poo
Program in Neuroscience
Section of Neurobiology, Division of Biology
University of California, San Diego
likely the result of an ontogenetic decrease in the intra-
La Jolla, California 92093
; Cherubini et al., 1990;
Luhmann and Prince, 1991; Chen et al., 1996; Owens et
al., 1996). Indeed, changes in the mRNA level for the
transporter KCC2 have been shown to
correlate with the modification of GABAergic transmis-
GABA is the main inhibitory neurotransmitter in the
sion (Lu et al., 1999; Rivera et al., 1999; Vu et al., 2000).
adult brain. Early in development, however, GABAergic
KCC2 increases the rate of Cl
extrusion, thus leading
synaptic transmission is excitatory and can exert
to a reduction in [Cl
and a consequent shift in E
widespread trophic effects. During the postnatal pe-
toward more hyperpolarized potentials (Jarolimek et al.,
riod, GABAergic responses undergo a switch from be-
1999; Kakazu et al., 1999; Rivera et al., 1999).
ing excitatory to inhibitory. Here, we show that the
This conversion of GABAergic transmission from de-
switch is delayed by chronic blockade of GABA
polarizing to hyperpolarizing is also accompanied by a
tors, and accelerated by increased GABA
change in GABA-mediated biochemical signaling. Only
activation. In contrast, blockade of glutamatergic
during this early developmental period, depolarizing
transmission or action potentials has no effect. Fur-
GABAergic potentials activate voltage-dependent Ca
thermore, GABAergic activity modulated the mRNA
channels (VDCCs) and elevate [Ca
(Connor et al.,
levels of KCC2, a K
cotransporter whose expres-
1987; Yuste and Katz, 1991; Wang et al., 1994). Such
sion correlates with the switch. Finally, we report that
GABA-induced elevation of [Ca
is likely to play a
GABA can alter the properties of depolarization-
critical role in the maturation of the nervous system.
influx. Thus, GABA acts as a self-limiting
For instance, GABA-mediated increases in [Ca
trophic factor during neural development.
induce BDNF expression (Berninger et al., 1995) and
promote neuronal survival and differentiation (LoTurco
In the adult central nervous system, ␥-amino-butyric
et al., 1995; Marty et al., 1996; Ikeda et al., 1997). GABA-
acid (GABA) is the primary inhibitory neurotransmitter.
induced elevation of [Ca
may also be required to
It regulates a neuron’s ability to fire action potentials
form, stabilize, and strengthen synaptic connections
either through hyperpolarization of the membrane po-
(Kirsch and Betz, 1998; Caillard et al., 1999; Kneussel
tential or through shunting of excitatory inputs. During
and Betz, 2000).
early postnatal development, however, GABAergic syn-
While the developmental transformation of GABAer-
aptic transmission is excitatory, able to elevate the intra-
gic transmission is well documented, little is known
), and even capable
about signals that induce this transformation. Since neu-
of triggering action potentials (Mueller et al., 1984; Luh-
ronal activity is known to increase during development,
mann and Prince, 1991; Yuste and Katz, 1991; Reichling
we examined in the present study whether synaptic ac-
et al., 1994; Wang et al., 1994; Leinekugel et al., 1995;
tivity can regulate the switch of GABAergic transmis-
Obrietan and van den Pol, 1995; Chen et al., 1996; Owens
sion. We found that the change in GABA signaling was
et al., 1996; Khazipov et al., 1997). Over a limited postna-
largely prevented by chronic blockade of GABA
tal period, in the hippocampus, neocortex, and hypo-
tors, and was accelerated by increased GABA receptor
thalamus, as well as other regions of the brain, there is
activation. Changes in the level of KCC2 mRNA tightly
a switch of the electrophysiological (depolarization to
correlated with the observed changes in GABA signal-
hyperpolarization) and biochemical (Ca
ing. In addition, we found that spontaneous GABAergic
naling) properties of GABAergic transmission (Mueller
activity regulated the activation of voltage-dependent
et al., 1984; Ben-Ari et al., 1989; Cherubini et al., 1991;
currents. These findings point to GABA as a critical
Luhmann and Prince, 1991; Owens et al., 1996).
maturation factor for the switch of the physiological and
receptor channel predominantly conducts
biochemical properties of GABA signaling.
ions. Consequently, the nature of GABAergic trans-
mission, excitatory versus inhibitory, is determined pri-
marily by the electrochemical gradient for Cl
depends on the intra- and extracellular concentrations
Switch of GABAergic Transmission from Depolarizing
. This electrochemical gradient sets the reversal
potential for GABAergic currents (E
; the membrane
To study the change in GABA signaling, we first moni-
tored GABA-induced elevations of [Ca
ment. GABA-mediated depolarization was reflected by
These authors contributed equally to this work.
an increase in [Ca
. Cultures of hippocampal neurons
Present address: Division of Neurobiology, Department of Molecu-
were loaded with the Ca
-sensitive dye Fluo-4 AM and
lar and Cell Biology, University of California, Berkeley, Berkeley
changes in fluorescence were measured using confocal
Figure 1. Developmental Changes in GABA-Induced Responses
(A) Pharmacological profile of GABA-induced elevations of [Ca
(BMI ⫹ PTX, 10 and 50 M; nimodipine, 10 M; thapsigargin ⫹ BHQ, 2 and
10 M; and baclofen, 10 M). Time course of changes in [Ca
, assessed by Fluo-4 fluorescence intensity. Traces are average fluorescence
intensity (% change shown by the scale) recorded from 30–40 randomly sampled neurons in response to pulses of GABA (open bar, 10 M,
15 s). The elevation of [Ca
during application of thapsigargin ⫹ BHQ was attributed to emptying of intracellular Ca
(B) Time course of the GABA switch. The percentage of neurons exhibiting Ca
elevation in response to GABA is shown for different days
after cell plating. Each point represents mean ⫾ sem (n ⫽ 3 to 21 experiments, 121 in total). Each experiment involved recording from 30–40
neurons. Insets: Sample recordings from 20 randomly selected neurons at the time points indicated by the arrows.
(C) Representative recordings of Ca
imaging of young (7 day) and old (13 day) neurons. First panels show bright-field images. The fluorescence
images on the right represent typical [Ca
in neurons before (“baseline”), during (“GABA”), and after (“recovery”) application of a pulse of
GABA (higher intensity represents higher [Ca
). Image field ⫽ 604 ⫻ 604 m.
(D) Left: Representative examples of peak GABA-induced currents versus membrane voltages (I-V relationship). The cells were voltage clamped
at ⫺70 mV and stepped to different potentials (⫺90 to ⫹40 mV, 10 mV steps). E
was calculated by fitting the I-V curve to a second-order
polynomial function. Right: Averaged I-V curves (n ⫽ 11 in both cases).
GABA as a Self-Limiting Trophic Factor
Figure 2. Role of Endogenous GABAergic
Transmission in the Developmental Switch
(A) Time course of the switch in GABA signal-
ing for neurons treated chronically with BMI ⫹
PTX (10 and 50 M; n ⫽ 31, 1085 neurons),
KCl (10 mM; n ⫽ 26, 910 neurons), and KCl ⫹
BMI ⫹ PTX (n ⫽ 14, 490 neurons), compared
to control neurons (n ⫽ 121, 4235 neurons).
