1Department of Neurology and 2The Center for Neurobiology and Behavior, College of Physicians and Surgeons of Columbia University, New York, New York 10032
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ABSTRACT |
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Owens, David F.,
Xiaolin Liu, and
Arnold R. Kriegstein.
Changing Properties of GABAA Receptor-Mediated
Signaling During Early Neocortical Development.
J. Neurophysiol. 82: 570-583, 1999.
Evidence from several
brain regions suggests -aminobutyric acid (GABA) can exert a trophic
influence during development, expanding the role of this amino acid
beyond its function as an inhibitory neurotransmitter. Proliferating
precursor cells in the neocortical ventricular zone (VZ) express
functional GABAA receptors as do immature postmigratory
neurons in the developing cortical plate (CP); however,
GABAA receptor properties in these distinct cell
populations have not been compared. Using electrophysiological techniques in embryonic and early postnatal neocortex, we find that
GABAA receptors expressed by VZ cells have a higher
apparent affinity for GABA and are relatively insensitive to receptor
desensitization compared with neurons in the CP. GABA-induced current
magnitude increases with maturation with the smallest responses found
in recordings from precursor cells in the VZ. No evidence was found that GABAA receptors on VZ cells are activated
synaptically, consistent with previous data suggesting that these
receptors are activated in a paracrine fashion by nonsynaptically
released ligand. After neurons are born and migrate to the CP, they
begin to demonstrate spontaneous synaptic activity, the majority of
which is GABAA mediated. These spontaneous
GABAA postsynaptic currents (sPSCs) first were detected at
embryonic day 18 (E18). At birth, ~50% of recordings from cortical
neurons demonstrated GABAA-mediated sPSCs, and this value
increased with age. GABAA-mediated sPSCs were action
potential dependent and arose from local GABAergic interneurons. GABA
application could evoke action potential-dependent PSCs in neonatal
cortical neurons, suggesting that during the first few postnatal days,
GABA can act as an excitatory neurotransmitter. Finally,
N-methyl-D-aspartate (NMDA)- but not
non-NMDA-mediated sPSCs were also present in early postnatal neurons.
These events were not observed in cells voltage clamped at negative
holding potentials (
60 to
70 mV) but were evident when the holding
potential was set at positive values (+30 to +60 mV). Together these
results provide evidence for the early maturation of GABAergic
communication in the neocortex and a functional change in
GABAA-receptor properties between precursor cells and early
postmitotic neurons. The change in GABAA-receptor
properties may reflect the shift from paracrine to synaptic receptor activation.
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INTRODUCTION |
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The amino acid GABA is considered the major
inhibitory neurotransmitter in the adult cortex (Connors et al.
1988; Krnjevic and Schwartz 1967
). GABA exerts
its influence in cortex by activation of GABAA
and GABAB receptors, which primarily activate
chloride (Cl
)- and potassium-dependent
conductances, respectively (Bormann 1988
; Connors
et al. 1988
; Kaila 1994
). At early stages of
cortical development, glutamatergic-mediated excitation is present, but there appears to be little functional inhibition (Agmon and
O'Dowd 1992
; Agmon et al. 1996
;
Burgard and Hablitz 1993b
; Kim et al. 1995
; Luhmann and Prince 1991
). The paucity of synaptic
inhibition in the immature neocortex has been interpreted as a
developmental delay in the maturation of the GABAergic signaling
system. However, evidence has accumulated that suggests that
GABA-mediated signaling may develop quite early in the cortex.
Messenger RNA (mRNA) and protein for GABA, the GABA biosynthetic enzyme
glutamate decarboxylase, and various
GABAA-receptor subunits have been localized in
embryonic cortical tissue (Cobas et al. 1991
;
Lauder et al. 1986
; Laurie et al. 1992
;
Ma and Barker 1995
; Van Eden et al.
1989
), and functional GABAA receptors
have been shown to be present on both proliferative and early
postmitotic cells (LoTurco et al. 1995
; Owens et
al. 1996
). In developing neocortex, activation of
GABAA receptors produces robust membrane
depolarization (Agmon et al. 1996
; LoTurco et al.
1995
; Owens et al. 1996
; Yuste and Katz
1991
) due to a relatively high concentration of intracellular
Cl
([Cl
]i) maintained in
immature cells (Clayton et al. 1998
; Owens et al.
1996
; Rivera et al. 1999
). Thus functional
inhibition may be less effective in neonatal cortex in part due to the
depolarizing actions of the principle inhibitory neurotransmitter GABA.
GABA can exert trophic influences during early CNS development, and
many of the developmental actions of GABA are mediated through
activation of GABAA receptors (Meier et
al. 1991). For example, GABAA-receptor
activation can influence DNA synthesis in proliferative cells
(LoTurco et al. 1995
) and cell motility and
morphological development in early postmitotic neurons (Barbin et al. 1993
; Behar et al. 1996
, 1998
;
Marty et al. 1996
). Whether the developmental effects of
GABA are mediated exclusively by synaptic or nonsynaptic (e.g.,
paracrine) receptor activation is not completely understood. However,
recent electrophysiological experiments and ultrastructural analysis
have demonstrated that GABAergic synapses are present perinatally in
the cortical plate (Agmon et al. 1996
; De Felipe
et al. 1997
; Micheva and Beaulieu 1996
;
Owens et al. 1996
), suggesting that synaptic
GABA-receptor activation, before the onset of mature inhibition, may
play a role in cortical development. To further explore the maturation of the GABAergic system in neocortex, we have compared the functional properties of GABAA receptors in both
proliferative and postmitotic neocortical cells in situ during the
embryonic and early postnatal period. In addition, we have investigated
the development of spontaneous GABAA-mediated
synaptic transmission.
