Conductance properties of GABA-activated chloride currents recorded from cultured hippocampal neurons

Brain Research, 410 (1987) 159-163 Elsevier

159

BRE 22231

Conductance properties of GABA-activated chloride currents recorded from cultured hippocampal neurons C.N. Allen* and E.X. Albuquerque Department of Pharmacology and Experimental Therapeutics, Universityof Maryland School of Medicine, Baltimore, MD 21201 (U.S.A.) (Accepted 20 January 1987)

Key' words: Cultured hippocampal pyramidal cell; y-Aminobutyric acid; Single chloride channel; Patch clamp recording; Single channel conductance; Amino acid

The conductance characteristics of y-aminobutyric acid-activated single channel currents from cultured hippocampal neurons were examined using patch clamp techniques. GABA-activated currents had amplitudes which were linearly correlated to the membrane potentials over a range of -80 to +70 mV and an open time and burst time of 2.2 and 4.3 ms, respectively. The conductance of the yaminobutyric acid-activated channels was 19 pS. These data demonstrate that cultured hippocampal neurons have channel eonductances which have characteristics different from those of adult neurons. 7-Aminobutyric acid ( G A B A ) is an important inhibitory neurotransmitter within the central nervous system of vertebrates ~4'17'18. In the hippocampus, G A B A released from interneurons inhibits the activity of the pyramidal cell neurons 4'13. A general inhibition of pyramidal cell firing is the result of hyperpolarization of the soma due to activation of an inward chloride current t'5. Blockade of these chloride currents by pharmacological agents generates synchronized hippocampal activity reminiscent of epileptic activity z'lS'L6. GABA-activated channels, through which the chloride current flows, have been studied using spinal cord neurons in primary culture 13'21-23"26. These neurons are easily visualized and the lack of glia facilitates the formation of a giga-ohm seal between a glass microelectrode and cell m e m b r a n e 26. Most primary neuronal cultures are isolated from embryonic brains and the neurons may, at different times in culture, have either adult or immature characteristics or a mixture of both. For example, the conductance and the lifetime properties of acetylcholine-activated

channels are modified during maturation of cultured embryonic muscle cells 6'19'26, and following denervation of muscles 3. Therefore, generalization of data from cultured and adult preparations must be done cautiously in the absence of direct comparative studies. Recently, Gray and Johnston used a modified hippocampal slice preparation to study the characteristics of single G A B A - a c t i v a t e d currents from adult CA~ pyramidal cells 2°, These G A B A - g a t e d currents had a linear current-voltage relationship at negative membrane potentials but showed a pronounced outward rectification at hyperpolarized membrane potentials. We tested the hypothesis that cultured hippocampal neurons would have GABA-activated chloride channels with conductance properties similar to those recorded from adult hippocampal neurons. All the cultures used were co-cultured with mouse astrocytes since hippocampal neurons require a substance released by glial cells for proper growth 9. Astrocyte cultures were prepared from the cerebral hemispheres of D u b : ( I C R ) random-bred mice and

* Present address: Wadsworth Center for Laboratories and Research, Empire State Plaza, Albany, NY 12201, U.S.A. Correspondence: E.X. Albuquerque, Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A. 0006-8993/87/$03.50 (~ 1987 Elsevier Science Publishers B.V. (Biomedical Division)

