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Received 8 September 1997; accepted after revision 10 March 1998.
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ABSTRACT |
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INTRODUCTION |
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Spinal astrocytes expressing glial fibrillary acid protein (GFAP) proliferate and differentiate during the embryonic and postnatal period (Yang, Lieska, Shao, Kriho & Pappas, 1993) when Cl--dependent GABAergic signals switch polarity in vivo (Wu et al. 1992). Here we have used dissociated cell-culture techniques to study the effects of GFAP+ astrocytes on the differentiation of GABAergic signal polarity and associated channel properties expressed by embryonic ventral horn neurons maintained in vitro for several weeks. The results indicate that in vitro, astrocytes accelerate the time-dependent switch in the polarity of GABAergic signals independently of developmental changes in the elementary properties of GABAA receptor/Cl- channels and these astrocyte-induced effects can be reproduced using astrocyte-conditioned medium (ACM). Some of these results have been reported in abstract form (Li, Schaffner, Walton & Barker, 1995).
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METHODS |
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Cell culture
Astrocyte monolayer. Rat pups (3-day-old) were rapidly decapitated. Cortices, free of hippocampi and striata and cleaned of meninges, were removed and placed in 10 ml of L-15 medium with 50 U ml-1 gentamicin. The tissue was triturated through a 5 ml pipette followed by mechanical dissociation through a series of small bore needles (3 × 19 g, 3 × 22 g, 1 × 25 g) and filtered through 62 µm nylon mesh (Nitex, TETKO, Inc., Kansas City, MO, USA). The dissociated cells were centrifuged and resuspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % v/v fetal calf serum (FCS) and 50 U ml-1 gentamicin and plated in 75 cm2 flasks at the equivalent of two brains per flask (all tissue culture supplies from Gibco, Grand Island, NY, USA). The medium was changed after 72 h and twice each week thereafter. When a confluent monolayer was present (after 
1 week) the flasks were tightly capped and placed overnight on a rotary shaker at 180 r.p.m. in an incubator at 37°C. The following morning the supernatant, containing microglia, loosely adherent O-2A progenitor cells and debris, was rapidly removed. Cultures were rinsed once with DMEM and re-fed with plating medium. To eliminate any surviving neurons and O-2A progenitor cells the flasks were then subjected to complement-mediated lysis, as described by Armstrong, Dorn, Kufta, Friedman & Dubois-Dalcq (1992). Briefly, the cultures were incubated with a 1 : 50 dilution of A2B5 ascites in DMEM with 1 % FCS or full strength A2B5 culture supernatant for 1 h at 37°C. Cultures were rinsed twice in DMEM-1 % FCS and treated with rabbit complement diluted 1 : 8 in DMEM-1 % FCS for 1 h at 37°C. To reduce the amount of antibody and expose a greater surface area for antibody binding the cells could also be trypsinized off the flask and resuspended in a small (1-2 ml) volume of antibody solution since A2B5 is a trypsin-resistant surface antigen. After cytolysis, the cultures were rinsed twice in DMEM-1 % FCS and held in DMEM-5 % FCS. The cultures could be maintained in flasks for an indefinite period or trypsinized and transferred to 35 mm2 dishes precoated with 5 µg ml-1 poly-D-lysine (PDL; 53 K, Sigma, St Louis, MO, USA). When cells reached confluency in the dishes they were exposed to 10 µM cytosine arabinoside for 2 days. If flasks or dishes were to be maintained for several weeks the medium was changed to DMEM-2·5 % FCS or Minimum Essential Medium (MEM)-5 % horse serum. Cultures prepared in this way contained 
95 % type-1 astrocytes as determined by GFAP, S100
, and A2B5 immunocytochemistry and morphological examination. Greater than 95 % of the cells on the monolayer were GFAP/S100 positive and A2B5 negative (data not shown).
Astrocyte-conditioned medium. ACM was prepared by the addition of MEM-5 % horse serum to flasks or dishes of 'purified' astrocytes for 24 h; 5 % FCS was added to ACM for incubation with newly dissociated neurons but was not added to ACM intended for neuronal cultures on or after day 4.
Neurons. Pregnant Sprague-Dawley rats were narcotized by CO2 inhalation followed by cervical dislocation. E15 embryos were removed from the uteri by Caesarean section, rapidly decapitated and placed in phosphate-buffered saline at room temperature (20-22°C). Ventral spinal cords were dissociated and placed into a solution of Earle's Balanced Salt Solution (EBSS) containing 20 U ml-1 papain (Worthington Biochemical, Freehold, NJ, USA), 0·01 % w/v DNase (Boehringer Mannheim, Indianapolis, IN, USA), 0·5 mM EGTA, and 1 mM L-cysteine for 45 min at 37°C (Huettner & Baughman, 1986) to initiate dissociation of the cells. After trituration the cells were spun at 300 g for 5 min, and resuspended in EBSS with 1 mg ml-1 bovine serum albumin (BSA; Sigma) and 1 mg ml-1 ovomucoid trypsin inhibitor (Sigma). The cell suspension was layered over 5 ml of EBSS with 10 mg ml-1 each of BSA and ovomucoid and centrifuged at 80 g for 7 min. The neurons were plated in 35 mm diameter plastic dishes (NUNC, Thomas Sci., Swedesboro, NJ, USA) that had been previously coated with high molecular weight PDL or on a confluent layer of astrocytes. Plating medium consisted of modified Eagle's medium (MEM) with 3·7 g l-1 sodium bicarbonate, 6 g l-1 glucose (Gibco), 5 % fetal calf serum and 5 % v/v horse serum. In some experiments medium conditioned by astrocytes for 24 h was used as the culture medium. Cultures were kept at 36°C in a CO2 incubator. The medium was changed twice weekly and the cells were maintained in MEM and 5 % horse serum.
