Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892-4066
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Liu, Qi-Ying,
Anne E. Schaffner,
Yoong H. Chang,
Dragan Maric, and
Jeffery L. Barker.
Persistent Activation of GABAA
Receptor/Cl Channels by Astrocyte-Derived GABA in
Cultured Embryonic Rat Hippocampal Neurons.
J. Neurophysiol. 84: 1392-1403, 2000.
Whole cell
patch-clamp recordings using Cl
-filled pipettes
revealed more negative levels of baseline current and associated current variance in embryonic rat hippocampal neurons co-cultured on a
monolayer of astrocytes than those cultured on
poly-D-lysine. These effects were mimicked by culturing
neurons on poly-D-lysine in astrocyte-conditioned medium
(ACM). The baseline current and variance decreased immediately in all
cells after either local perfusion with saline or exposure to
bicuculline, an antagonist of GABA at GABAA
receptor/Cl
channels. Baseline current and
variance in all cells reached a nadir at ~0 mV, the calculated
equilibrium potential for Cl
. Perfusion of ACM
rapidly induced a sustained current in neurons, which also reversed
polarity at ~0 mV. Bicuculline attenuated or eliminated the
ACM-induced current at a concentration that completely blocked
micromolar GABA-induced current. Quantitative analyses of
spontaneously occurring fluctuations superimposed on the ACM-induced
current revealed estimated unitary properties of the underlying
channel activity similar to those calculated for GABA's activation of
GABAA receptor/Cl
channels. Bicuculline-sensitive synaptic-like transients, which reversed at ~0 mV, were also detected in neurons cultured in ACM, and
these were immediately eliminated along with the negative baseline
current and superimposed current fluctuations by perfusion. Furthermore
bicuculline-sensitive synaptic-like transients were rapidly and
reversibly triggered when ACM was acutely applied. ACM induced an
increase in cytoplasmic Ca2+ in cultured
embryonic hippocampal neurons that was completely blocked by
bicuculline and strychnine. We conclude that astrocytes release
diffusible substances, most likely GABA, that persistently activate
GABAA receptor/Cl
channels in co-cultured neurons.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Astroglial cells proliferate and
differentiate throughout the CNS during the late embryonic/early
postnatal period, largely coinciding with the end of neurogenesis and
the differentiation of postmitotic neurons into fast-transmitting
networks. Astroglial cells have also been shown to modulate the
expression and distribution of ion channels, transmitter receptors, and
the Cl ion gradient in central neurons
(Chen et al. 1995
; Joe and Angelides 1992
; Liu et al. 1997b
;
Mandelzys and Cooper 1992
; Raucher and Dryer
1994
; Smith and Kessler 1988
; Wu and
Barish 1994
). In addition, astrocytes have been found to
influence the development of fast excitatory and inhibitory synaptic
signals among cultured neurons (Li et al. 1999
;
Pfrieger and Barres 1996
).
We reported previously that embryonic rat hippocampal neurons
grown either on a monolayer of astrocytes derived from postnatal tissue
or in medium conditioned for 24 h by astrocytes (as
astrocyte-conditioned medium) exhibit significantly greater membrane
surface areas and amino acid transmitter current densities than neurons
cultured on poly-D-lysine (PDL) (Liu et al. 1996,
1997b
, 1998
). Antagonism of GABA at GABAA
receptor/Cl
channels by bicuculline or
picrotoxin blocked the differentiating effects of both astrocytes and
ACM, suggesting the involvement of these amino acid receptors in
mediating the differentiating signals from astrocytes. However,
addition of the GABAA receptor agonist muscimol
to neurons on PDL could not by itself mimic the differentiating effects
attributed to astrocytes (Barbin et al. 1993
; Liu
et al. 1997b
). Thus the pharmacological sensitivity of the
astrocyte differentiating signals together with the latter results
reveals a necessary but not sufficient GABAergic component in the
astroglial-neuron communication. In this regard, it has been reported
that astrocytes can synthesize and secrete GABA (Bowery et al.
1976
; Pearce et al. 1981
; Wu et
al. 1979
) and/or GABA-like substances (Barbin et al.
1993
; Liu et al. 1997b
). Moonen and colleagues
reported that soluble astro-factors mimicked the effects of "inverse
agonists" at benzodiazepine receptors on neurons and reduced GABA's
activation of Cl
channels (Rigo et al.
1994, 1996). In the present study, we report that astrocytes
derived from postnatal hippocampal and cortical tissue release
diffusible factor(s), which directly activate
Cl
channels continuously in cultured embryonic
rat hippocampal neurons. The pharmacological sensitivity and
biophysical properties of the Cl
channels
activated by ACM lead to the conclusion that GABA accounts for most if
not all of the activity. Parts of this study have been published
previously in abstract form (Liu et al. 1997a
).
