Persistent Activation of GABAA Receptor/Clminus Channels by Astrocyte-Derived GABA in Cultured Embryonic Rat Hippocampal Neurons

Qi-Ying Liu, Anne E. Schaffner, Yoong H. Chang, Dragan Maric, and Jeffery L. Barker

Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892-4066


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Liu, Qi-Ying, Anne E. Schaffner, Yoong H. Chang, Dragan Maric, and Jeffery L. Barker. Persistent Activation of GABAA Receptor/Clminus 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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


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INTRODUCTION
METHODS
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REFERENCES

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 MOmega 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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 >1GOmega . 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).



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Fig. 1. Embryonic hippocampal neurons cultured on astrocytes exhibit enhanced levels of surface-accessible baseline current together with intensified fluctuations compared with neurons cultured on poly-D-lysine (PDL). The neurons were recorded in whole cell mode with Cl--filled patch pipettes to set ECl at ~0 mV. A1: when recorded in the incubation medium, neurons cultured on PDL typically exhibit a steady-state baseline current at a holding potential of -80 mV, which is superimposed with microscopic fluctuations or "noise" that disappears at ~0 mV and reappears at positive potentials. Superfusion with fresh saline rapidly lowers the level of inwardly directed, negative baseline current and markedly reduces the associated noise. A2: neurons cultured on astrocytes exhibit greater levels of negative baseline current that are superimposed with intensified fluctuations. Both baseline current and noise are markedly reduced by perfusion. A3: prior, 4-day treatment of astrocytes with 100 µM 3-mercaptopropionic acid (3-MPA), which blocks glutamic acid decarboxylase synthesis of GABA in neurons, does not diminish the astrocyte-derived effects on neurons. B1: the slope conductance calculated at both negative and positive potentials from the current-voltage plot is increased several-fold or more in neurons on astrocytes compared with those on PDL. B2: the noise, quantified as membrane current variance, disappears at ~0 mV, reappears at positive potentials, and is many-fold greater in neurons on astrocytes compared with neurons on PDL.

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.



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Fig. 2. Astrocyte-conditioned medium mimics the facilitatory effects of co-cultured astrocytes on the baseline current signals in a bicuculline-sensitive manner. A1: a neuron cultured on PDL for 24 h exhibits a perfusion-sensitive baseline current and noise similar to that illustrated in Fig. 1A, 1 and 2. A neuron cultured for 24 h on PDL in astrocyte-conditioned medium (ACM) exhibits a perfusion-sensitive steady-state baseline current and noise similar to that illustrated for co-cultured neurons (Fig. 1A2). A3: inclusion of 100 µM bicuculline (BIC) in ACM for the 24-h incubation period eliminates much of the baseline current and noise along with a sensitivity to superfusion with fresh saline. B1: the increase in slope conductance evident in ACM at negative and positive potentials is blocked by bicuculline. B2: the increase in membrane current variance in ACM is completely blocked by bicuculline at negative potentials and partially blocked at positive potentials.

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.



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Fig. 3. ACM generates nonlinear current-voltage relations in a hippocampal neuron. The neuron was cultured for 24 h on PDL in ACM, then patch-clamped with a Cl- -filled pipette. A: sequential steps in holding potential from -80 to +40 mV evoke step-wise changes in approximate steady-state current recorded in ACM before superfusion with Tyrode's. Large amplitude, inward fluctuations are synaptic-like transients and appear intermittently at -80 mV, while smaller ones of reversed polarity appear at positive potentials. After superfusion, almost all of the fluctuations disappear from the current trace and the same steps in voltage now evoke only modest changes in current. B: current-voltage (I-V) relations for 5 neurons in ACM show a nonlinear I-V relationship only at the most negative potentials. After superfusion with Tyrode's, the I-V relations become linear throughout. Slope conductance calculated over the linear portion of the I-V curve in ACM averages 2.8 nS, almost 10-fold greater than that calculated after superfusion (0.3 nS).

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.



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Fig. 4. Astrocyte-derived substances that sustain most of the steady-state current and superimposed noise reside in a low-molecular-weight (<10 kDa) fraction. ACM was fractionated before use in culturing neurons. A neuron cultured in ACM exhibits several hundred pA of inwardly directed current (A and E1) and superimposed noise (E2) that can be immediately perfused away while a neuron cultured in unconditioned medium shows little current or noise without sensitivity to perfusion. A neuron recorded in the low-molecular-weight fraction of ACM possesses significantly more perfusable current and noise (C, E1, and E2) than a neuron cultured in the high-molecular-weight fraction of ACM (D, E1, and E2).

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).