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METHODS |
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Tissue preparation
Results were obtained using brain slices and slabs from
embryonic (E15-20) and early postnatal (P0-5) Sprague-Dawley rats (Taconic, Germantown, NY). The day of birth was considered P0. For
embryonic tissue, gravid rats were anesthetized with an intraperitoneal injection of ketamine (50 mg/kg) and xylazine (10 mg/kg), and embryos
were exposed by cesarean section. Embryos were decapitated, and heads
were immediately placed in ice-cold artificial cerebrospinal fluid
[ACSF; it contained (in mM) 124 NaCl, 5 KCl, 1.25 NaH2PO4, 1 MgSO4, 2 CaCl2, 26 NaHCO3, and 10 glucose] oxygenated with 95% O2-5% CO2 (pH 7.4).
Cortical slabs (LoTurco et al. 1995) were prepared by
removing an entire cerebral hemisphere and trimming off the hippocampus
and striatal anlage. Most VZ recordings were obtained from slabs;
however, conventional coronal slices were used in some experiments. For
brain slices, whole embryonic brains were removed and embedded in warm
(28-30°C) 3-4% low-melting point agarose (Fisher Scientific, Fair
Lawn, NJ) in ACSF, hardened on ice, and cut into coronal sections
(300-400 µm) with a vibratome. Neonatal rat pups were decapitated
rapidly, and brains were removed and placed in ice-cold ACSF oxygenated
with 95% O2-5% CO2.
Coronal slices (300-400 µm), confined to the sensorimotor regions of
the cortex, were made with a vibratome. Tissue was kept in oxygenated ACSF at room temperature (RT; 21-25°C) for
1 h before recording.
Electrophysiological recordings and data analysis
Patch-clamp recordings were obtained at RT from cells in both
slices and slabs of neocortex continuously superfused with 95% O2-5% CO2 oxygenated ACSF
at a rate 1-2 ml/min (Blanton et al. 1989; Owens
et al. 1996
). For the majority of recordings, patch electrodes
were filled with (in mM) 100 CsCl, 30 Cs gluconate, 10 HEPES (pH 7.3),
2 CaCl2, and 11 EGTA. In some whole cell
recordings, a KCl filling solution was used [containing (in mM) 130 KCl, 5 NaCl, 0.4 CaCl2, 1 MgCl2, 10 HEPES (pH 7.3), and 1.1 EGTA]. In experiments calling for different concentrations of
Cl
in the pipette solution
([Cl
]p), gluconate
substituted for Cl
in the filling solution. The
liquid junction potential for the modified solution was determined
empirically (Neher 1992
) and data were corrected post
hoc. Recordings were digitized and analyzed with pClamp6 software (Axon
Instruments, Foster City, CA). Membrane resistance was calculated by
measuring the steady-state current deflection during 200-ms voltage
steps (±10-30 mV) from a holding potential of
60 mV. When using
voltage ramps, the ramp protocol consisted of stepping the cell from a
holding potential of
60 to +20 or +40 mV for 80 ms, then changing the
voltage at a rate of 150 mV/s to
100 mV. I-V curves were
plotted after subtracting the control response obtained in ACSF from
the response obtained during agonist application. GABA and glutamate
reversal potentials also were determined by applying agonists with the
membrane held at a series of potentials. Peak current responses for
each voltage were plotted, and the data were fitted using CA-Cricket
Graph III software (Computer Associates International). The
agonist-mediated reversal potentials were defined as the
x-intercept value of the fit. Spontaneous PSC reversal
potentials were estimated by holding the cell at a series of membrane
potentials and determining the potential when sPSCs were nullified.
This value then was compared with the reversal potential of the GABA-
or muscimol-induced currents and the Cl
equilibrium potential (ECl
) that was
determined by the Nernst equation of the form:
ECl
=
58 mV log10
([Cl
]o/[Cl
]i),
where [Cl
]o and
[Cl
]i are the
extracellular and intracellular Cl
concentrations, respectively. A cell was considered not to have sPSCs
if no synaptic potentials were observed after 5-10 min of recording.
Dose-response data were fit with the Hill equation of the form:
I/Imax = 1/[1+(EC50/[GABA])n],
where I is the GABA-induced current,
Imax is the maximal GABA-induced current, EC50 is GABA concentration producing a
half-maximal response, [GABA] is the GABA concentration, and
n is the Hill coefficient. GABA-induced current decays were
expressed as: percentage of apparent rate of desensitization = (Ipeak
I20/Ipeak) × 100. I20 was the level of current
20 s after the current peak
(Ipeak). The
I20 value was selected to normalize
data for comparisons. To isolate individual VZ cells, cortical slabs
were maintained in 0 calcium (Ca2+) ACSF for
30
min before recording and cell isolation. Using changes in membrane
resistance and GABA-induced current as an indicator of successful cell
isolation, we found that in ~40% of the attempts we were able to
isolate cells. This is close to the reported value of 50% found in
previous studies using this method (Mienville et al.
1994
). To uncouple VZ cell clusters pharmacologically, ACSF
bubbled with 100% CO2 was added to the bathing
solution, and membrane resistance was monitored to asses the decrease
in gap junction conductance (LoTurco and Kriegstein
1991
). In experiments examining the extent of bicuculline
methiodide (BMI) antagonism of GABA-induced currents, the data were
expressed as: percentage block = (Icontrol
IBMI/Icontrol) × 100. Where Icontrol is the peak
GABA-induced current and IBMI is the
peak current after the addition of BMI. All average values are
expressed as means ± SE. Cells were accepted for analysis only if
they maintained a stable access resistance throughout the recording.