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Culture of astrocytes and collection of conditioned media
The procedures for culturing hippocampal and cortical astrocytes
and collecting conditioned media were published previously (Liu
et al. 1997b). Briefly, 3-day-old rat neonates were quickly decapitated with surgical scissors. Cortices or hippocampi were removed, cleaned of meninges, and placed in 10 ml L-15 medium with 50 U/ml gentamicin. The tissues were mechanically triturated, and
dissociated cells were centrifuged at low speed. Cells were resuspended
in plating medium consisting of Dulbecco's modified Eagle medium
(DMEM) (GIBCO, Grand Island, NY) supplemented with 10% fetal calf
serum (FCS) and 50 U/ml gentamicin and plated at the equivalent of two
brains per flask in 75 cm2 flasks. When a
confluent monolayer formed (after ~1 wk), the flasks were tightly
capped and placed overnight on a rotary shaker at 180 rpm at 37°C to
remove microglia, O-2A progenitor cells, and debris. Cultures were
treated with A2B5 ascites and rabbit complement to kill any remaining
neurons and O-2A progenitors, resulting in nearly pure type 1 astrocyte
cultures (A2B5
GFAP+
epithelioid cells) as determined by immunocytochemical analysis (not
shown). Serum-free conditioned medium was generated by washing the
culture flasks twice, then incubating them with 12 ml of minimum essential medium (MEM) (GIBCO) plus (in µM) 109 putrescine, 0.04 progesterone, 0.06 sodium selenite, 0.03 T3, 0.12 corticosterone, 1.67 insulin, 0.001% albumin, and 0.02% transferrin
(N3 components) (Romijn et al. 1984
) for 24 h. The
conditioned media were collected and usually used the same day. In some
experiments, harvested media were frozen at ~70°C and tested later.
In other experiments, astrocyte-conditioned Tyrode's solution (ACT)
was used instead of ACM to eliminate any ambiguous effects of additives
(Liu et al. 1998
). For co-culture experiments of
astrocytes and neurons, the confluent astrocyte cultures were first
trypsinized, then replated onto 35-mm culture dishes precoated with
low-molecular-weight PDL (53K) (Sigma, St. Louis, MO). When the cells
reached confluence again they were transiently exposed to 10 µM
cytosine arabinoside for 2 days and then maintained for
7 days in
DMEM with 5% FCS before being used in co-culture.
Dissociation and culture of embryonic rat hippocampal neurons
Hippocampal neurons were dissociated at embryonic (E) day 19 and
cultured, as previously described (Liu et al. 1996).
Briefly, embryos were obtained by caesarian section from pregnant
mothers, which were anesthetized with CO2 and
killed by cervical dislocation. Embryos were quickly decapitated with
surgical scissors and hippocampal tissue was dissected, minced into
small pieces, transferred into 5 ml Earle's balanced salt solution
containing 20 U/ml papain, 0.01% DNase (both from Boehringer Mannheim,
Indianapolis, IN), 0.5 mM EDTA and 1 mM L-cysteine, and
rocked in an incubator for 35-40 min at 37°C. Single neurons,
obtained by triturating the tissue with a Pasteur pipette, were
resuspended in Earle's balanced salt solution with 1 mg/ml trypsin
inhibitor and 1 mg/ml bovine serum albumin and layered over 5 ml of
Earle's balanced salt solution with 10 mg/ml trypsin inhibitor and 10 mg/ml bovine serum albumin in a 15-ml plastic centrifuge tube. The
gradient was spun at ~80 g for 5 min, effectively removing
dead cells and debris from the suspension. The cell pellet was
resuspended in desired medium (e.g., conditioned Tyrode's solution or
conditioned MEM/N3) and plated at a density of 3.5-4 × 105 cells/dish on a monolayer of astrocytes or
directly on PDL in 35-mm plastic culture dishes. The cultures were kept
at 37°C in a humidified atmosphere containing 10%
CO2. Culture medium was changed once a week. All
animal procedures were done in accordance with the Guide for the
Care and Use of Laboratory Animals in the US.
Current recording and analysis
All recordings were made from neurons cultured for 1 day to 1 wk. In most experiments, the culture medium was not replaced immediately to detect possible direct effects of substances in the
culture medium on the electrical properties of embryonic neurons. In
some experiments, dishes were removed from the incubator and the
culture medium was completely replaced with Tyrode's solution containing (in mM) 145 NaCl, 5.4 KCl, 1.8 CaCl2,
0.8 MgCl2, 10 glucose, and 10 HEPES-NaOH, pH 7.4 and 310 mOsm. Standard patch-clamp recordings (Hamill et al.
1981) were made with pipettes pulled in three stages from 1.5 mm OD glass capillary tubes (WPI, Sarasota, FL) with a
computer-controlled pipette puller (BB-CH-PC, Mecanex SA, Switzerland).
These pipettes had a resistance of 3-5 M
when filled with internal
solution composed of (in mM) 145 CsCl, 2 MgCl2,
0.1 CaCl2, 1.1 EGTA, 5 HEPES, 5 ATP (potassium
salt), and 5 phosphocreatine (pH 7.2 and 290 mOsm). Whole cell currents
were recorded with a L/M EPC-7 patch-clamp amplifier (Medical Systems, Greenvale, NY) at a gain of 5 mV/pA. Series resistance was compensated for 70%. Current signals were stored on video cassettes via a videocassette recorder (VCR) and a VR-100 digital recorder (Instrutech, Port Washington, New York) for later off-line digitization with Digidata 1200 (Axon Instruments, Foster City, CA) and analysis with
Pclamp V6.0 (Axon Instruments) on a Pentium-based personal computer.