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Fig. 5. Pharmacological study of the current-voltage relations induced by astrocyte-conditioned Tyrode's solution (ACT) reveals bicuculline-sensitive and -resistant components. A: steady-state currents recorded at -80 mV and during 1-s voltage ramps (from -80 to +40 mV, top) under control (CTRL) and different experimental conditions are superimposed. ACT rapidly induces a steady-state current (IACT), approximating that induced by 3 µM GABA. In this cell, bicuculline (BIC) blocks IACT incompletely while BIC plus strychnine (STRY) together block IACT completely. B: current traces recorded during the ramp commands are superimposed to show the similar behavior of those evoked by ACT and GABA and the bicuculline- and strychnine-sensitive components. C: current traces during ramp commands after subtracting the current signal generated under control conditions reveal the same results due entirely to Cl- conductance.

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).



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Fig. 6. Pharmacology of currents generated by ACM. Bar graph summary of peak current responses recorded at -80 mV. IACM is ~70% of the Cl- current induced by 3 µM GABA, but not significantly different from it (P > 0.05) due to the variability in the response amplitudes. Bicuculline (BIC) blocks most, but not all of IACM (**P < 0.01), while BIC and strychnine (Stry) together completely eliminate it. Glycine (20 µM) induces a Cl- current response of relatively low amplitude (~100 pA), which is not statistically different from that induced by ACM in the presence of BIC. Numbers in parenthesis indicate numbers of neurons recorded in each group.



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Fig. 7. Close correspondence between glycine-induced current and BIC-insensitive IACT. A 1-s ramp command was applied under different conditions, as in Fig. 5. Currents during the ramps are plotted against the corresponding voltage values before (A) and after (B) subtracting that recorded under control conditions. There is a close correspondence between the currents induced by ACT in the presence of BIC and those induced by 20 µM glycine, but only over the negative range of membrane potential.

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.



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Fig. 8. Spectral analysis of baseline current signals recorded in neurons before perfusion under different experimental conditions reveal 2 Lorentzian components with similar values. Spectra were calculated for baseline current signals recorded at -80 mV in neurons before perfusion under different conditions (on PDL in ACM, on astrocytes, on PDL). Spectra calculated for baseline fluctuations before perfusion can be fitted by 2 Lorentzian terms (thin lines), indicating 2 exponentially distributed components with most of the power at low frequencies (<5 Hz) and an approximately parallel shift to higher levels of power in neurons on astrocytes or in ACM. Corner frequencies (fc, downward arrows) can be used to estimate burst-length durations, tau , from tau  = (2pi fc)-1, which are 100 and 2 ms (on PDL), 84 and 2.7 ms (in ACM), and 61 and 2.6 ms (on astrocytes). After perfusion or in the presence of 100 µM bicuculline the power drops in both components so that the entire spectrum can be fitted by a 1/f term, where power declines monotonically. Thus astrocytes and ACM boost the power in both components whose estimated burst-length duration values are generally similar to each other.


                              
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Table 1. Inferred unitary properties of channels underlying Cl- currents evoked in cultured embryonic rat hippocampal neurons recorded in different conditions and with different amino acids

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.



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Fig. 9. Elementary properties of single channels directly activated by GABA and ACM in outside-out patches are identical. Single-channel currents activated by 2 µM GABA and ACM were recorded at -80 mV in the same outside-patches excised from hippocampal neuron cell bodies cultured for 3 days. A, 1 and 2: all-or-none step-wise increases in current are evident in selected traces. C, closed; O, open. B, 1 and 2: the amplitude distributions can both be fitted by Gaussian functions whose means give similar averages. C, 1 and 2: open-states are biexponentially distributed with similar values for each.

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+.



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Fig. 10. ACM triggers GABAergic transients superimposed on the steady GABAergic baseline signal. The recordings were all made in the whole cell mode with Cl--filled pipettes from cells that had been cultured for 3-5 days. A: a neuron in ACM exhibits random, inwardly directed transients superimposed on a baseline exhibiting low-amplitude fluctuations. The transients are displayed at higher temporal resolution. Both tonic and transient signals disappear immediately on perfusion and are blocked by diffusion of bicuculline to the neuron (not shown), thus identifying all the activity as GABAergic. B: a neuron cultured in ACM exhibits both tonic and transient GABAergic signals. Addition of 20 µM bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) into the static bath rapidly eliminates the transients without significantly affecting the tonic baseline signal, which disappears on perfusion. C: applications of ACM derived from hippocampal (C1) and cortical astrocytes (C2) to a perfused neuron trigger immediate inward current responses, which decay to a variable extent and are superimposed with transients, both of which disappear rapidly on cessation of the ACM application.

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.



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Fig. 11. Ca2+ imaging reveals bicuculline-sensitive ACM-induced Cac2+ elevation. Four neurons representative of the results are illustrated. Under control conditions, baseline Cac2+ averages 41 ± 5 nM in 25 hippocampal neurons. ACM induces an elevation in Cac2+ that peaks in ~100 s and then recovers close to baseline levels during continued perfusion. The peak amplitude of the Cac2+ elevation averages 78 ± 10 nM (P < 0.01). Bicuculline (100 µM) attenuates or eliminates the ACM-induced rise in Cac2+ in all neurons, which can be readily recovered after washing.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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