Cultures used for digital videomicroscopy were in 35 mm diameter glass-bottom microwell dishes (MatTek Corp., Ashland, MA, USA).
Microelectrode recording
Cultures were removed from the incubator, medium was removed and replaced with recording saline. Recordings were made on the stage of an inverted phase microscope (Nikon). Patch-clamp recordings were carried out at room temperature either in the whole-cell configuration or with the perforated-patch technique using gramicidin. Ionic currents and voltages were measured with a patch-clamp amplifier (Axopatch 200A), band-pass filtered at 0·1 Hz to 2 kHz and monitored on a storage oscilloscope and displayed on a pen recorder. The electrical signals were recorded at 9 kHz on a four-channel VCR PCM system (Instrutech VR-100B, Great Neck, NY, USA). For fluctuation analysis of GABA-activated current responses, the currents were low-pass filtered at 0·4-250 Hz, digitized (12 bits, National Instrument LAB-PC card) at 2 kHz, stored on a 386 PC microcomputer and analysed using SPAN (software courtesy of J. Dempster, Strathclyde University, Glasgow, UK).
The patch pipettes for whole-cell recordings were filled with (mM): 145 CsCl, 5 Hepes, 2 Mg-ATP, 5 BAPTA, 5 sodium phosphocreatine, pH 7·2, and the osmolarity was adjusted to 310 mosmol l-1 with sucrose. The K2SO4 pipette solution contained (mM): 10 Hepes, 70 K2SO4, 10 KCl, 5 EGTA, 0·5 CaCl2, pH 7·2, with osmolarity at 310 mosmol l-1. Electrode resistance measured in the recording saline was between 4 and 6 M
. Series resistance was 15 M and was compensated for by 80-90 %. The standard external solution contained (mM): 145 NaCl, 10 Hepes, 10 D-glucose, 5·4 KCl, 1·8 CaCl2, 0·8 MgCl2, titrated to pH 7·4 with NaOH, and osmolarity adjusted to 330 mosmol l-1 with sucrose. The low Cl- solution was made by equimolar replacement of NaCl with sodium gluconate.
For perforated-patch recordings, the patch pipette solution contained (mM): 140 potassium gluconate, 10 KCl, 10 Hepes, titrated to pH 7·2 with NaOH, and osmolarity adjusted to 320 mosmol l-1 with sucrose. Gramicidin (Sigma) was initially dissolved in dimethyl sulphoxide (DMSO, 50 mg ml-1). The pipettes were first filled with gramicidin-free pipette solution by brief immersion of the tip. The remainder of the pipette was then backfilled with the solution containing gramicidin diluted to a final concentration of 100 µg ml-1. Series resistance was 
20 M and was compensated for by 80-90 %.
Dishes were superperfused continuously with recording medium at a rate of 1·2 ml min-1. Drugs were prepared and stored at -20°C in 10 mM aliquots and were applied from a puffer pipette.
To estimate intracellular Cl- concentration ([Cl-]i) a version of the Nernst equation was used:
[Cl-]i = 155·6 exp(EGABA/25·6),
where 155·6 is the concentration of extracellular Cl- and EGABA is the reversal potential for GABA-induced responses.
Digital video microscopy
Oxonol dye potentiometry. Details are described in Walton et al. (1993). Briefly, a Nikon Diaphot microscope was equipped with a xenon source and epi-illumination with a rhodamine filter was set appropriate for the oxonol dye DiBaC4(5) (Molecular Probes, Eugene, OR, USA). Dye (50 nM) was added to all superfusion media which consisted of a physiological saline with and without various test substances. Two glass inserts were placed inside the culture dish to decrease the volume to approximately 0·2 ml thus ensuring a rapid change in solutions. An image intensifier coupled to a CCD video camera was attached to the video output port of the microscope and fluorescent images were captured using a frame-grabber at the rate of one per minute. The anionic oxonol dyes diffuse across the membrane and achieve a Nernstian equilibrium (Apell & Bersch, 1987). Depolarization is recorded as an increase in fluorescence. Since fluorescence is directly related to the intracellular dye concentration and altered via diffusion, the fluorescence changes are quantitatively continuous rather than exhibiting a threshold. However, due to intrinsic electronic noise, the analysis procedure accepted only changes of at least 0·1 log units as an unequivocal change in membrane potential. Manipulation of extracellular [K+] indicated that a fluorescence change in the order of 0·1 log units corresponded to a potential difference of 8 mV, evidence that the technique is sensitive to discrete changes in membrane potential (Walton et al. 1993).
Calcium imaging. Neurons were loaded with the calcium indicator dye fura-2 by exposure to 4 µM fura-2 AM (Molecular Probes) in standard bath solution for 30 min and then washed and maintained for 45 min for ester hydrolysis at 37°C. As with oxonol recordings, rapid solution changes were obtained by decreasing the dish volume to 0·2 ml with glass inserts. Digital video imaging fluorescence microscopy was used for measuring the fluorescence of a chosen field of cells (× 40, using a Nikon Diaphot inverted microscope), and images using excitation wavelengths of 340 and 380 nm were captured and stored. The ratio of fluorescence at the two exciting wavelengths was calculated for each pixel within a cell boundary. Calibration of the ratio in terms of Ca2+ was carried out by adding ionomycin to the dish and recording images in calcium-free medium (no Ca2+ plus 5 mM EGTA) and in normal medium (1·8 mM Ca2+). The calcium concentration was derived from:
[Ca2+] = KDF0/F
where KD is the fura-Ca2+ binding constant (220 nM), R is a ratio of fluorescence at two wavelengths and Rmin and Rmax are values of R in calcium-free and normal medium, respectively. F0/F
Statistical tests
A two-tail t test was used to assess statistical significance in Figs 5 and 7. Differences were considered significant if P < 0·05 and have been indicated by *, while P < 0·01 are denoted by ** in these figures. The data points reflect means ± standard error of the mean. For electrophysiological studies n represents the number of neurons tested; for optical recordings n is the number of fields tested.