In all experiments involving KCl, TTX and
D-APV were included in the medium. Statisti-
cal comparisons were carried out for data
collected for day 8, 10, and 12 neurons.
BMI ⫹ PTX and KCl were significantly differ-
ent from the untreated control and KCl ⫹
BMI ⫹ PTX (p ⬍ 0.005, single-factor ANOVA
followed by a Bonferroni t test corrected for
multiple comparisons). Insets: I, Representa-
tive recordings of day 14 neurons grown in
the presence of BMI ⫹ PTX. II, Representative
results on day 10 neurons grown in the pres-
ence of KCl (10 mM).
(B) Comparison of the effects of chronic
blockade of GABA
and glutamate receptors
at day 13 neurons. Numbers of experiments
are indicated in parentheses. “**” denotes p ⬍
(C) Cumulative probability histograms of E
for day 7 and day 13 neurons (with or without
chronic treatment with BMI ⫹ PTX). Each
point represents E
measurement from one
cell. Data points with error bars below denote
mean ⫾ sem (n ⫽ 10 to 11). Data from both
control day 7 and BMI ⫹ PTX day 13 were
significantly different from the control day 13
(p ⬍ 0.001, Kolmogorov-Smirnov test).
microscopy. Neurons cultured for 4 to 9 days responded tures of hippocampal neurons provide a useful model
for studying the mechanism of the developmental switchto exogenous applications of GABA (100 M, 15 s pulse)
with a rapid and reversible increase in [Ca
. As shown in GABA signaling.
To directly assess the developmental shift in E
,in Figure 1A, these responses were largely blocked by a
combination of GABA
receptor antagonists (bicuculline whole-cell perforated patch recording was used to mea-
sure the reversal potential of currents elicited by localmethoiodide, “BMI”, 10 M; picrotoxin, “PTX”, 50 M)
or by the L-type Ca
channel antagonist nimodipine application of GABA (10 M, 50 ms pulse) in young (day
6–7) and mature (day 13–14) neurons. As shown in Figure(10 M), indicating that elevation of [Ca
is due to
influx through L-type Ca
channels activated by 1D, E
was significantly more hyperpolarized in ma-
ture neurons (⫺60.7 ⫾ 2.2 mV; mean ⫾ sem, n ⫽ 11)GABA
-receptor-mediated depolarization. In addition,
application of baclofen, a GABA
agonist, had no effect than in young neurons (⫺44.5 ⫾ 2.0 mV; n ⫽ 11, p ⬍
0.001), indicating that the developmental decrease ofon [Ca
. Furthermore, depleting intracellular Ca
stores with thapsigargin (2 M) and 2,5-di-t-butyl-1,4- E
contributes to the reduction of GABA-induced ele-
vations of [Ca
.benzohydroquinone (BHQ, 10 M) did not alter the
transients, indicating that the ele-
vation of [Ca
is a result of Ca
influx through VDCCs. GABA Itself Promotes the Developmental Switch
Based on the hypothesis that the transformation ofThe percentage of neurons that responded to GABA
with an elevation of [Ca
decreased with the age of GABA signaling depends on neuronal activity, we exam-
ined the effects of chronic blockade of ionotropic trans-the culture (Figures 1B and 1C). All neurons displayed
GABA-mediated depolarization at day 4 to 6, while most mitter receptors on the GABA-induced Ca
Interestingly, GABAergic, but not glutamatergic activityneurons exhibited no detectable response by day 13.
Regardless of whether a neuron responded to GABA, was essential for the developmental change (Figures
2A–2C). Chronic blockade of GABA
receptors with BMIdepolarization with a high concentration of extracellular
KCl (100 mM) always induced robust elevations of [Ca
(10 M) and PTX (50 M) prevented the transformation
in most neurons (note that similar effects were found(Figure 1C), suggesting that the lack of responsiveness
to GABA was due to a decrease in the extent of depolar- in the presence of BMI alone; Figure 6E). In contrast,
blockade of the N-methyl-D-aspartate (NMDA) sub-ization. These observations are consistent with previous
findings from brain slices and cultured neurons (Luh- type of glutamate receptors with D-APV (25 M), the
non-NMDA receptors with CNQX (15 M), or bothmann and Prince, 1991; Chen et al., 1996; Owens et al.,
1996; Wang et al., 1994), that GABA-induced elevations (CNQX⫹APV) did not affect the developmental time
course (Figure 2B). In addition, chronic activation of theof [Ca
are developmentally regulated and that cul-
Figure 3. Modulation of KCC2 mRNA Ex-
pression by GABAergic Activity
Levels of KCC2 mRNA assessed by the
RNase protection assay.
(A) Representative experiment displaying a
higher level of KCC2 mRNA in day 15 neurons
as compared to day 3 neurons.
(B) Representative experiment showing a de-
crease in KCC2 mRNA expression in day 15
neurons chronically treated with BMI ⫹ PTX,
as compared to untreated controls. The thin
band marked with “*” was due to residual
undigested ␤-actin probe, as shown by the
␤-actin probe lane alone.
(C) The expression of KCC2 mRNA was in-
creased by chronic depolarization with KCl
(10 mM), and decreased in day 9 neurons
following the BMI ⫹ PTX treatment. Replicate
samples of results are shown.
(D) Summary of all results on KCC2 mRNA
expression, assayed at day 3, 9, and 12–15.
For each condition, KCC2 mRNA levels were
normalized to that of ␤-actin and expressed
as fold induction over the band intensity ob-
served at day 3 in control neurons. “N” ⫽
number of experiments. Error bars ⫽ sem and
“**” denotes p ⬍ 0.005 (t test).
metabotropic glutamate receptor mGluR3 with L-AP4 slices (Rivera et al., 1999). Since GABAergic activity
drives this shift in E
, we examined whether the onto-did not modify the switch (d11; 0.49 ⫾ 0.07, n ⫽ 5, p ⫽
0.57, t test). Thus, the developmental switch of GABA- genetic increase in the levels of KCC2 mRNA is similarly
modulated. KCC2 mRNA levels were quantified in daymediated Ca
signaling is triggered by endogenous
GABAergic transmission. 3 and day 12–15 neurons using an RNase protection
assay and normalized to the levels of ␤-actin (Lee andThe maturational change in GABA-mediated Ca
naling may result directly from a shift in the reversal Costlow, 1987). Consistent with previous findings (Ri-
vera et al., 1999), we observed an ⵑ14-fold increase inpotential of GABAergic currents. We therefore examined
whether the ontogenetic modification of E
was also the level of KCC2 mRNA in mature neurons (Figures 3A
and 3D). At day 9 (during the onset of the switch), anregulated by GABAergic transmission. As shown in Fig-
ure 2C, chronic blockade of GABA
receptors prevented ⵑ6 fold increase in KCC2 expression was observed,
although only ⵑ20% of neurons failed to respond tothe shift of E
toward hyperpolarized potentials in day
13 neurons. E
remained at ⫺48.4 ⫾ 1.6 mV (n ⫽ 10), GABA at this time (Figure 1B). This suggests a nonlinear
relationship between the mRNA level of KCC2 and itsa value that was not significantly different from that of
young (day 7) neurons under control conditions (p ⫽ functional effects (see Discussion). Furthermore, we ex-
plored whether chronic blockade of GABAergic activity0.13, t test). These observations indicate that the devel-
opmental increase in GABAergic activity promotes the with BMI ⫹ PTX altered the level of KCC2 mRNA. As
shown in Figures 3B and 3D, KCC2 expression wastransformation of GABA signaling.