Well-established techniques in fluctuation analysis were used to
estimate the unitary properties of channels underlying baseline holding
currents and ACM-, ACT-, GABA-, and glycine-induced current responses
(Neher and Stevens 1977
). Briefly, membrane currents
were high-pass filtered at 0.1 Hz and low-pass filtered at 1 Hz with a
eight-pole Butterworth filter (Model 9002, Frequency Devices,
Haverhill, MA), then appropriately amplified to allow computer-assisted
analysis using Strachclyde electrophysiological software SPAN (Dr. John
Dempster, University of Strathclyde, Glasgow, Scotland). Spectra were
consistently well fitted with two Lorentzian functions. For outside-out
single-channel recordings, the extracellular solution contained (in mM)
142 NaCl, 8.1 CsCl, 1 CaCl2, 6 MgCl2, 10 glucose, and 10 HEPES-CsOH, pH 7.3 and
310 mOsm, while the pipette solution contained 153 CsCl, 1 MgCl2, 5 EGTA, and 10 HEPES-CsOH (pH 7.3) and 290 mOsm. All recordings were carried out at room temperature (22-25°C)
on a Nikon inverted microscope. For superfusing the cells, we used a
perfusion system composed of a locally made controller and miniature
electric solenoid valves (The Lee Co., Essex, CT) that allows fast
switching (<200 ms complete solution exchange time) among different
solutions (Liu et al. 1999
). The perfusion rate
(~0.3-0.5 ml/min) was controlled by the air pressure applied to the
solution reservoirs.
Calcium imaging
Neurons were loaded with 1 µM Fluo-3/AM (Molecular Probes,
Eugene, OR) in a standard bath solution for 30 min at 37°C, washed, and then maintained at 37°C for 45 min for ester hydrolysis. Digital imaging of Fluo-3-loaded cells was attained using the Zeiss Attofluor RatioVision workstation (Atto Instruments, Rockville, MD) equipped with
an Axiovert 135 inverted microscope (Carl Zeiss, Thornwood, NY), a ×40
Fluar objective (Carl Zeiss, Thornwood, NY) and an ICCD camera (Atto
Instruments, Rockville, MD). The Fluo-3 dye was excited at 500-ms
intervals with a 100-W mercury arc lamp filtered at 520-nm long-pass
filter set. All filters were obtained from Chroma Technology
Corporation (Brattleboro, VT). To collect the Fluo-3 fluorescence data,
either square or polygonal-shaped regions of interest (ROI) were
electronically drawn around each of 99 cells per recording field. The
fluorescence intensities from each ROI were digitized with a Matrox
image-processing board and plotted as line graphs using Attograph for
Windows analysis software (Atto Instruments, Rockville, MD). All
measurements were performed at room temperature (22-24°C).
Statistical tests
Data were shown as means ± SE. Two-tailed t tests were used to assess significance. Differences were considered significant if P < 0.05 (*) or P < 0.01 (**).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Embryonic rat hippocampal neurons cultured on astrocytes exhibit significantly different baseline properties than neurons on PDL
In previous studies, we found that astrocytes increase the density
of amino acid-evoked anion and cation current responses in neurons in
vitro (Liu et al. 1996, 1997b
, 1998
) and that the activation of GABAA
receptor/Cl
channels may be involved in the
differentiating effects of astrocytes (Liu et al.
1997b
). To test if GABAA receptors are
activated in neurons co-cultured with astrocytes, whole cell
patch-clamp recordings were first carried out in the growth medium to
investigate baseline properties of neurons grown on PDL or on
astrocytes. In all recordings, seals between pipette and membrane were
>1G
. When voltage-clamped at
80 mV, using
Cl
-filled patch pipettes, neurons cultured on
astrocytes exhibited considerably greater baseline negative holding
current and associated current variance than neurons cultured on PDL
(Fig. 1A, 1 and 3). Membrane current variance reached a nadir at ~0 mV,
which is the equilibrium potential for Cl
ions
(ECl) under these recording
conditions, and increased again at positive potentials (Fig.
1B2). The baseline holding current at
80 mV in neurons
cultured on PDL averaged
42 ± 4 pA (n = 26),
while that in neurons cultured on astrocytes averaged
177 ± 14 pA (n = 35; P < 0.01). Local perfusion
reduced the baseline holding current to
17 ± 3 pA in neurons on
PDL and
25 ± 3 pA in neurons on astrocytes. Thus local
perfusion immediately reduced the inwardly directed, negative current
and associated variance in all recorded cells, indicating that
surface-accessible sources accounted for much of the baseline
properties. On average, ~25 pA of the baseline current (~60%)
originated from surface-accessible sources in neurons on PDL, while
~150 pA (85%) was derived from these sites in neurons on astrocytes.
These results reproduce those previously reported for embryonic rat
hippocampal neurons, which have been attributed to GABA acting in
autocrine and/or paracrine ways (Valeyev et al. 1998
)
and, in addition, reveal a sixfold increase in the contribution of
surface-accessible signals to the baseline properties attributable to
astrocytes. The nadir in membrane current variance and reversal in
current polarity at ECl demonstrate
that the baseline properties of all neurons were dominated by
Cl
ion-dependent processes. These results imply
that the GABAergic component of baseline hippocampal neuron properties
has been enhanced, directly and/or indirectly. However, these astrocyte
effects were still present when neurons were co-cultured with
astrocytes, which were first treated for 24 h with 100 µM
3-mercaptopropionic acid (3-MPA) (Fig. 1, A3, B1,
and B2), an antagonist of the GABA-synthesizing enzyme
glutamic acid decarboxylase in neuronal tissue. Higher concentrations
of 3-MPA were deleterious to astrocytes and could not be used. Thus if
astrocyte-derived GABA does contribute to the enhanced signal recorded
on neurons, its synthesis is not effectively suppressed by this
concentration of 3-MPA, which does attenuate GABA synthesis and release
at this concentration in embryonic cortical neurons (unpublished
observations).