Unless otherwise noted, the holding potential for voltage clamp
recordings was
60 mV.
Filling Cells with Lucifer yellow
In some recordings a saturating concentration of Lucifer yellow (LY) (Sigma) was included in the pipette filling solution. After electrophysiological recording, the tissue was fixed in 4% paraformaldehyde for 30-60 min at RT or overnight at 4°C and then transferred to PBS. Tissue was dehydrated in ethanol, cleared with methyl salicylate (Sigma), and coverslipped. Filled cells then were viewed with scanning laser confocal microscopy using a Zeiss Axiovert microscope with illumination provided by a Zeiss argon crypton laser scanning confocal attachment or by conventional epifluorescence.
GABA immunohistochemistry
Whole animals were anesthetized with halothane and transcardially perfused with 4% paraformaldehyde. Heads were removed and postfixed in 4% paraformaldehyde overnight at 4°C, then placed in PBS. Brains were removed and sectioned at 50-100 µm on a vibratome. Sections were treated in 0.3% H2O2 in PBS for 30 min at RT to remove endogenous peroxidase activity. Sections then were permeabilized and blocked with 0.5% Triton X-100 and 10% normal goat serum (NGS) in PBS for 1 h at RT. After washing, sections were incubated in polyclonal anti-GABA primary antibody (Sigma) (1:1,000 dilution) in 0.1% Triton X-100 and 3% NGS in PBS for 1-2 h at RT or overnight at 4°C. Tissue was washed in PBS and then incubated in biotinylated anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA) (1:200 dilution) in 0.1% Triton X-100 and 3% NGS in PBS for 1 h at RT. After washing, sections were incubated with VECTASTAIN elite ABC reagent (Vector Laboratories) for 30 min at RT, washed in PBS, and then reacted with diaminobenzidine tetrahydrochloride substrate (Vector Laboratories) for 10-15 min at RT. Sections were rinsed with H2O, dehydrated in ethanol and xylene, and coverslipped. Tissue was viewed with a Zeiss Axioscope. In control experiments, no cellular staining was observed when the primary antibody was omitted (not shown).
Pharmacological agents and application
Muscimol, bicuculline methiodide (BMI), glutamate, and
tetrodotoxin (TTX) were obtained from Sigma (St. Louis). GABA,
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX),
6,7-dinitroquinoxaline-2,3-dione (DNQX), 2-amino-5-phosphonopentanoic (AP-5), N-methyl-D-aspartate (NMDA), and
cis-4-aminocrotonic acid (CACA) were obtained from RBI
(Natick, MA). Drugs were applied with a DAD-12 Superfusion System (ALA
Scientific Instruments, Westbury, NY). This application system
permitted more rapid drug exchange than the bath-perfusion system used
previously (LoTurco et al. 1995). Generally, a drug-free
ACSF wash was applied immediately before and after drug. In some
experiments CNQX and TTX were bath applied. Drugs were kept as
concentrated stock solutions at
20°C (Muscimol, BMI, CNQX, DNQX,
AP-5, NMDA, CACA, and GABA) or 4°C (TTX) and diluted to the desired
concentration on the day of the experiment.
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RESULTS |
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A summary of neocortical development is shown schematically in
Fig. 1A (see legend for
details). The cell populations investigated are in areas marked with
bold type in Fig. 1A and are displayed after filling of
cells with LY in Fig. 1, B-D. These include proliferating precursor cells in the VZ (VZ cells), immature neurons in the embryonic
CP (CP cells), and further differentiated postnatal cortical neurons
(P0-5, collectively termed PN cells). A hallmark of proliferating
precursor cells in the VZ is that they are coupled into discrete radial
cell clusters by gap junction channels (LoTurco and Kriegstein
1991). These clusters contain both proliferating precursor
cells and one or two radial glia (Bittman et al. 1997
; LoTurco et al. 1995
). Figure 1B shows an
example of a gap junction coupled cell cluster at E17. Due to coupling,
electrophysiological recordings from cells in clusters display lower
membrane resistances (LoTurco and Kriegstein 1991
) than
would be expected of single small cells (~5-10 µm in diameter).
The probability of encountering coupled cell clusters in the VZ
decreases with age so that by E19-20 one can obtain recordings from
both coupled precursor cells as well as from postmitotic neurons
(LoTurco and Kriegstein 1991
; LoTurco et al.
1991
). For this reason, we performed the majority of VZ
recordings at E15-17. Recordings were identified as deriving from gap
junction coupled clusters by their low membrane resistances (47-333
M
), and in all such recordings when including LY in the recording
pipette, we filled multicellular clusters (n = 11). After exit from the cell cycle, newly born neurons migrate to the CP.
Embryonic CP cells have higher membrane resistances (1-3 G
) than
coupled VZ cells, and intracellular filling with LY (Fig. 1C) or biocytin (Bittman et al. 1997
) reveals
cells with relatively immature neuronal morphology (n = 12). Because the CP contains both migratory and postmigratory neurons,
CP recordings could derive from either of these immature neuronal
types. During the early postnatal period, electrodes were targeted to
the upper cortical layers (i.e., presumptive layers II-IV). The
recorded cells displayed a relatively large range of membrane
resistances (range 0.2-5 G
) and, when filled with LY, had
pyramidal-like morphology (n = 15, Fig.
1D).
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Evidence suggests that postmitotic cortical neurons are uncoupled
during migration through the IZ (Bittman et al. 1997).
Once settled in the CP, neurons become coupled again into progressively larger clusters that peak in size during the first two postnatal weeks
(Kandler and Katz 1998
; Peinado et al.