decreased by 68 ⫾ 4% (n ⫽ 5, p ⬍ 0.001) in comparison
to age-matched control cultures (n ⫽ 10). To elevate
GABA Activates the Expression
GABAergic activity, we chronically depolarized the cul-
of the Cl
tures with 10 mM KCl (see Figures 6C and 6D). We
The hyperpolarizing shift in E
has been correlated
with an increase in KCC2 expression in hippocampal found that KCC2 message levels in day 9 neurons were
GABA as a Self-Limiting Trophic Factor
Figure 4. Regulation of Ca
Channel Properties during Development
) Representative recordings of changes in [Ca
induced by depolarization with 6, 8, or 10 mM KCl in day 7 and day 13 neurons. (A
Summary of normalized changes in Ca
fluorescence induced by KCl depolarization in young (day 7; n ⫽ 240 neurons), old (day 13; n ⫽ 260)
untreated neurons, and old neurons chronically treated with BMI ⫹ PTX (n ⫽ 111). (“*” and “**” denote p ⬍ 0.05 and p ⬍ 0.001, respectively,
when compared to the control day 13 treatment, single-factor ANOVA followed by a t test).
(B) GABA receptor blockade prevents the developmental shift in the activation of VDCCs. (B
) Representative voltage-clamp recordings of
currents. Inward Ca
currents (downward deflections) were elicited by step depolarizations (holding potential ⫽⫺80 mV, step ⫽ 10
) Normalized peak current versus voltage for day 7 (n ⫽ 12), day 13 (n ⫽ 9), and day 13 BMI ⫹ PTX (n ⫽ 11) neurons. On average,
currents measured between ⫺60 and ⫺40 mV are 1- to 2-fold larger in younger neurons than in mature neurons (with p ⬍ 0.05 for ⫺50
and ⫺40 mV; n ⫽ 9 for 7 day, and n ⫽ 12 for 13 day). (B
) Mean I-V curves for absolute peak current values (same data as in B
). As expected,
error bars were larger in these nonnormalized curves and were omitted for clarity.
enhanced by 69 ⫾ 6% in comparison to control condi- 4A). These measurements of KCl-induced changes in
fluorescence intensity most accurately reflect depolar-tions (Figures 3C and 3D), and most neurons in the KCl-
treated day 9 cultures had lost their responsiveness to ization-induced changes in “free” [Ca
these results indicate that while a mild depolarizationGABA (Figure 2A). Taken together, these results support
the notion that the expression of KCC2 can be regulated can robustly increase the free [Ca
in young neurons,
moderate depolarization is required to appreciably in-by GABA-mediated depolarization.
crease the levels of [Ca
in mature neurons. We thus
conclude that the switch in GABA-induced Ca
signal-Developmental Regulation of Depolarization-
Induced Elevations of [Ca
ing involves two independent ontogenetic modifica-
tions: a hyperpolarizing shift in E
and a reduction inGABA-induced Ca
transients depend both on the ex-
tent of membrane depolarization as well as the proper- the depolarization-induced elevation of free [Ca
Changes in free [Ca
are likely to depend on twoties of voltage-dependent Ca
influx. We thus examined
whether depolarization-induced Ca
transients are modi- factors: (1) the activation profile of voltage-dependent
), and (2) the Ca
buffering propertiesfied during neuronal development. We measured changes
in fluorescence intensity in response to increasing depo- of a neuron. We conducted voltage-clamp recordings of
from young and mature neurons. Step depolarizationlarization (induced by KCl) in young (day 7) and mature
(day 13) neurons. While young cells displayed a robust evoked fast inward currents that displayed slow and
partial inactivation (Figure 4B
). For young neurons, in-increase in [Ca
at all levels of depolarization, mature
neurons responded only to strong depolarization (Figure ward currents activated typically between ⫺60 and ⫺50
Figure 5. Spiking Is Not Required for the De-
Chronic blockade of action potentials with
TTX (2 M) did not affect the time course of
the GABA signaling switch. I and II: Typical
recordings of Ca
levels in neurons chroni-
cally treated with TTX, at the indicated time
points. Inset: Membrane currents recorded
from a day 6 neuron before and after applica-
tion of 2 M TTX.
mV, reaching a peak at about ⫺10 mV. In contrast, ma- ence of tetrodotoxin (TTX, 2 M), which blocks Na
dependent action potentials in these neurons (Figure 5).ture neurons displayed a shift in the activation profile
toward more hyperpolarized potentials, suggesting that Interestingly, blocking Na
spikes did not affect the time
course of the transformation, suggesting that spontane-the kinetics of I
are developmentally regulated (Figure
). However, the absolute amplitudes of the whole- ous miniature depolarizing GABAergic synaptic currents
(mGSCs) are sufficient to drive the developmental switchcell currents for young and mature neurons at the hyper-
polarized potentials (⫺70 to ⫺50 mV) were similar (Fig- (see Figure 6B). Spontaneous mGSCs were first de-
tected (at a low frequency) at day 7 (Figure 6A), and theure 4B
). These results suggest that additional factors
underlie the observation that mild depolarization in- frequency increased steadily to reach a plateau of about
0.5 Hz between days 11 and 12. Remarkably, the timeduces large increases in free [Ca
only in young neu-
rons (Figure 4A). One possible factor is that the cell course in which spontaneous GABAergic activity arises
closely parallels that of the switch in GABA signalingsurface of mature neurons is typically ⵑ2-fold larger
than that of young neurons. Thus, the current density at (compare Figures 5 and 6A). It is noteworthy that al-
though substantial glutamatergic activity was also pres-hyperpolarized potentials is likely to be higher in young
cells. Developmental changes in the Ca
-buffering ent during this period (Figures 6A and 6Bb), this excit-
atory activity did not contribute to the developmentalproperties of neurons are also likely to contribute
(Schierle et al., 1997; Rosenstein et al., 1998; Boukhad- switch in GABA signaling (Figure 2B).