|
Astrocyte-conditioned medium mimics the effects of co-cultured astrocytes on baseline properties of hippocampal neurons
The surface accessibility of the astrocyte-mediated enhancement in
baseline current and variance, as revealed by the dramatic decreases in
both inwardly directed current and variance on perfusion, prompted us
to compare baseline properties in neurons cultured on PDL with those
cultured on PDL in astrocyte-conditioned medium (ACM; Fig.
2). Like neurons cultured on a monolayer
of astrocytes, those cultured on PDL in ACM also exhibited
significantly greater negative baseline current (210 ± 31 pA;
n = 23) and associated current variance compared with
neurons cultured on PDL in the same medium, which had not been
conditioned (
40 ± l pA, P < 0.01; n = 18). Perfusion immediately reduced the baseline
currents to
26 ± 6 and
15 ± 3 pA in neurons cultured in
ACM or in control, respectively. These results closely parallel those
quantified for the set of experiments comparing currents recorded in
neurons on PDL with those recorded in co-cultured neurons. About 88%
of the baseline current signal in neurons grown in ACM (~184 pA) was
eliminated with perfusion, while ~60% of the holding current (~25
pA) was removed by perfusing neurons cultured on PDL.
|
In another set of culture experiments, bicuculline (BIC), an antagonist
of GABA at GABAA
receptor/Cl channels on hippocampal neurons,
was added to ACM. Neurons cultured in ACM with BIC exhibited
significantly less negative baseline current and current variance (Fig.
2A3). The baseline current at
80 mV averaged
171 ± 25 pA in neurons in ACM (n = 11) and
38 ± 5 pA
in ACM containing BIC (P < 0.01; n = 16). Collectively, these results suggest that much, if not all, of
baseline current and variance in neurons, with and without astrocytes
or their secretions, involves random activation of
GABAA receptor/Cl
channels by surface-accessible substances and that astrocytes enhance
this persistent activation via diffusible substances.
We quantified the steady-state properties of neurons over a 120-mV range of potential before and after removal of ACM (Fig. 3). Plots of the current-voltage relations showed inward rectification at the most negative membrane potentials in ACM, which disappeared on perfusion (Fig. 3B). Calculation of steady-state conductance over the 100-mV range in voltage where current-voltage relations were linear showed that before perfusion conductance averaged ~280 pS, while afterwards it averaged only 40 pS (Fig. 3B). Thus ACM enhanced the steady-state conductance over a wide range of physiologically relevant membrane potential about sevenfold.
|
We have shown previously that neurons grown on astrocytes or in
astrocyte-conditioned medium exhibit greater surface membrane areas and
a higher density of functional GABAA
receptor/Cl channels, as revealed by a higher
density of exogenous GABA-induced current recorded whole cell
(Liu et al. 1996
, 1997b
). However, the increased density
of GABAA receptor-coupled
Cl
conductance contributes only partly to the
greater (~318%) baseline current recorded in neurons cultured on
astrocytes because the density of macroscopic
Cl
current induced by exogenous GABA is only
109% greater in neurons cultured on astrocytes than that recorded in
neurons cultured on PDL (Liu et al. 1997b
). Hence, the
increased density of GABAA receptor-coupled
Cl
conductance induced in neurons and the
diffusible substances from astrocytes both factor into the
astrocyte-mediated baseline signal, with the latter contributing the
major share.
Low-molecular-weight fraction of ACM contains most of its facilitating activity
The discovery of diffusible activity secreted by cortical
astrocytes that dominated baseline properties led us to investigate whether substances released by astrocytes cultured from the postnatal hippocampus also induce a similar signal. Neurons cultured in ACM
conditioned by hippocampal astrocytes, like ACM from cortical astrocytes, facilitated the appearance of significant levels of inwardly directed baseline current (several hundred pA) and associated variance, which reversed polarity or reached a nadir in variance at
~0 mV (ECl) (Fig.
4). Neurons were also recorded on PDL in MEM/N3, which had not been astrocyte-conditioned, that exhibited baseline current less than 100 pA when clamped at
80 mV (Fig. 4B). Thus the whole cell recording strategy with
Cl
-filled patch pipettes did not, by itself,
facilitate the appearance of significant inwardly directed baseline
current in all of the cultured hippocampal neurons, which were
recorded. Furthermore there was no change in the baseline current
signal when these neurons with relatively low levels of negative
current were perfused with recording saline (Fig. 4B). This
eliminates mechanical effects of perfusion as contributing to the
phenomenology recorded in neurons with baselines greater than
100 pA.