160 grown to confluence using the method of Booher and Sensenbrenner l° as modified by Brookes and Yarowsky 12. The astrocytes were grown on acid-soluble calf skin collagen (Calbiochem) in plastic culture dishes (35 ram, Nunc) in modified Eagle's medium (MEM, Gibco) supplemented with 15% fetal calf serum (KC Biological) and incubated at 35,5-36.6 °C in 10% CO2/90% air. Plastic coverslips coated with 0.1% poly-L-lysine (Sigma) were added to the culture dishes immediately prior to addition of the hippocampal neurons. Female Sprague-Dawley rats, 12-14 days pregnant, were sacrificed by CO2 narcosis and cervical dislocation. The embryos were removed, the brains of 12-14 fetuses removed and placed in a cold dissecting solution containing (mM): NaCl 140, KCI 5.4, Na2HPO4 0.32, KH2PO 4 0.22, glucose 25 and N2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) 20 at pH 7.3 and adjusted to 325 mOsm with sucrose. The hippocampi were dissected free, minced with iridectomy scissors and incubated with trypsin (0.25%, Gibco) for 15 rain at 35.5 °C. The trypsin action was terminated by pipetting the brain sections into 6-7 ml of MEM with 10% fetal calf serum and 10% horse serum. The neurons were dissociated by trituration and plated at a concentration of 700,000 cells per dish. The cell cultures were incubated for 3-4 h and the medium changed to growth medium containing MEM plus 10% fetal calf serum (MEM 10). The cell cultures were incubated at 35.5-36.6 °C in 10% CO2/90% air and the media replaced with fresh MEM 10 every 3 days. For patch clamp recording the cells were grown in culture for 6-13 days. Since the hippocampal granule cells do not form until day 1 postnatally, the cultures contained primarily pyramidal cell neurons 7,s. The neurons had membrane potentials o f - 5 5 to -65 mV, generated action potentials and showed spontaneous synaptic potentials. Recordings of single channel currents were made using the patch clamp technique of Hamill et al. 21. The patch clamp microelectrodes were made from borosilicate capillary glass (A and M Systems) and had a resistance of 3-5 MQ when filled with recording solution. An LM-EPC-7 Patch Clamp System (List Electronic, F.R.G.) was used to record the single channel currents. The data were stored on FM magnetic tape (Racal) for later computer analysis.

The data were filtered at 1 kHz (-3 dB) with an 8pole Bessel filter (Frequency Devices), digitized at 10 kHz and stored on the hard disk of an IBM XT microcomputer. The IPROC-2 program was used for the analysis of current amplitudes and open-channel duration (open time) 25. The histograms of open times and burst durations, groups of channel openings separated by less than 3 ms, were fit using the NFITS program 21. Each digitized point was plotted relative to the mean baseline current and the single current amplitude was estimated from the current peaks. To reduce the background noise due to current flow through other ionic channels, all monovalent cations were replaced with choline. The single channel currents were recorded from cell-free patches (inside-out condition22). The intracellular portion of the membrane was exposed to a solution containing (mM): choline CI 135, MgC12 1, ethyleneglycol-bis(fl-ethylether)N,N'-tetraacetic acid (EGTA) 5 and Hepes 10. E G T A was added to 10 ml distilled water with 1.7 ml of 1 M Tris then neutralized with 0.5 ml of 1 M HCI in a final volume of 100 ml (the HC1 adds 5 mM CI-). The pH was adjusted to 7.4 with Tris base. The physiological solution used to fill the microelectrode had the following composition (mM): choline CI 136, MgCI 2 1, CaCI 2 2, Hepes 10 and G A B A (0.4-1 pM). The pH was adjusted to 7.4 with NaOH (NaCl was replaced by choline chloride). All experiments were performed at room temperature of 20-22 °C. ),-Aminobutyric acid (0.4-1 pM)-activated currents which flowed inward at hyperpolarized potentials, reversed at about 0 mV and flowed outward at depolarized potentials (Fig. 1A). These single channel currents were not observed if GABA was excluded from the microetectrode filling solution. The current amplitude was linearly related to the membrane potential from -80 mV to +70 mV (Fig. 1B). The channel conductance was estimated from the slope of a regression line, calculated by the leastsquares method, to be 19 pS. In contrast, adult pyramidal cells have GABA-gated CI- channels with a conductance of 20 pS at membrane potentials more negative than 0 mV 2°. However, as the membrane potential was made more positive the single channel conductance values increased 2°. Cultured spinal cord neurons had GABA-activated channels with a conductance of 19-21 pS -~3"27and a linear current-vol-