It has been reported that chronic blockade of GABA
daoui et al., 2000).
receptors may lead to increased activity even when
GABA is depolarizing (Lamsa et al., 2000). Thus, it isGABA Can Promote Developmental Changes
in Depolarization-Induced Ca
Transients possible that the effects of chronic blockade of GABA
receptors on the switch are the result of an increaseSince GABA itself promoted the shift in E
, we exam-
ined whether the properties of depolarization-induced in overall neuronal activity. To test this possibility, we
compared the effects of chronic blockade of GABA
transients are similarly regulated. As shown in Fig-
, chronic blockade of GABAergic transmission receptors alone (BMI) with those observed in the pres-
ence of BMI ⫹ TTX or BMI ⫹ CNQX in day 13 neuronsmarkedly reduced this developmental modification. In
mature neurons chronically treated with BMI ⫹ PTX (day (Figure 6E). As no significant differences were found
under these conditions (p ⬎ 0.2, ANOVA), the effects13–14), KCl triggered a robust elevation in the free [Ca
that was significantly larger than those in the untreated of chronic BMI treatment were not a consequence of
increased neuronal activity.parallel cultures. Although the ontogenetic change in
the activation profile for I
was reduced, the absolute The notion that spontaneous GABAergic synaptic ac-
tivity drives the developmental switch was further testedlevels of calcium influx were similar to that seen under
control conditions (Figures 4B
). Taken together, these with KCl-induced depolarization (in the presence of
TTX), which enhances the probability of transmitter re-findings suggest that GABA promotes the develop-
mental decrease in depolarization-induced Ca
tran- lease, thus increasing the frequency of mGSCs. As
shown in Figures 6C and 6D, KCl (10 mM) induced asients, but this decrease is not fully accounted for by
changes in voltage-dependent Ca
influx. rapid increase in the mGSC frequency (190 ⫾ 28%, n ⫽
4, p ⬍ 0.05). Furthermore, chronic treatment with KCl
(10 mM) markedly accelerated the time course of theThe Developmental Switch Does Not Require
Neuronal Spiking transformation of GABA signaling, as more than 90%
of all neurons failed to exhibit GABA-induced [Ca
To test whether spiking is required for the switch of
GABAergic signaling, neurons were cultured in the pres- elevation by day 10 (Figure 2A). We note that these same
GABA as a Self-Limiting Trophic Factor
Figure 6. Developmental Changes in Spontaneous Activity
(A) Developmental increase in the frequency of spontaneous GABAergic and glutamatergic currents, which were recorded in the presence of
TTX ⫹ CNQX/APV and TTX ⫹ BMI, respectively. Representative traces of mGSCs are shown (I and II). Scales: 10 pA, 0.5 s.
(Ba) Spontaneous depolarizing GABAergic potentials recorded in current clamp using gramicidin perforated patch in the presence of CNQX.
Resting potentials were approximately ⫺60 to ⫺70 mV. Scales: 10 mV, 0.3 s. (Bb) Voltage clamp recordings (⫺70 mV) of spontaneous
glutamatergic activity from cultures chronically treated with BMI ⫹ PTX. Scales: 100 pA, 0.3 s.
(C) An example of acute effects of 10 mM KCl on the frequency and amplitude of mGSCs. Each point represents a single mGSC.
(D) Changes in mGSC frequency after KCl treatment (the same experiment as shown in [C]). Inset: average mGSC frequency before and after
KCl treatment (n ⫽ 4). (p ⬍ 0.05, paired t test).
(E) Summary of experiments involving chronic blockade of GABAergic activity only (BMI), GABAergic activity and spiking (BMI ⫹ TTX), and
GABAergic and glutamatergic activities (BMI ⫹ CNQX).
(F) Comparison of chronic activation of GABA
receptors by muscimol (10–50 M; n ⫽ 8) versus control conditions (n ⫽ 21). “*” denotes p ⬍
0.05 (t test).
neurons did respond to acute depolarization with KCl Requirement for GABA-Mediated Ca
Early in development, GABA-induced depolarizationwith a Ca
transient. Moreover, the acceleration of the
switch by KCl was reversed in the presence of GABA
influx through voltage-dependent Ca
channels. Electrophysiological recordings from individ-receptor antagonists (KCl ⫹ BMI ⫹ PTX treatment in
Figure 2A). However, this time course was faster than ual neurons showed that I
was blocked by ⵑ50% by
the L-type Ca
channel antagonist nimodipine (10 M)the one observed for neurons treated only with BMI ⫹
PTX, suggesting that, in addition to increasing the mGSC and ⵑ90% by 10 M CdCl
(commonly used as a high-
channel blocker; Figure 7A). As chronicfrequency, KCl had additional effects (see Discussion).
Taken together, these results indicate that the frequency blockade of all I
was found to be deleterious for neu-
ronal survival, a partial block of I
was performed usingof mGSCs can determine the kinetics of the develop-
mental switch in GABA signaling. Consistent with this moderate concentrations of nimodipine (0.5–1 M). As
shown in Figure 7B, chronic treatment with nimodipinenotion, chronic activation of GABA
receptors with mus-
cimol (10–50 M) produced a significant acceleration of significantly delayed the developmental switch. To as-
sess whether this observation reflected a true shift inthe switch in day 9 neurons (Figure 6F).
Figure 7. Role of Ca
Influx for the Develop-
mental Switch of GABA Function
(A) Blockade of I
by nimodipine and by CdCl
(n ⫽ 6). Inset: Representative traces of Ca
currents elicited by depolarizing pulses in a
day 7 neuron, displaying the effects of nimo-
dipine (10 M) and CdCl
(B) Effects of chronic blockade of L-type
VDCCs on GABA-induced Ca
cultures of different ages. (“*” and “**” denote
p ⬍ 0.05 and p ⬍ 0.001, respectively, t test).
(C) Effects of chronic blockade of L-type
VDCCs on the shift in E
. Left: Representa-
tive example of a GABA-induced I-V relation-
ship. Cells were voltage clamped at ⫺70 mV
and stepped to different potentials (⫺70 to 0
mV, 10 mV steps). Right: Averaged I-V curve
(n ⫽ 9). Note that E
at day 11 in the pres-
ence of nimodipine is similar to that at day 7
in untreated cultures (Figure 1).
, we measured E
in cultures chronically treated tion (Figure 8). This suggests the notion that GABA-
mediated excitation is self-limiting.
with nimodipine (Figure 7C). We found that the shift in
was prevented at a time in which the effect of
nimodipine on the GABA-induced elevations of [Ca
The Role of GABAergic Activity and Depolarization
was most significant (day 11 neurons). Under these con-
We propose that the level of depolarizing GABAergic
was ⫺42.5 ⫾ 4.3 mV (n ⫽ 9), similar to that
activity determines the rate of transformation of neu-
observed in untreated neurons at day 7 (⫺44.5 ⫾ 2.0
ronal GABAergic responses. This is supported by the
mV; n ⫽ 11; p ⬎ 0.2). These findings support a model
following three lines of evidence: (1) Blockade of GABA
in which spontaneous GABAergic events elevate [Ca
receptors prevented the transformation; (2) elevating
and activate signaling pathways which can promote the
GABAergic activity or stimulating GABA
celerated it; and (3) the developmental increase in spon-
taneous GABA release tightly correlated with the time
course of the transformation. To elevate synaptic activity,
we chronically depolarized the neurons with KCl (10 mM)
in the presence of TTX. That KCl-depolarization had
The switch of GABA signaling from excitation to inhibi-
indeed elevated GABAergic activity was shown by (1)
tion occurs during early postnatal brain development,
the frequency of mGSCs was increased, and (2) the
when synaptic connections are being established,
accelerated rate of transformation was significantly re-
strengthened, and refined (Katz and Shatz, 1996). Using
duced when the KCl treatment was combined with block-
a model system of cultured hippocampal neurons, we
ade of GABA
receptors. However, the transformation
tested the hypothesis that this switch is regulated by
still occurred under conditions of KCl-induced depolar-
neuronal activity. We first characterized two aspects of
ization in the presence of GABA
GABAergic transmission—the electrophysiological (mem-
As evident from Figure 4A, 10 mM KCl acutely induces
brane depolarization) and the biochemical (Ca
a very robust elevation of [Ca
in immature neurons.