We fractionated the hippocampal astrocyte-conditioned medium into high
(>10,000 Da) and low (<10,000 Da) molecular weight fractions, then
cultured hippocampal neurons drawn from the same dissociate in
unfractionated and fractionated ACM. Neurons cultured in ACM restricted
to low molecular substances exhibited steady-state negative current
signals similar to those recorded in neurons cultured in unfractionated ACM (compare Fig. 4, A and C). There was also a
perfusion-sensitive, steady-state current detectable in neurons
cultured in fractionated ACM, which contained only high molecular
weight substances, but it was modest (
50 pA) in the three cells
tested (Fig. 4D). These results indicate that most of the
astrocyte-derived substances contributing to the baseline are <10,000
Da.
|
Quantitative comparisons of stepwise current-voltage relationships
recorded in neurons differentiating in the four different conditions revealed neurons in both ACM and ACM containing
low-molecular-weight substances exhibiting rectification at negative
potentials (40 to
80 mV; Fig, 4E1). Neurons recorded in
MEM/N3 or high-molecular-weight-containing ACM did not manifest
these characteristics. The slope conductance calculated over the linear
portions of the steady-state current-voltage relationship were ~7 nS
(unfractionated ACM), 4.4 nS (low-molecular-weight fraction), 1.6 nS
(high-molecular-weight fraction), and 1.4 nS (MEM/N3). The variances in
the baseline signals were significantly greater in neurons
cultured in ACM and low-molecular-weight-containing ACM, which is
consistent with the intensified activation of the currents
accounting for the elevated conductance.
ACT triggers rapidly reversible Cl currents most of
which are bicuculline-sensitive
If diffusible substances are secreted into the culture medium by
astrocytes during the conditioning period, which can be readily perfused away, then superfusing cells and acutely applying ACM might
rapidly induce a Cl current response that is
readily reversible. To eliminate possible ambiguities arising from the
inclusion of additives in the defined culture medium (MEM/N3), we
conditioned Tyrode's solution, the recording saline, using hippocampal
astrocytes (Liu et al. 1998
). In this way, we were able
to test ACT while holding the recording saline constant. Low pressure
application of ACT rapidly induced an inwardly directed current
response that peaked in <1 s and relaxed in several hundred
milliseconds. In 16 cells, ACT induced a current of
470 ± 97 pA, which was not significantly different from that induced by 3 µM
GABA tested in 7 of the 16 cells (
722 ± 235 pA;
P > 0.05). In three of these cells exhibiting both
responses, each was blocked completely by 50 µM BIC. In 12 other
cells, 50 µM BIC attenuated the ACT-induced current
(IACT) by 85 ± 3%
(P < 0.01) so that the residual
IACT averaged
58 ± 15 pA.
However, in the five cells tested IACT
was eliminated in the presence of both BIC and 20 µM strychnine
(Stry). The currents induced by GABA, ACT, and ACT in the presence of
blockers all reversed polarity at ~0 mV, the theoretical equilibrium
potential for Cl
in symmetrical
Cl
recordings (Fig.
5, B and C).
|
We compared the BIC-resistant, Stry-sensitive component of
IACT with that induced by glycine. In
embryonic rat hippocampal neurons cultured for 5 days, glycine 5 µM
did not induce any detectable current. However, 20 µM glycine induced
an average current of
120 ± 35 pA (Fig.
6) that was completely blocked by 20 µM
Stry (n = 7). Like that of the BIC-resistant component
of the ACT-induced current, the glycine-induced current also
reversed polarity at ~0 m V (Fig.
7). Taken together, these results
indicate that the majority of
IACT involved activation of
bicuculline-sensitive GABAA
receptor/Cl
channels, while in some cells a
minor component of IACT was
mediated via opening of strychnine-sensitive Cl
channels. Furthermore Stry partially blocked the GABA-induced Cl
current in these cells (results not shown)
(see also Shirasakik et al. 1991
) and
bicuculline-resistant GABA responses have previously been reported
(Park et al. 1999
).
|
|
Cl ion channel properties underlying baseline current
signals in different conditions are similar to those estimated for GABA
We analyzed the fluctuations in the baseline current signals with
spectral techniques to estimate the kinetics of the
Cl ion channels active in neurons under
different conditions. Power density spectra of baseline current
fluctuations calculated in neurons under the different experimental
conditions (on PDL, on astrocytes and on PDL in ACM) were well-fitted
by two Lorentzian components, indicating two populations of
exponentially distributed openings contributing to the baseline signal
(Fig. 8). The majority of the fluctuating
signals were conveyed by exponentially-distributed openings that
averaged ~75-85 ms, which accounted for ~70% of the power (Table
1). A minority of the power in the
fluctuating signals (~30%) was conveyed by exponentially-distributed
short-lasting openings of ~3 ms. We compared these estimated openings
with those inferred for GABA and glycine to reveal which amino acid
might contribute to the baseline current signals recorded under
different conditions. Spectra calculated for GABA-induced currents were similar to those resolved for the different baselines: long-lasting openings of ~73 ms, which accounted for ~72% of the signal, and short-lasting openings of ~3 ms (~20%). In contrast, spectral analyses of fluctuations superimposed on glycine-evoked
Cl
currents led to estimates of channel
kinetics that were significantly different: long-lasting openings were
~138 ms (~72%) while short-lasting openings were ~6 ms.
Collectively these results strongly suggest that GABA or a GABA- like
substance acting at GABAA
receptor/Cl
channels accounts for most, if not
all, of the surface-accessible baseline current signal recorded in all
of the experimental conditions.
|
|
ACM directly activates Cl channels in excised patches
To compare the properties of Cl channels
activated by ACM with those activated by GABA more directly and to
eliminate the possibility that ACM might trigger the tonic release of
GABA, thereby generating the enhanced baseline signal, we excised
patches from the cell bodies of cultured neurons and recorded
elementary Cl
channel activity in outside-out
patches perfused with ACM or GABA. In three patches, we found that ACM
induced all-or-none elementary current steps (Fig.