1993
; Rorig et al. 1996
). However, the sites of
contact and degree of junctional dye spread differ between VZ and CP
cells. For example, LY stains coupled VZ cells better than coupled
neonatal neurons (Fig. 1, B-D), while biocytin and
neurobiotin stain coupled cells in both regions well (Bittman et
al. 1997
; Peinado et al. 1993
). Additionally, coupling in cortical neurons is thought to occur at distal dendritic sites (Peinado et al. 1993
), whereas coupling in VZ
cells must occur more proximally because VZ cells have a relatively
simple bipolar morphology (Nadarajah et al. 1997
).
Changes in the functional properties of GABAA receptors between precursor cells and neurons
In whole cell recordings
([Cl]p = 104 mM), we
found that at ages
E15, application of GABA produced inward currents
in all VZ cells tested (Fig. 1B, inset). GABA-induced inward
currents also were observed in all recordings tested from CP and PN
neurons (Fig. 1, C and D, insets). These findings
are consistent with previous studies that have shown functional
GABAA receptors to be present in these cell
populations (LoTurco and Kriegstein 1991
; LoTurco
et al. 1995
; Owens et al. 1996
).
DOSE-RESPONSE PROPERTIES.
We first compared the dose-response properties of GABA currents in
recordings made from embryonic VZ (E16-17), embryonic CP (E19), and
early PN (P4-5) cells. In all cells GABA produced dose-dependent currents (Fig. 2A); however,
the properties were different between the distinct cell populations
(Fig. 2B). In VZ cells GABA produced a half-maximal response
at a concentration of 5.1 µM with a Hill coefficient of 1.9 (n = 16); this value is consistent with previous findings (LoTurco et al. 1995). In contrast, recordings
from CP and PN cortical neurons demonstrated that GABA was
approximately six- and eightfold less potent, respectively. GABA
produced a half-maximal response at a concentration of 28.2 µM with a
Hill coefficient of 1.0 in CP cells (n = 5) and 40.1 µM with a Hill coefficient of 1.3 in PN cells (n = 7). Also, consistent with receptors that have a higher apparent
affinity for GABA, VZ cell responses took longer to recover after
removal of GABA than CP and PN cell responses (see Fig.
3A). This effect was observed most readily after longer drug applications, and to be sure this was
not simply due to less effective drug clearance from the VZ environment, we compared the recovery after glutamate application. Consistent with effective drug clearance from the VZ, glutamate (300 µM)-induced currents in VZ cells, mediated by AMPA/kainate-type receptors with a EC50 of ~75 µM
(LoTurco et al. 1995
), displayed relatively rapid
recovery rates (Fig. 2C).
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RESPONSE MAGNITUDE.
The dose-response experiments indicate that GABA-induced current
increases in magnitude with development. In recordings from VZ
(E15-17) cells, the peak current induced by a saturating GABA concentration (50 µM) was 324 ± 36 pA (n = 19), whereas in CP (E19) and PN (P4-5) neurons saturating GABA
concentrations (500 µM) produced peak currents of
792 ± 161 pA (n = 8) and
1604 ± 229 pA (n = 13), respectively. This could reflect a developmental increase in the
GABAA channel conductance or an increase in the number of GABAA channels per cell. Considering
that the main conductance state of GABAA receptor
channels in VZ cells is similar to that reported for neurons
(LoTurco and Kriegstein 1991
; Rabow et al. 1995
; Xiang et al. 1998
), these results suggest
that the number of GABA receptors per cell increases with maturation.
Furthermore the number of receptors is likely to be particularly low in
VZ cells because GABA-induced currents in VZ recordings most likely reflect activation of receptors on multiple cells within a
gap-junction-coupled cell cluster (see following text).
RECEPTOR DESENSITIZATION.
In addition to differences in peak current and apparent affinity for
GABA between VZ cells and cortical neurons, GABA application to VZ
cells produced currents that persisted in the continued presence of
agonist, suggesting differences in receptor desensitization (Fig.
3A). To investigate this further, we applied 50 µM GABA to
VZ, CP, and PN cells for 20-30 s and measured the level of desensitization in each cell population. The percentage apparent rate
of desensitization (see METHODS) in VZ cells was 15.8 ± 2.4% (n = 9), whereas in CP and PN cells, these
values were 65.3 ± 3.1% (n = 10) and 64.1 ± 3.8% (n = 9), respectively (Fig. 3B). This difference could not be accounted for by differences in response magnitude because peak currents with similar amplitudes displayed differences in receptor desensitization in VZ and CP/PN cells. In VZ
recordings with a mean peak current of 418 ± 35 pA, the percentage apparent rate of desensitization was 14.9 ± 4.2%
(n = 4), whereas in CP/PN recordings with a mean peak
current of
415 ± 53 pA, the percentage apparent rate of
desensitization was 61.9 ± 3.1% (n = 3).
RECEPTOR PHARMACOLOGY.
The properties of the GABA-induced current in VZ cells (i.e., a
relatively high affinity for GABA and relative lack of receptor desensitization) are similar to those of the
GABAC subtype GABA receptor, a
Cl-selective ion channel distinguished
pharmacologically from GABAA receptors by being
insensitive to BMI (Bormann and Feigenspan 1995
).
However, previous results have demonstrated that GABA-induced currents
in VZ cells are sensitive to BMI (LoTurco and Kriegstein 1991
; LoTurco et al. 1995
), suggesting that
these are not GABAC receptors. In agreement with
these studies, we found that in eight of eight VZ (E16-17) recordings
currents induced by application of 20 µM GABA were blocked entirely
by 100 µM BMI (Fig. 4, A and D). In all cases, the antagonism produced by BMI was
reversible (not shown). In addition, CACA (100 µM), an agonist with
some specificity for GABAC receptors, produced
only small currents (
8.6 ± 1 pA) in four of six cells. In three
of three of the CACA-responding cells, 100 µM BMI blocked all of the
current (Fig. 4D). These results strongly suggest that all
of the GABA-induced current in VZ cells are mediated by
GABAA receptors.