nals)—which may serve different roles in the developing
Therefore, it is likely that chronic depolarization with 10
nervous system. We found that developmental changes
mM KCl induced a massive Ca
influx that bypassed the
in these two properties are related to a shift in E
required specificity for GABA-mediated depolarization.
toward hyperpolarized potentials as well as a change in
This notion is supported by the fact that physiological
depolarization-induced elevations of [Ca
. Our results
sources of depolarization (namely glutamatergic activity
also indicate that both of these changes can be triggered
and action potentials), although present at substantial
by GABAergic activity. The expression of the Cl
levels, are unable to regulate the time course of the
porter KCC2 was upregulated by increased spontane-
developmental switch (Figures 2B, 6A, and 6B).
ous GABAergic activity, and downregulated following
Interestingly, in spinal cord neurons, the switch of
blockade of GABA
receptors, indicating that GABA-
GABA signaling is accelerated when neurons are plated
mediated depolarization is itself able to increase the
on a higher density of astrocytes (Li et al., 1998). The
mechanism underlying this process remains unknown.expression of KCC2 and induce the maturation of inhibi-
GABA as a Self-Limiting Trophic Factor
Figure 8. A Model for the Developmental Switch of GABAergic Transmission
During embryonic development, GABA acts as an excitatory neurotransmitter. GABA-mediated excitation can trigger Ca
VDCCs, leading to an increase in the expression of the potassium-chloride cotransporter KCC2. This excitatory GABAergic activity can also
regulate the activation profile of VDCCs. During early postnatal development, the increase in KCC2 activity lowers [Cl
and reduces E
establishing GABA as an inhibitory neurotransmitter.
Recently, astrocytes have been shown to dramatically that either GABA-mediated elevations of [Ca
tially localized, or that additional Ca
-independent pro-increase the number and maturity of functional synapses
in cultured neurons (Ullian et al., 2001). Thus, it is possi- cesses are required. Consistent with the notion of
GABA-specific signaling is the growing evidence for anble that astrocytes might accelerate the maturation of
GABAergic synaptic transmission, thereby accelerating extensive protein network centered around gephyrin, a
protein that may link GABA
receptors to other signalingthe time course of the switch.
proteins within the inhibitory postsynaptic specialization
(Kneussel and Betz, 2000).
Specificity of GABA-Mediated Calcium Signaling
Spontaneous GABAergic and glutamatergic activity are
both present during the time window of the develop- GABA-Dependent Regulation of KCC2 Expression
In hippocampal slices, the temporal expression patternmental switch (Figures 6A and 6B; Hsia et al., 1998; van
den Pol et al., 1998; Lamsa et al., 2000; Palva et al., for KCC2 coincides with the postnatal switch of GABA-
signaling (Rivera et al., 1999; Vu et al., 2000). In these2000). GABAergic events are indeed depolarizing (3–20
mV), and the level of depolarization is enhanced by sum- cultures, there is a similar developmental increase in the
level of KCC2 mRNA (Figure 3). We found that the levelmation of unitary events. Such depolarizing responses
are likely to trigger Ca
influx through VDCCs (Koester of KCC2 mRNA can be regulated by GABAergic activity.
The level of KCC2 mRNA was reduced by chronic block-and Sakmann, 1998). Surprisingly, GABAergic but not
glutamatergic activity promotes the transformation of ade of GABA
receptors and increased by chronic depo-
larization with KCl (which enhanced spontaneous neuro-GABAergic transmission (Figures 2A and 2B).
What is the signal that is specifically activated by transmitter release, see Figures 6C and 6D). The observed
increase in KCC2 mRNA levels may result from en-GABA but not glutamate? Nimodipine delayed the
switch (Figures 7B and 7C), indicating that Ca
influx hanced transcription or an increase in the stability of
existing mRNAs. Given that the developmental increasethrough L-type VDCCs participates in the signaling cas-
cade initiated by GABA. Since glutamate-mediated de- in KCC2 mRNA levels will result in a decrease in the [Cl
and a reduction of the GABA-mediated depolarizationpolarization also activates L-type VDCCs (Yuste and
Katz, 1991; Deisseroth et al., 1998), this finding suggests (Jarolimek et al., 1999; Kakazu et al., 1999; Rivera et
al., 1999), these results further support the notion that activation of NMDA receptors and the maturation of
glutamatergic transmission (Ben-Ari et al., 1997).GABA-mediated excitation is self-limited.
While we have monitored the expression of KCC2, GABA-induced elevation of [Ca
is likely to promote
neuronal maturation. In the initial stages of brain devel-other genes may also contribute to the switch. The ex-
pression of NKCC1, a Na
transporter, is also opment, depolarizing GABAergic potentials can prevent
DNA synthesis, leading to the differentiation of corticaldevelopmentally regulated (Plotkin et al., 1997; Kakazu
et al., 1999; Lu et al., 1999). NKCC1 participates in Cl
progenitor neurons (LoTurco et al., 1995). Furthermore,
increases can induce BDNF ex-influx, and its expression is reduced during the same
time window in which KCC2 expression is increased, pression (Berninger et al., 1995) and the differentiation
into specific neuronal phenotypes (Marty et al., 1996).suggesting a mechanism for reducing Cl
influx as the
efflux is increased. It may be noted that at day 9, GABA-induced elevation of [Ca
may also be required
to form, stabilize, and strengthen synaptic connectionsthe expression of KCC2 was 6-fold higher than that at
day 3, while only 20% of the cells had failed to respond (Kirsch and Betz, 1998; Caillard et al., 1999; Kneussel
and Betz, 2000). For glycinergic transmission, there isto GABA with Ca
elevation (Figures 1B and 3D). This
could be attributed to a high rate of Cl
influx due to a similar developmental transformation from excitation
to inhibition (Reichling et al., 1994; Wang et al., 1994).NKCC1 activity during this early period of transfor-
mation. Interestingly, Ca
influx triggered by glycine-mediated
depolarization is required for clustering of postsynaptic
glycine receptors (Kirsch and Betz, 1998), an important
GABA-Induced Modifications of I
step in synaptogenesis. Similarly, Ca
In addition to the developmental transformation of
by GABA may also participate in postsynaptic differenti-
GABAergic transmission, we found that neuronal matu-
ation (Kneussel and Betz, 2000). Finally, there is increas-
ration involved a shift in the threshold for the activation
ing evidence that Ca
influx through synaptically acti-
(Figure 4). Pharmacological analysis of Ca
vated L-type Ca
channels is important for activation
rents suggested that, in both young and mature neurons,
of gene expression (Murphy et al., 1991; Berninger et
seems to reflect primarily the activation of “high-
al., 1995; Deisseroth et al., 1996, 1998).