9) that had a conductance of 27 ± 2 pS, reversed polarity at ~0 mV (ECl)
and were completely blocked by bicuculline (not shown). GABA applied to
the same patches also activated bicuculline-sensitive, all-or-none
current steps whose amplitude distributions and open-state kinetics
were similar, if not identical to those triggered by ACM (Fig. 9). The
conductance of GABA-induced currents was 26 ± 1 pS. These results
demonstrate that both ACM and GABA directly activate
GABAA receptor/Cl
channels in the same excised patches, which exhibit two exponentially distributed classes of openings that are consistent with the inferences drawn from spectral analyses of current fluctuations induced in whole
cell recordings by ACM and GABA.
|
ACM triggers GABAergic transients superimposed on tonic baseline signals in more differentiated neurons
A subpopulation of hippocampal neurons cultured on astrocytes or
in ACM for ~3-5 days or more exhibited spontaneous synaptic-like transients superimposed on the baseline current signal (Fig.
10A). These transients were
identified as GABAergic based on their exponential decay kinetics,
which matched the time constants summarized for GABA-activated channels
(Table 1), Cl ion selectivity and complete
block by BIC (data not shown) (see also Liu et al.
1998
). Both the GABAergic transients and the fluctuating baseline current signal disappeared immediately after perfusion with
Tyrode's solution, indicating that they were both due to diffusible
substance(s) in ACM. Furthermore the transients, but not the randomly
fluctuating baseline signal, were eliminated within ~2 min following
addition of
1,2-bis(2-aminophenoxy)ethane-N,N,N,N,-tetraacetic acid
acetoxymethyl ester (BAPTA-AM) to the ACM bathing neurons (Fig.
10B). This indicates that fluctuations in cytoplasmic
Ca2+ (Cac2+) near
the sites of GABA release are likely to underlie the transients. Furthermore the insensitivity of the baseline current signal to BAPTA-AM demonstrates the lack of an immediate requirement for fluctuating levels of Cac2+ increases in the
tonic release of GABA from neurons or in the activation of
GABAA receptor/Cl
channels by GABA derived from neurons or astrocytes. Applications of
ACM generated by hippocampal and cortical astrocytes to neurons immediately and reversibly induced GABAergic current transients superimposed on a sustained current signal (Fig. 10C). Thus
astrocytes secrete factors like GABA that activate
GABAA receptor/Cl
channels in a random manner, generating a tonic baseline signal, and in a nonrandom manner, triggering synaptic-like transients, which require elevation in Cac2+.
|
ACM triggers a bicuculline- and strychnine-sensitive Cac2+ elevation
The rapid elimination of the GABAergic transients induced by ACM
following exposure to BAPTA-AM led us to test whether ACM altered
Cac2+ levels in hippocampal neurons. Cells
loaded with Ca2+ indicator dye were perfused with ACM,
which immediately triggered a rise in Cac2+
that was attenuated or eliminated in a reversible manner by
co-application of bicuculline and strychnine (Fig.
11). These effects of ACM to elevate
Cac2+ were eliminated in
Ca2+-free saline, demonstrating that
extracellular Ca2+ was required (not shown).
These results demonstrate that in intact cultured hippocampal neurons
ACM stimulates a rise in Cac2+, which
involves activation of GABAA
receptor/Cl channels. This
Cac2+ response to ACM is consistent with the
depolarizing effects of GABA acting at GABAA
receptor/Cl
channels to stimulate
Ca2+ entry via voltage-dependent
Ca2+ channels. In some way, the sustained
elevation in Cac2+ is prerequisite to the
generation of GABAergic transients triggered by ACM.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Salient findings
Here we have demonstrated that in vitro astrocytes increase by
many-fold the steady activation of GABAA
receptor/Cl channels in embryonic hippocampal
neurons that dominates baseline membrane properties during the initial
phase of neurite outgrowth. Pharmacological and biophysical experiments
revealed that the astrocyte-derived effects are most likely mediated by
GABA present in ACM, which adds to the GABA synthesized and secreted by
embryonic hippocampal neurons during neuritogenesis.
Astrocytes sustain neuronal differentiation in vitro
It has been well established that astrocytes differentiate during
the late embryonic and early postnatal period throughout the CNS as
neurons extend processes and form synapses. In vitro, astrocytes have
long been used to sustain the differentiation of embryonic neurons into
functional circuits and networks (Banker and Goslin
1991). A large and growing body of literature based on results
obtained with "sandwich cultures" of well-differentiated confluent
astrocyte carpets out of direct contact with embryonic neurons has
accumulated over the past 20 yr, indicating that astrocyte secretions
per se are sufficient to support network formation. Hence direct
contact between the two cell types is not required even though they
intermingle and are in close apposition in vivo. That astrocyte-derived
secretions are critical in sustaining neuronal differentiation in a
serum-free, defined medium was revealed by the progressive death of all
neurons over several days in vitro in the absence of co-cultured
astrocytes. Therefore astrocytes supply diffusible substances that play
important, if not critical, roles in the differentiation of isolated
neurons into functional networks.