A Fisher exact probability test using 2 × 2 contingency tables was used to determine significance between responders and non-responders on the two surfaces in Fig. 3A; * denotes P < 0·05.
Animal procedures
All animals procedures were done in accordance with Public Health Service Policy on Humane Care and Use of Laboratory Animals and the Guide for the Care and Use of Laboratory Animals in the USA.
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RESULTS |
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Muscimol-induced depolarization disappears in vivo during the early postnatal period
We used oxonol dye potentiometry to record from intact ventral spinal cord cells dissociated at late embryonic and early postnatal days to assess the time course over which the GABAA agonist muscimol is able to depolarize neurons. We found that about 80 % of embryonic ventral horn cells dissociated and recorded within 2-3 h of plating on PDL (79 ± 2 %; n = 3 fields/129 cells) or after 24 h in culture (75 ± 6 %; n = 6 fields/201 cells) depolarized to 2 µM muscimol (Fig. 1A). All the responding cells exhibited processes and were TuJ1 or tetanus-toxin positive (data not shown), identifying them as neurons (Koulakoff, Bizzini & Berwald-Netter, 1983; Moody, Quigg & Frankfurter, 1989). At this age and subsequent ages it is unlikely that non-responders are dead or traumatized. These states should result in a very high level of baseline oxonol fluorescence due to leaky plasma membranes and this was not found to be the case. A similar percentage of neurons dissociated at birth or at different postnatal days depolarized to muscimol when the cells were recorded within 2-3 h of plating. However, progressively fewer neurons depolarized after recovery in culture for 24 h (Fig. 1A). Thus, by postnatal day 14 few neurons recovered in culture for 24 h depolarized to muscimol (4 ± 3 %; n = 7 fields/98 cells). This was not due to selective loss of muscimol-responsive neurons. When the same neuron was recorded after several hours in culture and again after 24 h, the disappearance of a depolarizing response was demonstrated in the same cell. In contrast, the percentage of neurons that depolarized to 50 µM kainate remained high at all times whether they were derived from the embryonic or postnatal period or whether they were recorded several hours or 24 h after dissociation (Fig. 1A). A similar percentage of embryonic neurons depolarizing at 2 and 24 h in response to kainate strongly suggests that the depolarizing effects are not due to the trauma of cell dissociation, but reflect the physiological state of the cells that exists in vivo. These potentiometric results recorded in vitro on ventral spinal cord neurons indicate that in vivo the depolarizing effects of muscimol and, by inference GABA, disappear during the early postnatal period. The results with acutely recorded postnatal neurons also imply that the trauma of cell dissociation leads to Cl- loaded cells whose intracellular Cl- decreases dramatically during 24 h recovery in vitro.
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A, depolarizing responses to muscimol, examined with the potentiometric dye oxonol, disappear in postnatal dissociates recovered for 1 day in vitro (1 Div). Eighty percent of the embryonic cells depolarize to muscimol after 2-3 h in vitro and after a 1 day recovery in vitro. Progressively fewer postnatal cells depolarize to muscimol after 24 h with only rare cells from postnatal day (PN) 14 responding. The percentage of neurons that depolarize to kainate remains high at all times independent of age at dissociation or time after dissociation. Each data point represents the mean ± | ||
GABA triggers a sustained rise in Ca2+c in embryonic ventral spinal cord neurons
We recorded the effects of GABA on ventral spinal cord neurons dissociated at E15 and recovered for 24 h in culture on PDL. GABA triggered a rise in Ca2+c from about 50 nM to about 200 nM (Fig. 1B-D) in the same percentage of neurons (80 %) as depolarized to muscimol. The Ca2+c response to GABA was blocked almost completely and in an entirely reversible manner by co-application of bicuculline, which by itself had no effect (Fig. 1B). The steady-state level of Ca2+c was noticeably decreased in Ca
-free medium while the response to GABA was completely eliminated in a reversible manner (Fig. 1C). The Ca2+c rise to GABA was also markedly, but not completely, blocked by nifedipine in a reversible manner (Fig. 1D). Virtually identical effects were recorded regarding bicuculline and nifedipine sensitivity and Ca dependency when muscimol was used instead of GABA (data not shown). Co-application of 5 µM 
-conotoxin GVIA, 3 µM -conotoxin MVIIC or 100 nM tetrodotoxin with muscimol did not affect the Ca2+c response (n = 4; data not shown). These results implicate bicuculline-sensitive GABAA receptor/Cl- channels, which, when activated, depolarize neurons and trigger extracellular Ca2+ entry via nifedipine-sensitive, voltage-dependent Ca2+ channels.
Ca2+cresponse is sensitive to changes in extracellular chloride
Since our results implicated Cl--dependent depolarization in the GABA- and muscimol-evoked Ca2+ responses, we changed extracellular Cl- (Clo-) and recorded Ca2+c responses. Virtually all neurons that exhibited Ca2+c responses of 100 nM to muscimol in normal saline responded to muscimol co-incidentally delivered with low Clo- saline with about a 50 % increase in the amplitude (150 nM) of the sustained signal (Fig. 2A,B and D). When neurons were allowed to equilibrate in low Clo- saline and then exposed to muscimol delivered in normal saline, most neurons (75 %) no longer responded (Fig. 2A and B (
)). Those that did respond manifested markedly reduced responses (Fig. 2B ()). Neurons were also recorded that did not respond to muscimol in normal saline but upon switching to low Clo- did express quite detectable Ca2+c responses indistinguishable from neuronal responses in normal saline (Fig. 2C). These results are consistent with the idea that the observed Ca2+ responses are triggered via voltage-dependent Ca2+ channels activated by the depolarization of the cell in response to opening of GABAA receptor/Cl- channels.