threshold” VDCCs, with ⵑ50% of those channels corre-
During development, these two aspects of GABAergic
sponding to the L-type (Figure 7A). Our findings suggest
synaptic transmission—depolarization and Ca
that either the subunit composition of Ca
are transformed. We have demonstrated that GABA it-
modified or that the expression of low-threshold Ca
self promotes this transformation. Such a self-limiting
channels decreases with age. Consistent with this idea,
trophic action of GABA allows for an activity-dependent
the expression of Ca
channel genes appears to be
transition of the nervous system from its early depen-
developmentally regulated (Gruol et al., 1992; Hilaire et
dence on global excitation to the requirement of mature
al., 1996; Desmadryl et al., 1998). In fact, the functional
neural circuits, where inhibition plays a critical role in
expression of low threshold T-type Ca
its development and function.
been shown to decrease in cultured hippocampal neu-
rons between day 4 and day 16, a time window similar
to that studied here (Chameau et al., 1999). Since chronic
blockade of GABA
receptors (Figure 4B) reduced the
Cell Culture and Chronic Treatments
developmental shift in the threshold for the activation
Hippocampi from E18 rats were trypsinized (15 min, 37⬚C), washed,
, GABAergic transmission can trigger the observed
and gently triturated by passing the tissue through a Pasteur pipette
developmental changes in Ca
with a fire-polished tip. Neurons were plated at 500,000 cells/ml on
poly-L-lysine coated coverslips in 35 mm dishes. The plating me-
dium was DMEM (BioWhittaker, Walkersville, MD) containing10%
Two Faces of GABA Signaling: Membrane
heat-inactivated fetal bovine serum (Hyclone, Logan, UT) and 10%
Depolarization and Ca
Ham’s F12 with glutamine. Twenty four hours after plating, the cul-
GABA is able to induce electrophysiological (membrane
ture medium was completely replaced by Neurobasal medium with-
depolarization) and biochemical (Ca
out glutamine (Gibco, Life Technologies) with B-27 supplement
which may serve different roles in the developing ner-
(Gibco). These serum-free conditions supported the growth of neu-
vous system. While the extent of GABA-mediated depo-
rons but not glial cells. Cultures were fed once a week by adding
ⵑ0.4 ml of neurobasal medium. Chronic treatments were started
larization depends primarily on E
2 days after plating. BMI, D-APV, TTX, GABA, baclofen, CNQX,
signaling through GABA is dependent both on E
Nimodipine, PTX (Sigma), thapsigargin, and BHQ (Calbiochem) were
the properties of depolarization-induced elevation of
added daily from fresh stocks prepared at 1000-fold the final con-
; these two aspects may be independently regu-
centrations (indicated in the Results section), except for KCl, which
lated (Obrietan and van den Pol, 1997). Previous work
was added only once. All chronic treatments involving KCl (10 mM)
has suggested that the GABA-induced depolarization
were done in the presence of TTX and D-APV (2 and 50 M).
may serve to either facilitate or inhibit neuronal activity
under different circumstances in the developing brain
Neurons were loaded with the Ca
-sensitive fluorescent dye Fluo-4
(Ben-Ari et al., 1989; Chen et al., 1996; Garaschuk et al.,
for 20–40 min at room temperature in HEPES buffered saline solution
2000; Lamsa et al., 2000). A depolarizing GABAergic
(HBS, in mM: NaCl 150, KCl 3, CaCl
2, HEPES 10, and
potential can result in a shunting inhibition or can facili-
glucose 5; pH 7.4; osmolarity 310 mOsm) containing 15 M CNQX
tate excitation, depending on the relative timing and
and 10 M BMI in the presence of 5 M Fluo-4AM dissolved in
spatial distribution of GABAergic and glutamatergic in-
DMSO (Molecular Probes, Eugene, OR). HEPES-buffered solution
puts (Chen et al., 1996). In addition, GABA-mediated
(in the absence of HCO
) was used throughout the experiments,
thus consistent with the notion that changes in [Cl
depolarization has been proposed to play a role in the
GABA as a Self-Limiting Trophic Factor
for the switch in GABA signaling (Staley et al., 1995). All recordings Acknowledgments
were carried out at room temperature, using HBS containing 10 M
CNQX. Solutions were exchanged using a multibarreled perfusion The Rat KCC2 clone was a generous gift from John Payne. We thank
Xiao-yun Wang for the preparation of the hippocampal cultures andsystem (at 1–2 ml/min). Recordings were performed using a confocal
laser scanning microscope BioRad MRC1024MP (Biorad, Hemel Benedikt Berninger, Fernanda Ceriani, Miguel Morales, Madhu Rao,
and David Sykes for helpful comments on the manuscript. We alsoHempstead, England). The scanhead was connected to an Olympus
BX50WI upright microscope and images were collected using a 20⫻ thank Miguel Morales and Yuki Goda for their invaluable help during
the last stages of this work, and Luca for his support. This workwater-immersion objective. A total of 30–40 neurons were studied
in each experiment. Somatic regions of ⵑ20–50 pixels were chosen was supported by grants from the NIH (NS 37831 and NS 36999).
for quantification of fluorescence intensity. Sampling of average
pixel intensity was performed at 0.5 Hz. Transmitted and fluorescent
Received September 29, 2000; revised April 18, 2001.
images reflecting Ca
dynamics were acquired (picture size of 512 ⫻
512 pixels) using the Biorad Lasersharp acquisition program. Data
were analyzed using custom made LabView Software (National In-
struments) on a PC. A neuron was considered responsive to GABA
Ben-Ari, Y., Cherubini, E., Corradetti, R., and Gaiarsa, J.L. (1989).
if the peak ⌬F/F
was larger than a threshold, defined as six standard
Giant synaptic potentials in immature rat CA3 hippocampal neu-
deviations (SD) of the baseline noise. This “six SD” criterion was
rones. J. Physiol. 416, 303–325.
developed by repeatedly comparing visual analysis on blind experi-
Ben-Ari, Y., Khazipov, R., Leinekugel, X., Caillard, O., and Gaiarsa,
ments with threshold analysis, until the results matched.
J.L. (1997). GABA
, NMDA and AMPA receptors: a developmentally
regulated ‘menage a trois’. Trends Neurosci. 20, 523–529.