161 A *50

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Membrane

Potential

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Fig. 1. Characteristics of GABA-activated chlc,ride channels. A: examples of single GABA-activated currents recorded from insideout patches. The values are the membrane potentials in mV. c, baseline current level; o, current level following opening of single chloride channels. B: single channel current amplitude plotted versus membrane potential. Each point represents the mean of the current amplitudes recorded from 4-7 patches. The vertical bars represent the standard deviations. The slope conductance was determined to be 19 pS from the slope of a line calculated by the least-squares method of linear regression. C: plot of single channel conductance and membrane potential. The channel conductance was calculated from the amplitude as shown in B. The vertical bars represent the standard deviation of the mean. The mean values are not significantly different from each other as determined by analysis of variance.

tage c u r v e o v e r a r a n g e f r o m - 1 0 0 mV22,24.

m V to + 7 0

In o u r study, G A B A - a c t i v a t e d c u r r e n t s of the cult u r e d h i p p o c a m p a l n e u r o n s did n o t rectify at any of the m e m b r a n e

tials 22'24. A n a l t e r n a t i v e e x p l a n a t i o n for this lack of rectification was that the culture e n v i r o n m e n t was

p o t e n t i a l s studied. T h e a b s e n c e of

not c o n d u c i v e to the d e v e l o p m e n t of c h a n n e l s which rectify at positive potentials. T h e m e a n c h a n n e l lifetimes of the o p e n state w e r e

o u t w a r d rectification was also o b s e r v e d w h e n the single c h a n n e l c o n d u c t a n c e was p l o t t e d v e r s u s m e m b r a n e p o t e n t i a l (Fig. 1C). T h e c o n d u c t a n c e of the single c h a n n e l s

was not significantly d i f f e r e n t

at

m e m b r a n e p o t e n t i a l r a n g e o f - 8 0 m V to + 7 0 m V ( P

TABLE I The open times anti burst times for GA BA-activated chloride currents

Values are means _+ S.D.

< 0.05, analysis of variance2S).The rectification of G A B A - a c t i v a t e d c u r r e n t s m a y reflect a m o d i f i c a t i o n of the c h a n n e l which occurs as the n e u r o n s m a t u r e f r o m e m b r y o s to adults. Six to 14 days in culture was not e n o u g h t i m e for the a p p e a r a n c e of rectification in these channels.

I n t e r e s t i n g in this r e g a r d was the

o b s e r v a t i o n that c u l t u r e d spinal c o r d n e u r o n s did not show

rectification

at

positive

membrane

poten-

Membrane potential (m V)

Open time (ms)

Burst duration (ms)

+60 +70 +80 -60 -70 -80

1.9±0.3 1.7±0.1 1.5±0.1 3.1±0.6 2.3±0.2 2.7±0.2

3.7±0.2 3.5±0.2 4.6±0.3 4.7±0.5 4.4±0.2 5.0±0.3

162 between 1.5 and 3.1 ms at the m e m b r a n e potentials tested (Table I). Due to the low amplitude of the ion-

rons. Additionally, the conductance values were sim-

ic currents and the inability to u n a m b i g u o u s l y resolve

and adult hippocampal n e u r o n s recorded from hip-

the open durations from the noise of the baseline, the open and burst times at m e m b r a n e potentials be-

pocampal slices. The cultured n e u r o n s did not have

tween - 5 0 and 50 mV were not included. The open

brane potentials in the adult neurons. This lack of rectification may reflect the physiological condition

time and burst time histograms were both fitted by curves which were the sum of two exponential functions. The first function had a decay time constant of 0.5 ms which was less than the rolloff of the filter and its significance was not determined. The lifetime estimates were not corrected for missed fast events. The open times (see Table I) were similar to the 2.5 ms open times of G A B A - a c t i v a t e d channels of adrenal chromaffin cells 11. These data demonstrate that channels activated by

ilar to those reported for cultured spinal cord n e u r o n s

the p r o n o u n c e d rectification seen at positive m e m -

of channels at an early stage of d e v e l o p m e n t or the effect of growth in culture. We are indebted to Dr. Neville Brookes for the use of the cell culture facilities and many helpful suggestions. We thank Mrs. Y v o n n e Logan for skillful technical assistance and Ms. Mabel Zelle for expert computer programming. This project was supported by U.S. A r m y Medical Research D e v e l o p m e n t Com-

G A B A were present on cultured hippocampal neu-

mand Contract DAMD-17-84-C-4219.

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