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Embryonic neurons were plated on poly-D-lysine (PDL), then recorded for their Ca2+c responses to 10 µM muscimol under control conditions and in salines with altered [Cl-]. Representative recordings are shown on the left and plots summarizing all the results from multiple recordings are depicted on the right (means ± | ||
Astrocytes accelerate time-dependent change in GABA-induced Ca2+cresponses occurring in vitro
We recorded Ca2+c responses to GABA in E15 neurons cultured on a commonly used surface (PDL) in a standard medium and compared them to responses of neurons plated on confluent cortical astrocytes in the same medium. After one day in culture, similar percentages of neurons (80 %) differentiating on PDL or on astrocytes exhibited Ca2+c responses of identical amplitude (Fig. 3A). At 4 days, both the percentage of cells exhibiting GABA-induced Ca2+c responses and their mean amplitude had decreased considerably more in neurons differentiating on astrocytes compared with neurons growing on PDL. At 1 week, more than 60 % of neurons cultured on PDL still exhibited Ca2+c responses to GABA. In those neurons that responded the mean elevation of Ca2+c was greater than 90 nM. Less than 20 % of neurons on astrocytes expressed Ca2+c signals and in those neurons the mean Ca2+c response was less than 60 nM (Fig. 3A and B). By 2 weeks in culture, neurons on astrocytes did not respond to GABA with an elevation in Ca2+c while one-third of those grown on PDL still manifested Ca2+c signals with a mean about 60 nM. Thus, relative to cultivation of ventral spinal neurons on PDL, co-culture of neurons on astrocytes accelerated the developmental disappearance in the cellular distribution of GABA-induced Ca2+c responses.
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A, the percentage of neurons responding to 10 µM GABA with an increase in [Ca2+]c of at least 15 nM is plotted as a function of days in culture. The number of cells responding decreases with time in culture on both PDL ( | ||
Astrocytes enhance developmental changes in EGABA that involve diffusible factors
We used gramicidin-perforated patch recordings to record the resting membrane potentials and equilibrium potential for GABA-induced conductance and polarization (EGABA). GABA depolarized neurons cultured for 1 day on either PDL or astrocytes in a bicuculline-sensitive manner (n = 7) (Fig. 4A). Bicuculline by itself did not alter resting membrane potential. Depolarizing responses evoked under these experimental conditions usually did not provoke regenerative action potential activity (Fig. 4A) except in several neurons cultured on PDL for 1-2 weeks (data not shown). We recorded brief pulses to GABA under voltage clamp in normal saline and low Clo--containing saline. In the cell illustrated (Fig. 4B and C), GABA-induced current reversed polarity at approximately -38 mV in normal saline and -2 mV in low Clo-, consistent with a Nernstian shift in EGABA. Similar results were obtained in six out of six cells tested.
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A, a depolarizing response to GABA was recorded under current clamp (I = 0) and could be reversibly blocked by 100 µM bicuculline. Note that neither depolarizing response triggers an action potential. B, the reversal in polarity of superimposed current responses activated by 10 µM GABA (bar at bottom) occurs at different potentials in the two salines. C, current-voltage curves constructed from the data shown in B (same cell) reveal the Cl-dependency of the reversal potential in current polarity. | ||
We recorded steady-state membrane potential and GABA-induced polarization of neurons cultured on PDL or on astrocytes using gramicidin-perforated patch-recording techniques. Resting membrane potential recorded in this manner progressively increased from an initial level of about -40 mV within hours of plating to about -55 mV at 1 week in vitro in all neurons studied (Fig. 5A; PDL, -40 ± 0·6 to -56 ± 1·0 mV; astrocytes, -42 ± 1·6 to -55 ± 1·6 mV). EGABA was about -30 mV at 1 day in culture in all neurons recorded (Fig. 5B). Astrocytes accelerated the time course of changes in EGABA, which hyperpolarized to -60 ± 2·5 mV by 1 week in culture, eventually reaching -64 ± 1·0 mV by 3 weeks. In contrast, EGABA recorded in neurons on PDL, was between -48 ± 0·8 mV by 1 week, eventually reaching -53 ± 1·8 mV by 3 weeks (Fig. 5B). Medium conditioned by astrocytes (ACM) completely mimicked the initial rate of change in EGABA recorded in co-cultures of neurons with astrocytes (Fig. 5B). However, the effectiveness of ACM in promoting hyperpolarization became limiting such that EGABA levelled off at -58 ± 1·4 mV, a value intermediate between those recorded in the two other conditions. When the data were plotted relative to the resting potential, both astrocytes and ACM stimulated changes in EGABA that, by 4 days in vitro, led to no potential response or hyperpolarization relative to the resting potential. Hyperpolarizing responses took 3 weeks to manifest in neurons on PDL alone (Fig. 5C). Intracellular chloride, [Cl-]i (estimated from EGABA), was calculated to change from 45-50 mM at the time of plating to 12-15 mM after 4-7 days in culture in neurons on astrocytes or in ACM. A similar decrease in [Cl-]i was evident in neurons on PDL but took 3 weeks to drop to 20 mM (Fig. 5D).