Berninger, B., Marty, S., Zafra, F., da Penha Berzaghi, M., Thoenen,
All experiments were carried out using the perforated-patch whole
H., and Lindholm, D. (1995). GABAergic stimulation switches from
cell recording technique. Neurons were visualized using phase-con-
enhancing to repressing BDNF expression in rat hippocampal neu-
trast on a Nikon inverted microscope. Recordings were carried out
rons during maturation in vitro. Development 121, 2327–2335.
using patch clamp amplifiers (Axopatch 200B, Axon Instruments,
Boukhaddaoui, H., Sieso, V., Scamps, F., Vigues, S., Roig, A., and
Foster City, CA). Signals were filtered at 2 kHz, sampled at 10 kHz
Valmier, J. (2000). Q- and L-type calcium channels control the devel-
and analyzed using Pclamp 6.0 software (Axon Instruments). Series
opment of calbindin phenotype in hippocampal pyramidal neurons
resistance was compensated at 80% (lag 30–60 s). Micropipettes
in vitro Eur. J. Neurosci. 12, 2068–2078.
were made from borosilicate glass capillaries (KG-33, Garner Glass)
Caillard, O., Ben-Ari, Y., and Gaiarsa, J.L. (1999). Long-term potenti-
with a resistance of ⬇2M⍀. For recordings of E
, pipettes were
ation of GABAergic synaptic transmission in neonatal rat hippocam-
tip filled with internal solution and then back filled with internal
pus. J. Physiol. 518, 109–119.
solution containing 20 g/ml gramicidin A (dissolved in methanol,
Chameau, P., Lucas, P., Melliti, K., Bournaud, R., and Shimahara,
Sigma). The internal solution contained (in mM): K-gluconate 154,
T. (1999). Development of multiple calcium channel types in cultured
NaCl 9, MgCl
1, HEPES 10, and EGTA 0.2; pH 7.4; osmolarity 300
mouse hippocampal neurons. Neuroscience 90, 383–388.
mOsm. The bath solution contained HBS with 15 M CNQX, and
was constantly perfused at a rate of ⵑ1 ml/min. GABA (100 M, 50
Chen, G., Trombley, P.Q., and van den Pol, A.N. (1996). Excitatory
ms, 5 psi) was locally applied every 10 s through a micropipette
actions of GABA in developing rat hypothalamic neurones. J. Phys-
connected to a Picospritzer (General Valve Corporation). For re-
iol. 494, 451–464.
cordings of Ca
currents, pipettes were back filled with a high CsCl
Cherubini, E., Rovira, C., Gaiarsa, J.L., Corradetti, R., and Ben Ari, Y.
internal solution containing 200 g/ml amphotericin B (dissolved in
(1990). GABA mediated excitation in immature rat CA3 hippocampal
DMSO, ICN). The high CsCl
internal solution contained (in mM):
neurons. Int. J. Dev. Neurosci. 8, 481–490.
154, NaCl 9, MgCl
1, HEPES 10, and EGTA 0.2; pH 7.4;
Cherubini, E., Gaiarsa, J.L., and Ben-Ari, Y. (1991). GABA: an excit-
osmolarity 300 mOsm. The bath solution was a modified HBS con-
atory transmitter in early postnatal life. Trends Neurosci. 14,
taining (in mM): NaCl 150, KCl 3, BaCl
2, HEPES 10,
glucose 5, and tetraethylammonium 10; TTX 2 M. Leak and capaci-
Connor, J.A., Tseng, H.Y., and Hockberger, P.E. (1987). Depolariza-
tive currents were subtracted using the average of four hyperpolariz-
tion- and transmitter-induced changes in intracellular Ca
ing pulses. Recordings of miniature synaptic currents were per-
cerebellar granule cells in explant cultures. J. Neurosci. 7, 1384–
formed using amphotericin B and high CsCl
internal solution. The
bath solution was HBS with CNQX (15 M) for GABA-mediated
Deisseroth, K., Bito, H., and Tsien, R.W. (1996). Signaling from syn-
currents, and with BMI (10 M) for glutamate-mediated currents.
apse to nucleus: postsynaptic CREB phosphorylation during multi-
Events were filtered at 2 kHz and detected online using the WCP
ple forms of hippocampal synaptic plasticity. Neuron 16, 89–101.
software (kindly provided by J. Dempster, University of Strathclyde,
Scotland). All experiments were performed at 22–24⬚C. Experiments
Deisseroth, K., Heist, E.K., and Tsien, R.W. (1998). Translocation
were rejected if the leak current exceeded 100 pA.
of calmodulin to the nucleus supports CREB phosphorylation in
hippocampal neurons. Nature 392, 198–202.
Quantitation of KCC2 mRNA in Cultured Neurons
Desmadryl, G., Hilaire, C., Vigues, S., Diochot, S., and Valmier, J.
Total RNA was harvested from neurons treated with vehicle, BMI ⫹
(1998). Developmental regulation of T-, N- and L-type calcium cur-
PTX or 10 mM KCl. Cells were lysed with guanidine isothiocyanate
rents in mouse embryonic sensory neurones. Eur. J. Neurosci. 10,
(600 l) and purified (RNeasy, Qiagen). An antisense RNA template
was generated using 316 bp of noncoding sequence residing at the
Garaschuk, O., Linn, J., Eilers, J., and Konnerth, A. (2000). Large-
distal 3⬘ end of the mRNA sequence (Payne et al., 1996). An anti-
scale oscillatory calcium waves in the immature cortex. Nat. Neu-
sense ␤-actin probe (245 bp) was mixed with the KCC2 probe and
rosci. 3, 452–459.
used as an internal standard in all assays.
Gruol, D.L., Deal, C.R., and Yool, A.J. (1992). Developmental changes
were generated using Maxiscript (Ambion), and RNase protection
in calcium conductances contribute to the physiological maturation
assays were performed using Hybspeed (Ambion). Briefly, 3.5 micro-
of cerebellar Purkinje neurons in culture. J. Neurosci. 12, 2838–2848.
grams of total RNA were treated with a mixture of the KCC2 and
Hilaire, C., Diochot, S., Desmadryl, G., Baldy-Moulinier, M., Richard,
␤-actin antisense probes (5 ⫻ 10
cpm each) and annealed at 68⬚C
S., and Valmier, J. (1996). Opposite developmental regulation of
for 10 min. The samples were then immediately treated with RNase
P- and Q-type calcium currents during ontogenesis of large diameter
T1 (200 U/ml) for 30 min at 37⬚C. The protected RNA species were
mouse sensory neurons. Neuroscience 75, 1219–1229.
separated over a 5% acrylamide gel containing 8 M urea, and then
dried. Radioactive band intensities were then measured (Molecular Hsia, A.Y., Malenka, R.C., and Nicoll, R.A. (1998). Development of
excitatory circuitry in the hippocampus. J. Neurophysiol. 79, 2013–Dynamics Phosphorimager) and quantified (NIH image v1.62,
Bethesda MD). 2024.
Ikeda, Y., Nishiyama, N., Saito, H., and Katsuki, H. (1997). GABA
slices demonstrated by gramicidin perforated-patch recordings and
calcium imaging. J. Neurosci. 16, 6414–6423.receptor stimulation promotes survival of embryonic rat striatal neu-
rons in culture. Brain Res. Dev. Brain Res. 98, 253–258.
Palva, J.M., Lamsa, K., Lauri, S.E., Rauvala, H., Kaila, K., and Taira,
T. (2000). Fast network oscillations in the newborn rat hippocampus
Jarolimek, W., Lewen, A., and Misgeld, U. (1999). A furosemide-
in vitro. J. Neurosci. 20, 1170–1178.
cotransporter counteracts intracellular Cl
mulation and depletion in cultured rat midbrain neurons. J. Neurosci.