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GABAergic synaptic transmission
DEVELOPMENTAL ONSET OF SPONTANEOUS GABAA-MEDIATED
SYNAPTIC TRANSMISSION.
Spontaneous PSCs were observed in recordings from early neocortical
neurons consistent with previous reports (Luhmann and Prince
1991; Owens et al. 1996
). These sPSCs were
mediated by GABAA receptors because they were reversibly
blocked by BMI (n = 21; Figs.
5, A and
B; 6, A and
C; 7A; and
8, A and
C3), but not by the glutamate-receptor antagonists CNQX,
DNQX, or AP-5 (n = 6; Fig. 5B).
Glutamate-receptor antagonists could never eliminate the
sPSCs; however, modulation of GABAA-mediated
sPSCs by glutamate-receptor blockade (Salin and Prince
1996
) was not examined. The properties of the sPSCs varied from
cell to cell (Fig. 6, A and B). The
frequency ranged from 0.02 to 1.4 Hz, and in many of the cells, sPSCs
occurred in relatively high-frequency bursts (Fig. 6B).
Also, consistent with the sPSCs being GABAA mediated, sPSCs
reversed near ECl
and matched the reversal potential
found for exogenously applied GABA and muscimol but not glutamate or
NMDA (Fig. 6C). Considering that
[Cl
]i is relatively high in immature
cortical neurons (Ben-Ari et al. 1989
; Owens et
al. 1996
) and the resting membrane potential is generally more
negative than ECl
(Burgard and Hablitz
1993b
; Luhmann and Prince 1991
; Owens et al. 1996
), these synaptic currents would serve to depolarize
the membrane.
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NMDA-MEDIATED SPSCS.
Our results indicate that in early postnatal cortical neurons the
majority of sPSCs in cells voltage clamped at negative holding potentials (60 to
70 mV) are mediated by activation of
GABAA receptors. Glutamatergic sPSCs were not
observed; however, previous studies have demonstrated that
glutamate-receptor-mediated synaptic currents can be evoked by at
least P0 (Kim et al. 1995
) and are detected
spontaneously by at least P3 in neocortical cells (Burgard and
Hablitz 1993a
). Because nearly all immature cortical neurons express NMDA receptors (LoTurco et al. 1991
), it is
possible that glutamate-receptor-mediated sPSCs are dominated by NMDA
receptors, making them functionally silent at negative membrane
potentials (Isaac et al. 1997
). To test this, we applied
BMI to recordings held at positive potentials (+30 to +60 mV) and
monitored the sPSCs. In these recordings, we found that the sPSCs were
not always eliminated with BMI (Fig. 7A), and consistent
with the BMI-insensitive events being mediated by NMDA receptors, they
were blocked by AP-5 (n = 4; Fig. 7B).
SOURCE OF GABAERGIC SPSCS.
Most BMI-sensitive sPSCs could be abolished by application of TTX
(n = 6; Fig. 8A), indicating that the
majority of GABAA-mediated sPSCs resulted from
action potential dependent activity of GABAergic neurons. Consistent
with previously published reports (Cobas et al. 1991;
Lauder et al. 1986
; Van Eden et al.
1989
), we observed GABA immunopositive cells in the developing
cortical layers that could be the presynaptic source of the sPSCs (Fig.
8B). To investigate whether activity of local GABAergic
interneurons could generate PSCs in neighboring neurons, we used focal
applications of glutamate to locally excite neurons while recording
from nearby pyramidal cells. ECl
was
set at
45 mV ([Cl
]p = 22 mM) and cells were voltage clamped at ~0 mV, close to the
glutamate reversal potential. Pulses of glutamate (100 µM) induced
BMI-sensitive PSCs (n = 5; Fig. 8C2). This
result suggests that local activation of GABA-containing cells can
evoke GABAA-mediated PSCs. Additionally, we
isolated small pieces of neocortex by making two complete radial cuts
2-3 mm apart and a horizontal cut above the white matter
(n = 3; Fig. 8C3).
GABAA-mediated sPSCs were still present after
this microdissection. Collectively these results suggest that the sPSCs
are mediated by local spontaneously active GABAergic neurons that
reside in the cortex and that cortical afferents are not required to
drive these cells.
GABAA RECEPTOR ACTIVATION CAN BE EXCITATORY.
Previous studies have demonstrated that during the early postnatal
period GABAA-receptor activation is depolarizing
when [Cl]i is kept
intact (Owens et al. 1996
). It is possible that
GABAA-receptor activation is excitatory and may
depolarize cells sufficiently to induce action potential activity. To
test this idea, we attempted chemical microstimulation with GABA
instead of glutamate. Local application of GABA (1-5 µM) could
induce PSCs (Fig. 9A). In 52% of recordings from cells at P3/4, GABA application either induced PSCs
or increased the frequency of sPSCs (n = 25). In all
cells tested (n = 7), application of TTX (2 µM)
before the GABA application blocked the ability of GABA to induce PSCs
(Fig. 9B). These data suggest that during the early
postnatal period GABAA-receptor activation can
elicit action potential activity in a subset of cortical neurons and
that GABA can function as an excitatory neurotransmitter.
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DISCUSSION |
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The early development of the GABAA signaling system was explored in neocortex. The major findings are that GABAA receptors in proliferative VZ cells and postmitotic neurons display significant functional differences in dose-response properties, receptor number, and receptor desensitization. We also find that most of the spontaneous synaptic activity in the perinatal neocortex is GABAA-receptor mediated. This form of synaptic transmission begins to develop in the CP during the late embryonic period, requires action potential activity, and appears to arise from local GABAergic interneurons. Also during the perinatal period, GABAA-receptor activation will depolarize cortical neurons and in some cases induce action potential firing.