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A, the resting membrane potentials (Vm) recorded with perforated-patch techniques become more negative over time in culture independently of the presence of astrocytes. All the shift in EGABA occurs during the first week. B, EGABA hyperpolarizes during long-term culture and the rate and extent is greater on astrocytes or in ACM relative to that recorded in neurons on PDL. C, the differences between the absolute values of EGABA and Vm are plotted, with positive values representing the depolarizing responses and the negative values representing hyperpolarizing responses. D, intracellular chloride ([Cl-]i) derived from measurements of EGABA, changes over time in culture more rapidly in neurons on astrocytes and in neurons exposed to ACM compared with changes occurring on PDL alone. The change in [Cl-]i occurs over the first week with differences becoming statistically significant at 2 days. The data points represent the means ± | ||
GABAA receptor/Cl- channel properties change in neurons independently of astrocytes
We used the whole-cell patch-clamp recording technique with Cl--filled pipettes to set ECl near or at 0 mV and clamped cells at -80 mV to optimize 10 µM GABA-induced Cl--dependent current signals. Macroscopic GABA-induced currents were always superimposed with microscopic fluctuations characteristic of underlying Cl- channel activity (Fig. 6A and B). We used fluctuation analysis methods to estimate the elementary properties of the Cl- channels activated by GABA in the whole cell (Neher & Stevens, 1977). At the recording bandwidth used under our experimental conditions we consistently calculated two components in every spectrum of current fluctuations in all neurons (Figs 6 and 7). We computed the relative areas of the two components and found that the long-lasting component accounted for about 80-85 % of the spectrum in all neurons at all time points (Fig. 7B). At day 1 for example, PDL = 82 ± 3·5 %, astrocytes = 79 ± 4·3 %, ACM = 79 ± 1·7 %; and by day 21, PDL = 77 ± 3·0 %, astrocytes = 81 ± 2·1 %, ACM = 80 ± 2·0 %; n = 6-8 cells. Since the estimated mean channel open time, or burst duration, of the greater component (
long) was considerably longer than the shorter component at all times in culture, more than 95 % of the pharmacologically induced current reflects Cl- channel activity with these kinetics. long decreased progressively in all neurons from 75 ms in acutely recorded cells to 50 ms in cells cultured for 3 weeks (Fig. 7A). In contrast, a short-lasting component averaging 3-4 ms was recorded in all neurons at all times (Fig. 7D). The estimated elementary conductance progressively increased from 16 to 22 pS over the first week in culture in all neurons recorded (Fig. 7C). These results demonstrate that unitary GABAA receptor/Cl- channel properties inferred from analysis of fluctuations in GABA-evoked Cl- current recorded from whole cells change in a concerted manner primarily during the first week in vitro and that these changes occur independently of astrocytes.
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Cells were recorded within hours of plating (0 Div) or after 7 days in culture (7 Div). At both times GABA macroscopic currents superimposed with microscopic fluctuations. Representative traces reveal the presence of low frequency fluctuations at 0 Div (A), which are less apparent at 7 Div (B). Spectral analysis of the fluctuations show that both spectra can be adequately explained with two components indicated by the corner frequencies, fc1 and fc2. The mean open times ( | ||
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Unitary properties of channels were estimated at different times in neurons cultured under three different experimental conditions: on PDL; on astrocytes; or in astrocyte-conditioned medium (ACM). Each data point reflects the mean ± | ||
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DISCUSSION |
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The present study shows that: (1) pharmacological activation of GABAA receptor/Cl- channels depolarizes cultured ventral spinal cord neurons and triggers sustained elevation of Ca2+c ; (2) both responses disappear in long-term culture, with GABAA receptor/Cl- channel activation eventually hyperpolarizing cells relative to the resting potential; (3) astrocytes in co-culture and medium conditioned by them for 24 h accelerate these changes relative to the rate recorded in neurons cultured on PDL; and (4) the unitary properties of GABAA receptor/Cl- channels change independently of astrocytes, as channels shorten in open time and increase in conductance. Thus, in vitro, astrocytes regulate changes in the Cl- gradient extrinsic to GABAA receptor/Cl- channels without modulating their intrinsic properties, which change in parallel.
GABA depolarizes embryonic neurons
Depolarization via GABAA receptor/Cl- channels has been recorded in cells dissociated from the cervical region of the embryonic rat spinal cord using dye potentiometry (Walton et al. 1993). Depolarizing responses have also been recorded electrically using perforated-patch techniques in the majority of dorsal horn neurons plated at E15-16 and studied during the first week in culture (Wang et al. 1994). Embryonic, rat lumbar motoneurons in slices of spinal cord prepared from E16 and then recorded at PN2 with high resistance microelectrodes also depolarized in response to GABA (Wu et al. 1992). In the latter preparation, depolarizing synaptic potentials mediated by GABA can be evoked in motoneurons by electrical stimulation of primary afferents in dorsal roots. These depolarizing GABAergic potentials did not trigger action potentials. This may be due to the peak of the depolarizing potential lying subthreshold to action potential activation as well as to the associated conductance increase to Cl- ions. Depolarizations evoked pharmacologically in perforated-patch recordings of cultured dorsal horn neurons (Wang et al. 1994) or ventral cervical cord neurons (this study) only rarely triggered action potentials. However, in on-cell patch-clamp recordings of spinal neurons, GABA's activation of GABAA receptor/Cl- channels enclosed in the patch triggered action potentials and promoted their occurrence in the remainder of the membrane (Serafini et al. 1995). These excitatory effects of single-channel openings may result from charge transfer throughout the intact membrane that effectively depolarizes the cell outside the patch. Thus, differences in experimental conditions and/or electrical recording configurations may account for the variability in recording action potentials.
The Cl--dependent depolarization of embryonic and early postnatal spinal cord neurons, and presumably embryonic neurons from brain as well, is due to high intracellular [Cl-]i and disappears with age as the Cl- gradient across the membrane increases. In spinal motoneurons GABA generates membrane depolarization until at least 1-2 days after birth but it has not been determined when the response becomes hyperpolarizing (Wu et al. 1992). CA3 and CA1 neurons of the hippocampus show the transition from Cl--dependent depolarizations to hyperpolarizations at the beginning of the second postnatal week (Ben-Ari, Cherubini, Corradetti & Gaiarsa, 1989). In the present study the number of ventral spinal cord neurons exhibiting depolarizations to muscimol (after a 24 h recovery) dropped from 80 % at E17 to 40 % at PN0/PN7 and to 4 % at PN14. Neurons dissociated at E15 and grown on astrocytes or PDL exhibit the transition to GABA-induced hyperpolarizations after 4 days in vitro or 3 weeks in vitro, respectively. These results indicate that the transition occurring in vitro follows roughly the same time course as has been seen for spinal and hippocampal neurons in vivo.