Payne, J.A., Stevenson, T.J., and Donaldson, L.F. (1996). Molecular
characterization of a putative K-Cl cotransporter in rat brain. A neu-
ronal-specific isoform. J. Biol. Chem. 271, 16245–16252.
Kakazu, Y., Akaike, N., Komiyama, S., and Nabekura, J. (1999). Regu-
lation of intracellular chloride by cotransporters in developing lateral
Plotkin, M.D., Snyder, E.Y., Hebert, S.C., and Delpire, E. (1997).
superior olive neurons. J. Neurosci. 19, 2843–2851.
Expression of the Na-K-2Cl cotransporter is developmentally regu-
lated in postnatal rat brains: a possible mechanism underlying
Katz, L.C., and Shatz, C.J. (1996). Synaptic activity and the construc-
GABA’s excitatory role in immature brain. J. Neurobiol. 33, 781–795.
tion of cortical circuits. Science 274, 1133–1138.
Reichling, D.B., Kyrozis, A., Wang, J., and MacDermott, A.B. (1994).
Khazipov, R., Leinekugel, X., Khalilov, I., Gaiarsa, J.L., and Ben-Ari,
Mechanisms of GABA and glycine depolarization-induced calcium
Y. (1997). Synchronization of GABAergic interneuronal network in
transients in rat dorsal horn neurons. J. Physiol. 476, 411–421.
CA3 subfield of neonatal rat hippocampal slices. J. Physiol. 498,
763–772. Rivera, C., Voipio, J., Payne, J.A., Ruusuvuori, E., Lahtinen, H.,
Lamsa, K., Pirvola, U., Saarma, M., and Kaila, K. (1999). The K
Kirsch, J., and Betz, H. (1998). Glycine-receptor activation is re-
co-transporter KCC2 renders GABA hyperpolarizing during neuronal
quired for receptor clustering in spinal neurons. Nature 392,
maturation. Nature 397, 251–256.
Rosenstein, J.M., More, N.S., Mani, N., and Krum, J.M. (1998). Devel-
Kneussel, M., and Betz, H. (2000). Clustering of inhibitory neuro-
opmental expression of calcium-binding protein-containing neurons
transmitter receptors at developing postsynaptic sites: the mem-
in neocortical transplants. Cell Transplant. 7, 121–129.
brane activation model. Trends Neurosci. 23, 429–435.
Schierle, G.S., Gander, J.C., D’Orlando, C., Ceilo, M.R., and Vogt
Koester, H.J., and Sakmann, B. (1998). Calcium dynamics in single
Weisenhorn, D.M. (1997). Calretinin-immunoreactivity during post-
spines during coincident pre- and postsynaptic activity depend on
natal development of the rat isocortex: a qualitative and quantitative
relative timing of back-propagating action potentials and subthresh-
study Cereb. Cortex. 7, 130–142.
old excitatory postsynaptic potentials. Proc. Natl. Acad. Sci. USA
Staley, K.J., Soldo, B.L., and Proctor, W.R. (1995). Ionic mechanisms
of neuronal excitation by inhibitory GABA
receptors. Science 269,
Lamsa, K., Palva, J.M., Ruusuvuori, E., Kaila, K., and Taira, T. (2000).
Synaptic GABA(A) activation inhibits AMPA-kainate receptor-medi-
Ullian, E.M., Sapperstein, S.K., Christopherson, K.S., and Barres,
ated bursting in the newborn (P0–P2) rat hippocampus. J. Neuro-
B.A. (2001). Control of synapse number by glia. Science 291,
physiol. 83, 359–366.
Lee, J.J., and Costlow, N.A. (1987). A molecular titration assay to
van den Pol, A.N., Gao, X.B., Patrylo, P.R., Ghosh, P.K., and Obrie-
measure transcript prevalence levels. Meth. Enzymol. 152, 633–648.
tan, K. (1998). Glutamate inhibits GABA excitatory activity in devel-
Leinekugel, X., Tseeb, V., Ben-Ari, Y., and Bregestovski, P. (1995).
oping neurons. J. Neurosci. 18, 10749–10761.
activation induces Ca
rise in pyramidal cells and
Vu, T.Q., Payne, J.A., and Copenhagen, D.R. (2000). Localization
interneurons from rat neonatal hippocampal slices. J. Physiol. 487,
and developmental expression patterns of the neuronal K-Cl co-
transporter (KCC2) in the rat retina. J. Neurosci. 20, 1414–1423.
Li, Y.-x., Schaffner, A., Walton, M.K., and Barker, J.L. (1998).
Wang, J., Reichling, D.B., Kyrozis, A., and MacDermott, A.B. (1994).
Astrocytes regulate developmental changes in the chloride ion gra-
Developmental loss of GABA- and glycine-induced depolarization
dient of embryonic rat ventral spinal cord neurons in culture. J.
transients in embryonic rat dorsal horn neurons in culture.
Physiol. 509, 847–858.
Eur. J. Neurosci. 6, 1275–1280.
LoTurco, J.J., Owens, D.F., Heath, M.J., Davis, M.B., and Kriegstein,
Yuste, R., and Katz, L.C. (1991). Control of postsynaptic Ca
A.R. (1995). GABA and glutamate depolarize cortical progenitor cells
in developing neocortex by excitatory and inhibitory neurotransmit-
and inhibit DNA synthesis. Neuron 15, 1287–1298.
ters. Neuron 6, 334–344.
Lu, J., Karadsheh, M., and Delpire, E. (1999). Developmental regula-
tion of the neuronal-specific isoform of K-Cl cotransporter KCC2 in
postnatal rat brains. J. Neurobiol. 39, 558–568.
Luhmann, H.J., and Prince, D.A. (1991). Postnatal maturation of the
GABAergic system in rat neocortex. J. Neurophysiol. 65, 247–263.
Marty, S., Berninger, B., Carroll, P., and Thoenen, H. (1996). GABA-
ergic stimulation regulates the phenotype of hippocampal interneu-
rons through the regulation of brain-derived neurotrophic factor.
Neuron 16, 565–570.
Mueller, A.L., Taube, J.S., and Schwartzkroin, P.A. (1984). Develop-
ment of hyperpolarizing inhibitory postsynaptic potentials and hy-
perpolarizing response to gamma-aminobutyric acid in rabbit hippo-
campus studied in vitro. J. Neurosci. 4, 860–867.
Murphy, T.H., Worley, P.F., and Baraban, J.M. (1991). L-type voltage-
sensitive calcium channels mediate synaptic activation of immedi-
ate early genes. Neuron 7, 625–635.
Obrietan, K., and van den Pol, A.N. (1995). GABA neurotransmission
in the hypothalamus: developmental reversal from Ca
to depressing. J. Neurosci. 15, 5065–5077.
Obrietan, K., and van den Pol, A.N. (1997). GABA activity mediating
rises in developing neurons is modulated by cAMP-
dependent signal transduction. J. Neurosci. 17, 4785–4799.
Owens, D.F., Boyce, L.H., Davis, M.B., and Kriegstein, A.R. (1996).
Excitatory GABA responses in embryonic and neonatal cortical