Astrocytes intensify activation of GABAA
receptor/Cl channels
Previously we reported that astrocyte-derived secretions
upregulated the membrane surface area and the densities of amino acid-evoked anion and cation currents recorded in embryonic hippocampal neurons and that these effects were blocked by antagonists of GABA at
GABAA receptor/Cl
channels and of glutamate at ionotropic receptors (Liu et al. 1998
). In the present study, we recorded a detectable
activation of GABAA
receptor/Cl
channels in embryonic hippocampal
neurons, which were cultured in defined medium on PDL in the initial
absence of well-differentiated astrocytes. The random activation of
these channels on isolated neurons was immediately eliminated by
perfusion or by exposure to bicuculline, thus replicating previous
findings regarding a surface-accessible source of GABA steadily
activating GABAA
receptor/Cl
channels via autocrine and
paracrine mechanisms (Valeyev et al. 1998
). The
GABAergic contribution to the steady-state properties involved
activation of small numbers of Cl
channels
(<20) whose random openings superimposed to generate low-amplitude
(<
50 pA) baseline current signals when the neurons were recorded
with Cl
-filled pipettes and clamped at
80 mV.
After eliminating the endogenous signal with perfusion, the intensity
of this channel activity and the level of DC current were closely
mimicked by applying ~200-500 nM GABA, which did not desensitize but
induced a steady macroscopic current superimposed with microscopic
fluctuations identical to the endogenous GABAergic baseline signal.
Together these results lead us to conclude that during neuritogenesis
in vitro GABA steadily emerges at the neuronal surface of embryonic hippocampal neurons in an unstirred layer where it equilibrates with,
and randomly activates, GABAA
receptor/Cl
channels to dominate baseline conductance.
Astrocyte-derived substances intensified this autocrine/paracrine
activation of GABAA
receptor/Cl channels in neurons ~10- to
20-fold, as reflected in comparative analysis of membrane current
variance quantified in neurons on PDL or on astrocytes or on PDL in
ACM. The intensified Cl
channel activity
resulted in a more negative baseline current (>
100 pA), which
could be mimicked by perfusing micromolar levels of GABA (1-3 µM).
Almost all of the intensifying activity in ACM was present in the
low-molecular-weight fraction (<10 kDa), and this activity was
markedly attenuated by bicuculline and eliminated completely by
bicuculline and strychnine. Although the majority of GABA-evoked
Cl
currents in hippocampal neurons were blocked
completely by bicuculline, some Cl
current
responses to GABA exhibited bicuculline-resistant components, which
could be eliminated by the inclusion of strychnine (unpublished observations). The parallelism in pharmacological antagonism of ACM-
and GABA-induced Cl
currents strongly suggests
that all of the astrocyte-mediated effects involve intensified
activation of GABAA
receptor/Cl
channels.
Astrocyte-derived GABA mediates the intensifying activity
We used fluctuation analysis of baseline current signals recorded
whole cell in neurons on PDL, on astrocytes, or on PDL in ACM to
estimate the unitary properties of the GABAA
receptor/Cl channel activity under the
different experimental conditions. Two Lorentzian components
contributed to each of the spectra and astrocyte-derived substances
intensified the power in both components more or less equally, thus
shifting the spectrum to higher levels in an approximately parallel
manner. Estimated unitary properties were similar to each other and,
after perfusion, to those calculated for GABA, while those calculated
for glycine were significantly different from these. Furthermore higher
concentrations of glycine (~20 µM) were required to activate
Cl
currents than were measured in samples of
ACM using biochemical techniques (submicromolar-micromolar
concentrations), which revealed submicromolar-micromolar levels of GABA
(unpublished observations).
We compared the elementary properties of Cl
channels directly activated by ACM with those activated by GABA in
excised, outside-out patches. We found that the directly measured
Cl
channel properties activated by ACM and by
GABA to be in close agreement. Collectively, these biophysical findings
together with the pharmacological results identify GABA as the most
likely candidate mediating the astrocyte-induced intensification of
GABAA receptor/Cl
channel
activity. In our experiments, the ability of confluent astrocytes to
synthesis and secrete GABA was not affected by 100 µM 3-MPA included
during the 24-h conditioning period. This concentration of 3-MPA
effectively eliminated enzymatic decarboxylation of glutamate and
immunohistochemically detectable GABA in embryonic hippocampal and
cortical neurons (unpublished observations). Thus it is likely that
alternative synthetic pathways known to be expressed by astrocytes (Laschet et al. 1992
) are involved. Our results support
previous findings regarding the presence of GABA in astrocytes
(Blomqvist and Broman 1988
; Holopainen and Kontro
1989
; Lin et al. 1993
; Yang et al.
1998
) and its release (Gallo et al. 1991
;
Holopainen and Kontro 1989
).
Astrocyte-induced GABAergic transients
We have previously reported that astrocytes facilitate the
appearance of GABAergic, then glutamatergic transients in cultured embryonic spinal cord neurons and that these effects are mediated via
diffusible substances (Li et al. 1999). In the present
study on hippocampal neurons, we found that after ~3 days in culture GABAergic Cl
transients emerged superimposed on
the steady baseline signal when the cells were cultured on astrocytes
or on PDL in ACM but not on PDL alone. These results are consistent
with those involving differentiating spinal cord neurons, which showed
that astrocytes facilitated the appearance of transients relative to
their emergence on neurons cultured on PDL (Li et al.