Functional properties of GABAA receptors in proliferative and postmitotic cells
The differences in GABAA-receptor properties
in precursor cells and postmitotic neurons are likely due to
differences in subunit composition. GABAA
receptors are thought to be heteropentameric proteins constructed of
subunits derived from five related gene families (Macdonald and
Olsen 1994). Presently, six
, three
, three
, one
,
and one
subunit subtypes have been identified (Davies et al.
1997
; Macdonald and Olsen 1994
; Whiting
et al. 1997
), providing an enormous potential number of subunit
combinations. However, it has been suggested that the actual number of
different subunit combinations in native channels is more restricted
(McKernan and Whiting 1996
). Immunohistochemical and in
situ hybridization methods have been used to localize a variety of
GABAA-receptor subunits in developing neocortex
(Araki et al. 1992
; Fritschy et al. 1994
;
Laurie et al. 1992
; Ma and Barker 1995
;
Poulter et al. 1992
, 1993
). In the neocortical
proliferative zone, the most prominently expressed subunits appear to
be
4,
1, and
1 (Araki et al. 1992
;
Laurie et al. 1992
; Ma and Barker 1995
;
Poulter et al. 1992
, 1993
), however mRNA for other
GABAA-receptor subunits also has been localized
to the VZ (Laurie et al. 1992
; Poulter et al.
1993
, 1993
). Presently, it is uncertain which of the detected subunits form native channels and whether there is a differential expression of specific subunits in proliferative and newly born postmitotic neurons. Nevertheless, comparison of the physiological properties of GABAA receptors examined in
expression systems to the properties of the native receptors may permit
tentative conclusions concerning possible subunit combinations. For
example, expression studies have demonstrated that addition of the
subunit to
1/
1 and
1/
1/
2L combinations produces
receptors that have relatively high affinity for GABA, little receptor
desensitization, and slow recovery (Saxena and Macdonald 1994
,
1996
). These properties resemble those described here for
GABAA receptors in VZ cells. The
subunit may
contribute to the distinct properties of native
GABAA receptors in proliferative cells because
subunit mRNA has been detected in the neocortical VZ
(Poulter et al. 1993
), and recent evidence has shown
that the
subunit selectively associates with the
4 subunit,
which is expressed at high levels in the VZ (Homanics et al.
1998
; Ma and Barker 1995
).
In the embryonic CP 3,
2/3, and
3 appear to be the predominant
subunits expressed (Ma and Barker 1995
); however, other
subunits (
1,
2, and
5), the
subunit (Poulter et
al. 1993
), and the
2 subunit (Laurie et al.
1992
) also have been localized to the CP. Interestingly,
expression studies have shown that addition of the
2 subunit can
increase the desensitization rate of recombinant receptors
(Saxena and Macdonald 1994
). Expression of the
2
subunit by neurons in the CP could account for some of the differences in receptor properties observed between cells in the VZ and CP. Furthermore the
2 subunit has been shown to be critical for the postsynaptic clustering of GABAA receptors and
synaptogenesis in cultured cortical neurons (Essrich et al.
1998
).
Although differences in subunit composition may underlie the
differences in receptor properties, we cannot dismiss the possibility that the physiological state of the cell can influence receptor function. Levels of intracellular Ca2+
concentration
([Ca2+]i) and
degree of receptor phosphorylation have been shown to change the peak
current and rate of desensitization of GABAA
receptors (Moss and Smart 1996; Mozrzymas and
Cherubini 1998
). Thus differences in intrinsic cell properties
and not simply subunit composition may contribute to the observed
differences between GABAA receptors in
proliferative and postmitotic cells.
Endogenous mode of GABAA-receptor activation
It is unlikely that synaptic activation of
GABAA receptors occurs in VZ cells. Anatomically
defined synaptic contacts have not been detected in the cortical
proliferative zone, in contrast to the CP where they have been observed
as early as E16 (Balslev et al. 1996). Consistent with
the anatomic studies, physiological studies have failed to detect
synaptic potentials in either gap junction coupled (Fig. 6)
(LoTurco et al. 1995
) or uncoupled, presumably
postmitotic, VZ cells (LoTurco et al. 1991
).
Nevertheless, evidence has suggested that endogenous
GABAA-receptor activation does occur in the
proliferative zone (LoTurco et al. 1995
). Whole cell
recordings of VZ cells display outward current shifts on application of
BMI, suggesting the presence of a tonically released endogenous ligand
(LoTurco et al. 1995
). The physiological properties of
GABAA receptors in VZ cells, namely the
relatively high apparent affinity for GABA and the relative lack of
desensitization, would increase the likelihood of tonic receptor
activation from low levels of nonsynaptically released ligand.
Immunostaining has demonstrated that GABA positive cells are localized
directly above as well as within the VZ (Behar et al.
1996
; Cobas et al. 1991
; Lauder et al.
1986
; Van Eden et al. 1989
). Growth cones
arising from these cells could be the source of endogenous GABA release (Taylor et al. 1990
).
Synaptically mediated GABAA-receptor activation
is also unlikely to occur during neuronal migration because no synapses
have been detected in the intermediate zone (Balslev et al.
1996; Bourgeois and Rakic 1993
). However,
GABAA receptors expressed by migrating neurons
could be activated by nonsynaptically released agonist. Studies of
neocortical cells in culture have shown that immature neurons migrate
toward a localized source of GABA (Behar et al. 1996
).
