Neuroscience Letters, 138 (1992) 67-71
67
© 1992ElsevierScientificPublishers Ireland Ltd. All rights reserved0304-3940/92/$05.00 NSL 08527
Two classes of spontaneous GABA-mediated miniature synaptic currents in cultured rat hippocampal neurons J e a n Yautrin b, A n n e E. S c h a f f n e r a a n d Jeffery L. B a r k e r a "Laboratory of Neurophysiology, National Institute of Neurological and Communicative Disorders and Stroke, National Institute of Health, Bethesda, MD 20892 (USA) and bLaboratoire de Physiologie GOnOrale, UniversitO Paris XII, CrOteil (France)
(Received 8 October 1991;Revised version received 3 January 1992;Accepted 13 January 1992) Key words." Quantum; GABA; Hippocampal neuron; Transmitter release; Dissociatedcell culture; CI conductance
Amplitudeand time course of spontaneous ?-aminobutyricacid (GABA)-mediatedminiaturepostsynapticcurrents (MPSCs), recordedin cultured embryonichippocampalneuronsin presenceof either tetrodotoxin(TTX)or increasedexternal[Mg2÷/Ca2+]ratio, revealedthat they form two classes. The distribution of the most commonlyrecordedMPSCs was skewedboth in terms of peak amplitudeand rise-time(skew-MPSCs,mode: 70-120 pS). Another, less frequent class (mode: 1-3 nS) formed bell-shaped (bell-MPSCs) amplitude and rise-time distributions. MPSC initial slope did not correlate with rise time, indicating that smaller MPSCs were not electrotonicallyattenuated. BeII-MPSCsdid not result from the integration of skew-MPSCs and both classes appeared to be composed of subunits.
The observation of spontaneous, 'miniature' endplate potentials with constant time-course and amplitude revealed that the transmitter acetylcholine is released from spinal motoneuron terminals in uniform packets called quanta [7, 10]. Equally spaced peaks in the amplitude distributions of stimulus-evoked endplate potentials have been attributed to variations in the numbers of acetylcholine quanta and the endplate potential to the postsynaptic integration of miniatures evoked by the presynaptic action potential [7]. Recent recordings of synaptic signals generated by hippocampal GABAergic neurons in culture [2,8,14] and in slices [9, 19, 20] suggest that ),-aminobutyric acid (GABA) is also released in a quantal form. GABA quanta were estimated to activate only 3-20 C1- channels [8, 14, 19, 20], which is 2-3 orders of magnitude less than that calculated for cholinergic quanta. However, the variance of the peaks in multimodal amplitude distributions of 1-3 nS GABAergic synaptic events does not increase with amplitude as expected if they are due to the simultaneous, near-synchronized effects of independent quanta [7]; and the amplitudes of spontaneous miniature postsynaptic currents (MPSCs) form skewed rather than bell-shaped gaussian Correspondence: J.L. Barker, Laboratory of Neurophysiology, National Institute of Neurological and Communicative Disorders and Stroke, National Institute of Health, Bldg. 36, Room 2C02, Bethesda, MD 20892, USA. Fax: (1) (301) 402 15 65.
distributions, as expected for a quantal process [8, 14, 19, 20]. Here we have resolved two classes of GABAergic MPSCs at cultured hippocampal synapses that remain in T T X or elevated [Mg2+/Ca 2+] ratios and can be differentiated according to their conductance and rise time distributions. Hippocampi from 18-day-old rat embryos were enzymatically dissociated with papain (Worthington) [12]. Cells were plated onto 35 mm plates coated with 20 pg/ ml high molecular weight poly-D-lysine at a density of 2.1- 5.2 × 1 0 4 cells/cmz. Plating medium consisted of minimal essential medium (MEM) with 3.7 g/l sodium bicarbonate, 6 g/1 glucose (Gibco), 5% fetal calf serum and 5% horse serum. Cultures were kept at 36°C in a CO2 incubator and after 4 days the cells were maintained with M E M and 5% horse serum. Postsynaptic currents were recorded at room temperature (23-25°C) using the patch clamp recording technique in the whole-cell mode [11]. The electrode contained (in mM): CsCI 145, CaCIE 0.1, EGTA 1.1, MgC12 2, ATP 5, HEPES 5 adjusted to pH 7.2 and 310 mOsm with sucrose. The bath contained (in mM): NaC1 115, NaHCO3 25, KC1 5, CaCI2 2, MgC12 1, HEPES 10, o-glucose 10, adjusted to pH 7.4 and 295 mOsm. One-third of the 80 neurons included in the study exhibited spontaneous synaptic currents and were analyzed in further detail. The synaptic signal was filtered at 1 kHz (8-Pole Bessel) and digitized (12 bits) at 20-10 kHz. All events detected via internal-triggering
68
were visually selected. Frequent negative slopes during the °rising' phase were attributed to closely clustered multiple events; these were discarded from further analysis. Amplitude and rise-time (10-90% of peak amplitude) properties of more than 20,000 MPSCs from 13 of the 20-odd neurons exhibiting spontaneous synaptic activity were analyzed with the SCAN program, courtesy of Dr.