Cl--dependent depolarization activates Ca2+ entry
Previous studies on cultured embryonic dorsal horn neurons have revealed that GABA evokes a sustained elevation in Ca2+c, which has been attributed to voltage-dependent Ca2+ channels activated by depolarization (Reichling et al. 1994). In this study, we have found similar effects of GABAA receptor/Cl- channel activation on Ca2+c levels in embryonic ventral cervical spinal cord neurons. The sensitivity of the Ca2+c response to nifedipine in the present study implicates voltage-dependent L-type Ca2+ channels as the primary pathway for Ca2+ entry. Thus, GABA induces Ca2+c responses indirectly via its Cl--dependent depolarizing actions. Quite similar conclusions regarding L-type Ca2+ channel involvement in GABA-evoked Ca2+c responses have been reported in cultured embryonic hypothalamic neurons (Obrietan & van den Pol, 1995). Perforated-patch recordings of hypothalamic neurons cultured for several days show that in about half of the cells GABA evoked depolarizing responses that triggered one or several action potentials. Bicuculline blocked many of these action potentials occurring spontaneously together with most, but not all of the depolarizing synaptic activity (Chen, Trombley & van den Pol, 1996). The coupling of bicuculline-sensitive, GABA-mediated depolarization and Ca2+c elevation has also been reported in recordings of cultured rat hippocampal neurons (Segal, 1993), pyramidal neurons in early postnatal hippocampal slice preparations (Leinekugel, Tseeb, Ben-Ari & Bregestovski, 1995) and in tissue print preparations of embryonic neocortex (Owens, Boyce, Davis & Kriegstein, 1996). In the hippocampal slice preparation, the coupling between GABAA receptor/Cl- conductance, depolarization and activation of voltage-dependent Ca2+ channels disappears during the postnatal period coincident with a progressive shift in ECl to more hyperpolarized potentials.
Astrocyte effects
Changes in the functional effects of GABAA receptor activation recorded in slice preparations during the postnatal period parallel the developmental appearance of astrocytes in the hippocampus (Nixdorf-Bergweiler, Albrecht & Heinemann, 1994). In the developing rat spinal cord, GFAP+ elements appear at E16 and their numbers increase during the late embryonic and early postnatal period (Yang et al. 1993). In the present study, accelerated changes in the polarity of the GABA response and the progressive loss of a concomitant elevation in Ca2+c occur in ventral spinal cord neurons dissociated at E15 and cultured over a 3 week period in the presence of astrocytes. This is the same time period during which these neurons would be subject to the influence(s) of proliferating astrocytes in vivo. Hence, in vivo astrocytes could play a role in regulating these developmental changes. There have been numerous studies on the profound effects of astrocytes on neuronal proliferation, morphology, electrophysiological properties and homeostasis (Silver, Lorenz, Wahlsten & Coughline, 1982; Sivron, Eitan, Schreyer & Schwartz, 1993; Travis, 1994; Wu & Barish, 1994; Barish, 1995; Sontheimer, 1995; Tsacopoulos & Magistretti, 1996).
The effects of astrocytes on GABA-induced, chloride-dependent potential in vitro were mimicked by exposure to medium conditioned for 24 h by astrocytes. Thus, confluent astrocytes derived from postnatal cortex secrete substances independently of the presence of embryonic neurons that serve to signal developmental changes in ECl and, in turn, Ca2+c responses. There are several possible targets for astrocyte factor regulation that could influence levels of intracellular Cl-. These include a Cl-/HCO3- exchanger driven by the intracellular production of CO2/HCO3- (Kobayashi, Morgans, Casey & Kopito, 1994), a Cl-/cation transporter driven by the transmembrane gradient for Na+ (Misgeld, Deisz, Dodt & Lux, 1986; Rohrbough & Spitzer, 1996), or an ATP-dependent Cl- pump (Inagaki, Hara & Inoue, 1992). Interestingly, brain astrocytes and their conditioned media have recently been shown to stimulate Na+-K+-Cl- cotransporter activity in brain endothelial cells (Sun, Lytle & O'Donnell, 1997). In the latter study the cytokine, IL-6 appeared to be the stimulatory factor. Further work should help to identify which transporter, if any, is a target for astrocyte regulation in developing spinal cord neurons.
Developmental changes in GABAA receptor/Cl- channel properties
We found that the unitary properties of GABAA receptor/Cl- channels inferred from fluctuation analyses of Cl- current responses changed in parallel with the hyperpolarization of ECl and loss of GABA-induced Ca2+c responses. However, these changes occurred with the same time course whether or not neurons were cultured with astrocytes. Thus, in vitro, astrocytes do not regulate the intrinsic biophysical properties of GABAA receptor/Cl- channels. The developmental changes in Cl- channel properties could involve a switch in the subunit composition of the putative pentameric GABAA receptors. In this regard, we have carried out a preliminary study of GABAA receptor subunit transcript expression using in situ hybridization applied to cells dissociated at E17 and P12 (data not shown). We found cells expressing transcripts for 
2, 3, 4, 5, 1, 2, 3, 
1 and 2 in E17 dissociates, and in P12 dissociates, transcripts for 2, 3, 4, 2, 3 and 2, consistent with a previous study in vivo (Ma et al. 1993), were found.
It is significant that GABAA receptor subtypes have been shown to change during development (Poulter, Barker, O'Carroll, Lolait & Mahan, 1992; Fritschy, Paysan, Enna & Mohler, 1994; Chang, Luntz-Leybman, Evans, Rotter & Frostholm, 1995; Ma et al. 1993) and different combinations of subunits are associated with different physiological properties (Mathews et al. 1994; Saxena & Macdonald, 1994). Hence, the shortening in the open time derived from the major component in the spectral analyses of GABA-induced fluctuations and the coincident increase in estimated unitary conductance occurring over time in vitro may reflect changes in subunit composition. This remains to be determined.