In addition, the migratory promoting effects of GABA occur at lower
concentrations for VZ-derived cells than for CP-derived cells
(Behar et al. 1998
). Our data concerning the greater
sensitivity of VZ cell GABAA receptors are
consistent with this observation. There are several potential cellular
sources for GABA as a migration signal in the developing cortex.
Immunohistochemical studies have demonstrated a differential
distribution of GABA-positive cells throughout the embryonic cortical
wall. For example, there are a large number of GABA-positive cells at
the top of the VZ and in the SP and MZ at E17 in rat (Behar et
al. 1996
; Cobas et al. 1991
). These cells may
provide GABA gradients in the cortical environment that a cell may
sense as it migrates from the VZ to the CP.
Not until cells settle in the CP would synaptic transmission play a
role in GABAA-receptor activation. As shown here,
sPSCs first are detected at E18. Additionally, recent
immunocytochemical analyses of the developing somatosensory cortex of
mice and rats have identified GABAergic synaptic contacts at the
earliest ages studied (P4 in mouse and P5 in rat), demonstrating that
these synapses are anatomically as well as physiologically developed in
the neonate (De Felipe et al. 1997; Micheva and
Beaulieu 1996
). Our results indicate that during early cortical
development, the majority of spontaneous synaptic activity is mediated
by activation of GABAA receptors. This contrasts
with a previous study in which GABAA-mediated
sPSCs were not observed in recordings from P3 to 8 layer II/III
pyramidal neurons (Burgard and Hablitz 1993b
). However,
as emphasized by these authors, the recordings were preformed with
ECl
set near the resting membrane
potential, which would diminish the driving force for
Cl
through the GABAA
channel, making the detection of these events difficult (Burgard
and Hablitz 1993b
). These early sPSPs most likely arise from
intrinsic GABAergic neurons in the developing cortex (Fig.
8B). GABAergic cells in marginal zone (MZ) and subplate (SP)
cannot be ruled out as contributing to the sPSCs recorded in CP and PN
cells; however, the majority of subplate projections to CP are
glutamatergic (Finney et al. 1998
). Additionally, while GABAergic cells are present in the MZ and SP at E16, the earliest sPSCs
are detected at E18, the same age as GABA-positive cells appear in the
CP (Cobas et al. 1991
). Although immature GABAergic cortical neurons are spontaneously active, they are not easily activated by afferent stimulation. Little or no
GABAA-dependent synaptic potentials are evoked
with stimulation of cortical afferents in the perinatal cortex
(Agmon et al. 1996
; Burgard and Hablitz 1993a
; Kim et al. 1995
; Luhmann and
Prince 1991
). Using high-intensity stimulation, it was found
that activation of cortical afferents during the first few postnatal
days can produce polysynaptic responses that contain a
GABAA-mediated component (Agmon et al.
1996
). However, the GABAA-mediated
polysynaptic responses fatigued easily with repeated stimulation,
suggesting that afferent drive of cortical GABAergic interneurons is
weak. Therefore although GABAergic interneurons form functional
synapses and are spontaneously active in early neocortex, afferent
activation of these cells, both by feedback and feedforward pathways,
is poorly developed. During the second postnatal week, activation of
cortical inputs can reliably evoke GABAergic synaptic potentials
(Agmon and O'Dowd 1992
; Luhmann and Prince
1991
).
Downstream effects of GABAA-receptor activation during development
In addition to serving an inhibitory function in more mature
cells, GABAA-receptor activation may play a
maturational role. Many of the developmental effects of GABA are
thought to occur during the embryonic and early postnatal periods when
GABAA-receptor activation produces membrane
depolarization (Berninger et al. 1995; Cherubini
et al. 1991
). In proliferative cells in the VZ, activation of
GABAA receptors has been shown to downregulate
DNA synthesis measured by thymidine or 5-bromo-2'-deoxyuridine
incorporation assays (LoTurco et al. 1995
). The GABA
effect on DNA synthesis was abolished by furosemide, which negatively
shifted ECl
, suggesting that GABA-induced
depolarization is the signal that influences DNA synthesis. Likewise,
activation of GABAA receptors inhibits the proliferative
effects of basic fibroblast growth factor in cortical progenitor cells
in culture (Antonopoulos et al. 1997
).
GABAA-receptor activation also has been shown to influence the morphology and motility of young hippocampal and neocortical neurons in cell culture (Barbin et al. 1993
;
Behar et al. 1996
, 1998
; Marty et al.
1996
). Additionally, activation of GABAA receptors has effects on cell survival (Ikeda et al. 1997
) and
gene expression (Marty et al. 1997
). The depolarizing
action of GABAA-receptor activation has been shown to
increase [Ca2+]i through activation of
voltage-gated Ca2+ channels (LoTurco et al.
1995
; Yuste and Katz 1991
), suggesting Ca2+-dependent second-messenger pathways may mediate the
developmental effects of GABA. For example, GABA-induced depolarization
has been shown to upregulate brain-derived neurotrophic factor
expression (Berninger et al. 1995
). The mechanism may
involve downstream activation of the transcription factor CREB because
a pathway involving depolarization, voltage-dependent Ca2+
increase, and activation of CREB has been shown to induce BDNF expression (Shieh et al. 1998
; Tao et al.
1998
).
Early GABAergic communication also may function in concert with
NMDA-receptor activation to regulate synaptogenesis and/or synaptic
consolidation. It is thought that NMDA receptors underlie the robust
synaptic and developmental plasticity seen in immature animals;
however, in many cases NMDA receptors are silent at negative holding
potentials in developing cortical neurons (Fig. 7) (Durand et
al. 1996; Isaac et al. 1997
). Therefore an
endogenous depolarizing influence must be present at immature synapses
to relieve the Mg2+ block of the NMDA receptor, a role
attributed to AMPA/kainate-receptor activation in more mature cortex.