1999
). However, in the previous study, the GABAergic and later
the glutamatergic transients were consistently recorded both after the
ACM had been replaced by physiological saline as well as in perfused
neurons, which were co-cultured with astrocytes. Thus the source of
transmitter mediating the synaptic transients recorded in cultured
spinal cord neurons was not readily accessible to disturbances of the
unstirred layer at the neuronal surface. In the present study, the
GABAergic transients were as accessible as the steady baseline signal
to perfusion; both types of signal were eliminated immediately on
perfusion. In addition, both random steady activation and nonrandom
synchronized, interrupted activation of GABAA
receptor/Cl
channels, which generated tonic and
transient signals, respectively, could be rapidly induced in perfused
neurons by applying ACM. These results indicate that similar to the
tonic baseline signal the transients can readily and reversibly be
induced and may involve surface-accessible sources or compartments of
GABA. Furthermore the two forms of GABAergic signaling at
GABAA receptor/Cl
channels have different requirements for fluctuations in cytosolic Ca2+ (Cac2+) since
exposure to BAPTA-AM, which effectively clamps
Cac2+ at low levels and prevents its
elevations, rapidly eliminated the transient but not the tonic signal.
In intact hippocampal neurons, we found that ACM elevated
Cac2+ levels in a bicuculline-sensitive
manner, indicating that the depolarizing effects of GABA present in ACM
were sufficient to activate Ca2+ entry. Thus an
elevated level of Cac2+ may be prerequisite
to triggering GABAergic transients.
In independent studies on cultured embryonic thalamic neurons, we
reported that submicromolar-micromolar concentrations of GABA
immediately and reversibly induced both tonic and transient signals
(Liu et al. 1997c). The ability of GABA to induce
GABAergic transients was not mimicked by muscimol, which simply
produced a tonic signal reflecting random activation of
Cl
channels. The lack of an effect with
muscimol eliminates a mechanism involving GABAA
receptor/Cl
channel activation and
depolarization of physiologically intact GABAergic neurons putatively
innervating the recorded cell. Rather it implies a structural
requirement for contributing to transients that does not include
muscimol with its planar configuration. Furthermore the effects of GABA
to induce GABAergic transients were present at concentrations that by
themselves did not evoke summating Cl
channel
activity and a sustained DC current signal capable of depolarizing
intact innervating neurons. However, the ability of GABA to induce
Cl
transients but not its ability to randomly
activate GABAA
receptor/Cl
channels was eliminated in
Ca2+-free saline or in saline
containing either Co2+ or verapamil, which blocks
L-type Ca2+ channels. These results demonstrate a
role for extracellular Ca2+ and possibly
Co2+-sensitive Ca2+ entry
via L-type Ca2+ channels in the phenomenology.
We also found that in embryonic thalamic neurons, exogenous GABA could
be loaded into a surface-accessible saturable compartment, which was
not affected by a potent blocker of GABA uptake (tiagabine) and did not
exhibit either voltage sensitivity or a requirement for extracellular
Ca2+ (Liu et al. 1995). Taken
together, these earlier results on embryonic thalamic neurons
differentiating in vitro suggest that GABA-induced GABAergic transients
may involve a surface-accessible compartment, which has some structural
requirements but does not require extracellular Ca2+ to be loaded yet does require
Ca2+ entry and local fluctuations in
Cac2+ for all-or-none unloading. A similar
mechanism may help to explain the ACM-induced GABAergic transients,
which could involve loading of GABA present in ACM followed by
Cac2+-dependent discharge. In this regard, we
have recently found that another GABAmimetic isoguvacine can readily be
loaded onto the surface of embryonic hippocampal neurons in the
presence of a GABA uptake blocker (NO-711) where it immediately and
reversibly replaces endogenous GABA in both tonic and transient forms
of signaling (Vautrin et al. 2000
).
Conclusions
Astrocyte-released GABA intensifies GABAergic autocrine/paracrine
signaling at GABAA
receptor/Cl channels, thus effectively
polarizing differentiating hippocampal neurons near or at
ECl. During morphogenesis,
ECl is sufficiently depolarized (about
50 mV) (Ben-Ari et al. 1989
) that activation of
GABAA receptor/Cl
channels by GABA derived from neuronal and astrocyte sources stimulates
Ca2+ entry via L-type Ca2+
channels (Reichling et al. 1994
). In preliminary
experiments, we have found that ACM can rescue neurite outgrowth in
embryonic hippocampal and cortical neurons, which have been treated
with 3-MPA (to block GAD-derived GABA synthesis), via bicuculline- and
nitrendipine-sensitive mechanisms (D. Maric and J. L. Barker, unpublished observations). These results indicate that
astrocyte-derived GABA provides a critical depolarizing signal that
indirectly stimulates Ca2+ entry, thereby
supporting neuritogenesis.
![]() |
FOOTNOTES |
---|
Address for reprint requests: J. L. Barker, Laboratory of Neurophysiology, NINDS, National Institutes of Health, Bldg. 36, Rm. 2C-02, 36 Convent Dr., MSC 4066, Bethesda, MD 20892-4066 (E-mail: barkerj{at}ninds.nih.gov).
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 28 January 2000; accepted in final form 9 May 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|