Conclusion
In summary, astrocytes facilitate developmental changes in the Cl- gradient of spinal neurons extrinsic to GABAA receptor/Cl- channels that profoundly influence the functional effects of GABAA receptor/Cl- channel activation. These astrocyte effects are mediated by diffusible substances and occur independently of parallel changes in the intrinsic properties of the receptor.
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REFERENCES |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Apell, H.-J. & Bersch, B. (1987). Oxonol VI as an optical indicator for membrane potentials in lipid vesicles. Biochimica et Biophysica Acta 903, 480-494.
[Medline] Armstrong, R. C., Dorn, H. H., Kufta, C. V., Friedman, E. & Dubois-Dalcq, M. E. (1992). Pre-oligodendrocytes from adult human CNS. Journal of Neuroscience 12, 1538-1547.
[Abstract] Barish, M. E. (1995). Modulation of the electrical differentiation of neurons by interactions with glia and other non-neuronal cells. Perspectives on Developmental Neurobiology 2, 357-370.
[Medline] Behar, T., Schaffner, A., Laing, P., Hudson, L., Komoly, S. & Barker, J. L. (1993). Many spinal cord cells transiently express low molecular weight forms of glutamic acid decarboxylase during embryonic development. Developmental Brain Research 72, 203-218.
[Medline] Ben-Ari, Y., Cherubini, E., Corradetti, R. & Gaiarsa, J.-L. (1989). Giant synaptic potentials in immature rat CA3 hippocampal neurones. The Journal of Physiology 416, 303-325.
[Abstract]
Chang, C. C. A., Luntz-Leybman, V., Evans, J. E., Rotter, A. & Frostholm, A. (1995). Developmental changes in the expression of -aminobutyric acidA/benzodiazepine receptor subunit mRNAs in the murine inferior olivary complex. Journal of Comparative Neurology 356, 615-628.
[Medline] Chen, G., Trombley, P. Q. & van den Pol, A. N. (1996). Excitatory actions of GABA in developing rat hypothalamic neurones. The Journal of Physiology 494, 451-464.
[Abstract] Fritschy, J.-M., Paysan, J., Enna, A. & Mohler, H. (1994). Switch in the expression of rat GABAA-receptor subtypes during postnatal development: an immunohistochemical study. Journal of Neuroscience 14, 5302-5324.
[Abstract] Huettner, J. E. & Baughman, R. W. (1986). Primary culture of identified neurons from the visual cortex. Journal of Neuroscience 6, 3044-3060.
[Abstract] Inagaki, C., Hara, M. & Inoue, M. (1992). Neuronal intracellular chloride ion concentration and their regulatory mechanisms. Nippon Yakurigaku Zasshi 99, 307-315.
[Medline] Kobayashi, S., Morgans, C. W., Casey, J. R. & Kopito, R. R. (1994). AE3 anion exchanger isoforms in the vertebrate retina: developmental regulation and differential expression in neurons and glia. Journal of Neuroscience 14, 6266-6279.
[Abstract] Koulakoff, A., Bizzini, B. & Berwald-Netter, Y. (1983). Neuronal acquisition of tetanus toxin binding sites: relationship with the last mitotic cycle. Developmental Biology 100, 350-357.
[Medline] Lauder, J. M. (1993). Neurotransmitters as growth regulatory signals: role of receptors and second messengers. Trends in Neurosciences 16, 233-240.
[Medline] Leinekugel, X., Tseeb, V., Ben-Ari, Y. & Bregestovski, P. (1995). Synaptic GABAA activation induces Ca2+ rise in pyramidal cells and interneurons from rat neonatal hippocampal slices. The Journal of Physiology 487, 319-329.
[Abstract] Li, Y.-X., Schaffner, A. E., Walton, M. K. & Barker, J. L. (1995). Cortical astrocytes affect the differentiation of cultured embryonic rat spinal cord neurons. Journal of Neuroscience 21, A1298.
Ma, W., Saunders, P. A., Somogyi, R., Poulter, M. O. & Barker, J. L. (1993). Ontogeny of GABAA receptor subunit mRNAs in rat spinal cord and dorsal root ganglia. Journal of Comparative Neurology 338, 337-359.
[Medline] Mathews, G., Bolossy, A. M., Holland, K. D., Isenberg, K. E., Covey, D. F., Ferrendelli, J. A. & Rothman, S. M. (1994). Developmental alteration in GABAA receptor structure and physiological properties in cultured cerebellar granule neurons. Neuron 13, 149-158.
[Medline] Misgeld, U., Deisz, R. A., Dodt, H. U. & Lux, H. D. (1986). The role of chloride transport in postsynaptic inhibition of hippocampal neurons. Science 232, 1413-1415.
[Medline] Moody, S. A., Quigg, M. S. & Frankfurter, A. (1989). Development of the peripheral trigeminal system in the chick revealed by an isotype-specific anti-beta-tubulin monoclonal antibody. Journal of Comparative Neurology 279, 567-580.
[Medline] Neher, E. & Stevens, C. F. (1977). Conductance fluctuations and ionic pores in membranes. Annual Reviews of Biophysics and Bioengineering 6, 345-381.
[Medline] Nixdorf-Bergweiler, B. E., Albrecht, D. & Heinemann, U. (1994). Developmental changes in the number, size and orientation of GFAP-positive cells in the CA1 region of rat hippocampus. Glia 12, 180-195.
[Medline] Obrietan, K. & van den Pol, A. N. (1995). GABA activity mediating cytosolic Ca 2+ rises in developing neurons is modulated by cAMP-dependent signal transduction. Journal of Neuroscience 17, 4785-4799.