Possibly GABA could play this role because GABAergic synapses develop
early in cortex and have depolarizing effects (Agmon et al.
1996
; Owens et al. 1996
). Support for this idea
has come from studies in the developing hippocampus in which
GABA-mediated synaptic activity has been shown to have synergistic
actions with NMDA-receptor activation by providing the depolarization
necessary to relieve the Mg2+ block of the NMDA channel
(Ben-Ari et al. 1997
). Furthermore activation of NMDA
receptors may be a signal for synapse stabilization of non-NMDA
receptors (Durand et al. 1996
; Isaac et al.
1997
).
Excitation versus inhibition
It often is stated that during cortical development GABA-mediated
synaptic inhibition lags behind the development of glutamate-mediated excitation (Burgard and Hablitz 1993a; Kim et al.
1995
). The present results demonstrate that
GABAA-mediated synaptic transmission is present
in the perinatal cortex. During early development, spontaneous or
evoked GABAA-mediated synaptic potentials
depolarize postsynaptic cells (Agmon et al. 1996
;
Owens et al. 1996
). Depolarizing effects of GABA have
been reported in developing neurons from a number of brain regions
including the hippocampus (Ben-Ari et al. 1989
;
Cherubini et al. 1990
), spinal cord (Reichling et
al. 1994
; Rohrbough and Spitzer 1996
;
Wang et al. 1994
; Wu et al. 1992
),
cerebellum (Brickley et al. 1996
; Connor et al.
1987
), olfactory bulb (Serafini et al. 1995
),
hypothalamus (Chen et al. 1996
), and retina
(Yamashita and Fukuda 1993
), strongly suggesting a
general role for GABA-mediated depolarization during development. This
has led to the suggestion that, in the immature brain, fast synaptic
transmission is mediated by GABAA receptors
(Cherubini et al. 1991
). However, it should be
emphasized that depolarization and excitation are not necessarily
equivalent. Data presented here suggest that in the developing
neocortex GABA-induced depolarization can excite, that is, trigger
action potential discharge, in presynaptic cells because GABA-evoked
PSCs can be blocked by TTX. The excitatory effect of
GABAA-receptor activation is likely due to
ECl
, which can be above spike
threshold in some immature neurons (Owens et al. 1996
).
Whether GABAA-receptor activation inhibits postsynaptic cells depends on ECl
and the resting membrane potential. In cases where
ECl
is more positive than the resting
potential but more negative than action potential threshold,
depolarizing GABAA-mediated responses can produce
inhibition by shunting other conductances (Kaila 1994
; Mody et al. 1994
). However, even when
GABAA currents produce shunting inhibition in
immature neurons, the resulting membrane depolarization can facilitate
action potential discharge to a subsequent excitatory input that occurs
during the falling phase of the GABAA response (Chen et al. 1996
; Gao et al. 1998
). As
[Cl
]i decreases
perinatally, the excitatory effects of GABA diminish, and the net
effect of synaptic GABAA-receptor activation
becomes inhibitory even though GABA may still depolarize the cell
membrane. This is reflected by the ability of BMI to induce
epileptiform activity, which develops during the latter part of the
first postnatal week (Burgard and Hablitz 1993a
;
Kriegstein et al. 1987
). Therefore in the early
postnatal cortex, GABAA-receptor activation may
excite cells, but it is also likely to inhibit or facilitate other
excitatory inputs depending on their temporal pattern.
Concluding remarks
The current report adds to a growing body of literature that
suggests GABA-mediated signaling plays a role in the development of
neural structures. This role of GABA may be unrelated to its importance
as a mediator of fast synaptic inhibition in the mature nervous system.
Initially, the trophic effects of GABA were thought to be mediated by
membrane hyperpolarization or inhibitory action (Meier et al.
1991). It now appears that GABA-mediated developmental effects
are dependent on membrane depolarization (Berninger et al.
1995
; LoTurco et al. 1995
). A role for GABA in
nervous system development is supported by observations in cortex that
show that GABAergic signaling occurs well before the onset of synaptic
inhibition. Many of the growth-related effects may rely on nonsynaptic
or paracrine receptor activation. Interestingly, GABA signaling systems are present in invertebrates including flatworms and snails
(Bargmann 1998
; Morse et al. 1980
),
demonstrating that a signaling role for GABA has been conserved through
evolution. Moreover, in mollusks GABA exerts its effects through
membrane depolarization (Trapido-Rosenthal and Morse
1986
). It is thus possible that a GABA signaling pathway arose
in ancient organisms to serve a trophic role the effects of which on
growth or gene expression were dependent on depolarizing membrane
effects. As neural assemblies evolved, GABA may have acquired a new
role as an inhibitory synaptic transmitter possibly as a consequence of
a change in neuronal Cl
homeostasis.
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ACKNOWLEDGMENTS |
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We thank Dr. Gareth Tibbs for providing the fitting routine used in the dose-response experiments and Drs. Alexander Flint, Kai Kaila, and Joseph LoTurco for helpful comments on the manuscript.
This work was supported in part by Research Grant FY95-0879 from the March of Dimes Birth Defects Foundation and Grants NS-21223 and NS-35710 from the National Institute of Neurological Disorders and Stroke.
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FOOTNOTES |
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Address for reprint requests: A. R. Kriegstein, Dept. of Neurology, College of Physicians and Surgeons of Columbia University, 630 West 168th St., Box 31, New York, NY 10032.
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 5 February 1999; accepted in final form 16 April 1999.
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REFERENCES |
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