[Abstract/Full Text] Owens, D. F., Boyce, L. H., Davis, M. B. & Kriegstein, A. R. (1996). Excitatory GABA responses in embryonic and neonatal cortical slices demonstrated by gramicidin perforated-patch recordings and calcium imaging. Journal of Neuroscience 16, 6414-6423.
[Abstract/Full Text]
Poulter, M. O., Barker, J. L., O'Carroll, A. M., Lolait, S. J. & Mahan, L. C. (1992). Differential and transient expression of GABAA receptor -subunit mRNAs in the developing rat CNS. Journal of Neuroscience 12, 2888-2900.
[Abstract] Reichling, D. B., Kyrozis, A., Wang, J. & MacDermott, A. B. (1994). Mechanisms of GABA and glycine depolarization-induced calcium transients in rat dorsal horn neurons. The Journal of Physiology 476, 411-421.
[Abstract] Rohrbough, J. & Spitzer, N. C. (1996). Regulation of intracellular Cl- levels by Na(+)-dependent Cl- cotransport distinguishes depolarizing from hyperpolarizing GABAA receptor-mediated responses in spinal neurons. Journal of Neuroscience 16, 82-91.
[Abstract] Saxena, N. C. & Macdonald, R. L. (1994). Assembly of GABAA receptor subunits: role of delta subunit. Journal of Neuroscience 14, 7077-7086.
[Abstract] Segal, M. (1993). GABA induces a unique rise of [Ca2+]i in cultured rat hippocampal neurons. Hippocampus 3, 229-238.
[Medline] Serafini, R., Valeyev, A. Y., Barker, J. L. & Poulter, M. O. (1995). Depolarizing GABA-activated Cl- channels in embryonic rat spinal and olfactory bulb cells. The Journal of Physiology 488, 371-386.
[Abstract] Silver, J., Lorenz, S. E., Wahlsten, D. & Coughline, J. (1982). Axon guidance during development of the great cerebral commissures: descriptive and experimental studies, in vivo, on the role of preformed glial pathways. Journal of Comparative Neurology 210, 10-29.
[Medline] Sivron, T., Eitan, S., Schreyer, D. J. & Schwartz, M. (1993). Astrocytes play a major role in the control of neuronal proliferation in vitro. Brain Research 629, 199-208.
[Medline] Sontheimer, H. (1995). Glial neuronal interactions: a physiological perspective. The Neuroscientist 1, 328-337.
Sun, D., Lytle, C. & O'Donnell, M. E. (1997). IL-6 secreted by astroglial cells regulates Na-K-Cl cotransport in brain microvessel endothelial cells. American Journal of Physiology 272, C1829-1835.
[Medline] Travis, J. (1994). Glia: the brain' s other cells. Science 266, 970-972.
[Medline] Tsacopoulos, M. & Magistretti, P. J. (1996). Metabolic coupling between glia and neurons. Journal of Neuroscience 16, 877-885.
[Medline] Walton, M. K., Schaffner, A. E. & Barker, J. L. (1993). Sodium channels, GABAA receptors, glutamate receptors develop sequentially on embryonic rat spinal cord cells. Journal of Neuroscience 13, 2068-2084.
[Abstract] Wang, J., Reichling, D. B., Kyrozis, A. & MacDermott, A. B. (1994). Developmental loss of GABA- and glycine-induced depolarization and Ca2+ transients in embryonic rat dorsal horn neurons in culture. European Journal of Neuroscience 6, 1275-1280.
[Medline] Wu, R. L. & Barish, M. E. (1994). Astroglia modulation of transient potassium current development in cultured mouse hippocampal neurons. Journal of Neuroscience 14, 1677-1687.
[Abstract] Wu, W. L., Ziskind-Conhaim, L. & Sweet, M. A. (1992). Early development of glycine and GABA-mediated synapses in rat spinal cord. Journal of Neuroscience 12, 3935-3945.
[Abstract] Yang, H. Y., Lieska, N., Shao, D., Kriho, V. & Pappas, G. D. (1993). Immunotyping of radial glia and their glial derivatives during development of the rat spinal cord. Journal of Neurocytology 22, 558-571.
Corresponding author
A. E. Schaffner: Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
Reprint requests
J. L. Barker: Building 36, Room 2C02, 36 Convent Drive, MSC 4066, Bethesda, MD 20892-4066, USA.
Email: jlbarker{at}codon.nih.gov
Authors' present addresses
Y.-X. Li: Division of Biology, 156-29, California Institute of Technology, Pasadena, CA 91125, USA.
M. K. Walton: Division of Clinical Trial Design and Analysis, OTRR, CBER, FDA, HFM 576, 1401 Rockville Pike, Rockville, MD 20852, USA.
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, B). C, neurons were also recorded that did not respond to muscimol in normal saline but did so when exposed to muscimol delivered coincidentally in low Cl-o saline. These responses were indistinguishable from those typically recorded in normal saline. D, all the cells that responded to muscimol delivered in standard saline with an increase in Ca2+c were defined as the starting population (100 %). The vast majority of these cells responded similarly when the saline was changed to one with low Cl-. However, when neurons were allowed to equilibrate in low Cl-o saline a much smaller percentage responded with an increase in Ca2+c when muscimol was subsequently delivered in normal saline. In A and C, smaller ticks are drawn at 5 s intervals, taller ticks at 1 min intervals.
) but astrocytes accelerate this change. The numbers above each column are the number of responding cells/number of cells tested. * denotes P < 0·05. B, the mean amplitude of the Ca2+ c response evoked by 10 µM GABA decreases with time in culture on both surfaces, but the decrease is magnified in the presence of astrocytes. * P significant at the 0·05 level, ** P significant at the 0·01 level.
fc)-1 shorten from ~70 ms to ~50 ms for the long-lasting openings, but remain at about 3 ms for the short-lasting component.