A
J. Dempster. All of the synaptic events reversed polarity at ~0 mV (Eel) and at - 8 0 mV had an averaged decay fitted by a single exponential with a time constant ->20 ms [3]. Either 50/aM bicuculline or 10/aM picrotoxin blocked all of this spontaneous synaptic activity. We conclude that all of this activity was mediated by GABA. Amplitude vs. rise-time plots revealed that
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69 spontaneous MPSCs group as two classes, which could be either quite distinct (Fig. 1A) or overlapping (Fig. 2B) regardless of the holding potential. One class of tetrodotoxin (TTX)-resistant MPSCs exhibited amplitude distributions typically more skewed than bell-shaped (Figs. 1El and 2B1). Their rise-time distribution was also skewed (Fig. IF1). Some skew-MPSC rise-time distributions showed clearly recognizable peaks involving up to 50% of the counts (Fig. 1F1). The mode of skew-MPSCs was usually about 120 pS; but in some cases the resolution of the smallest MPSCs (
67
© 1992ElsevierScientificPublishers Ireland Ltd. All rights reserved0304-3940/92/$05.00 NSL 08527
Two classes of spontaneous GABA-mediated miniature synaptic currents in cultured rat hippocampal neurons J e a n Yautrin b, A n n e E. S c h a f f n e r a a n d Jeffery L. B a r k e r a "Laboratory of Neurophysiology, National Institute of Neurological and Communicative Disorders and Stroke, National Institute of Health, Bethesda, MD 20892 (USA) and bLaboratoire de Physiologie GOnOrale, UniversitO Paris XII, CrOteil (France)
(Received 8 October 1991;Revised version received 3 January 1992;Accepted 13 January 1992) Key words." Quantum; GABA; Hippocampal neuron; Transmitter release; Dissociatedcell culture; CI conductance
Amplitudeand time course of spontaneous ?-aminobutyricacid (GABA)-mediatedminiaturepostsynapticcurrents (MPSCs), recordedin cultured embryonichippocampalneuronsin presenceof either tetrodotoxin(TTX)or increasedexternal[Mg2÷/Ca2+]ratio, revealedthat they form two classes. The distribution of the most commonlyrecordedMPSCs was skewedboth in terms of peak amplitudeand rise-time(skew-MPSCs,mode: 70-120 pS). Another, less frequent class (mode: 1-3 nS) formed bell-shaped (bell-MPSCs) amplitude and rise-time distributions. MPSC initial slope did not correlate with rise time, indicating that smaller MPSCs were not electrotonicallyattenuated. BeII-MPSCsdid not result from the integration of skew-MPSCs and both classes appeared to be composed of subunits.
The observation of spontaneous, 'miniature' endplate potentials with constant time-course and amplitude revealed that the transmitter acetylcholine is released from spinal motoneuron terminals in uniform packets called quanta [7, 10]. Equally spaced peaks in the amplitude distributions of stimulus-evoked endplate potentials have been attributed to variations in the numbers of acetylcholine quanta and the endplate potential to the postsynaptic integration of miniatures evoked by the presynaptic action potential [7]. Recent recordings of synaptic signals generated by hippocampal GABAergic neurons in culture [2,8,14] and in slices [9, 19, 20] suggest that ),-aminobutyric acid (GABA) is also released in a quantal form. GABA quanta were estimated to activate only 3-20 C1- channels [8, 14, 19, 20], which is 2-3 orders of magnitude less than that calculated for cholinergic quanta. However, the variance of the peaks in multimodal amplitude distributions of 1-3 nS GABAergic synaptic events does not increase with amplitude as expected if they are due to the simultaneous, near-synchronized effects of independent quanta [7]; and the amplitudes of spontaneous miniature postsynaptic currents (MPSCs) form skewed rather than bell-shaped gaussian Correspondence: J.L. Barker, Laboratory of Neurophysiology, National Institute of Neurological and Communicative Disorders and Stroke, National Institute of Health, Bldg. 36, Room 2C02, Bethesda, MD 20892, USA. Fax: (1) (301) 402 15 65.
distributions, as expected for a quantal process [8, 14, 19, 20]. Here we have resolved two classes of GABAergic MPSCs at cultured hippocampal synapses that remain in T T X or elevated [Mg2+/Ca 2+] ratios and can be differentiated according to their conductance and rise time distributions. Hippocampi from 18-day-old rat embryos were enzymatically dissociated with papain (Worthington) [12]. Cells were plated onto 35 mm plates coated with 20 pg/ ml high molecular weight poly-D-lysine at a density of 2.1- 5.2 × 1 0 4 cells/cmz. Plating medium consisted of minimal essential medium (MEM) with 3.7 g/l sodium bicarbonate, 6 g/1 glucose (Gibco), 5% fetal calf serum and 5% horse serum. Cultures were kept at 36°C in a CO2 incubator and after 4 days the cells were maintained with M E M and 5% horse serum. Postsynaptic currents were recorded at room temperature (23-25°C) using the patch clamp recording technique in the whole-cell mode [11]. The electrode contained (in mM): CsCI 145, CaCIE 0.1, EGTA 1.1, MgC12 2, ATP 5, HEPES 5 adjusted to pH 7.2 and 310 mOsm with sucrose. The bath contained (in mM): NaC1 115, NaHCO3 25, KC1 5, CaCI2 2, MgC12 1, HEPES 10, o-glucose 10, adjusted to pH 7.4 and 295 mOsm. One-third of the 80 neurons included in the study exhibited spontaneous synaptic currents and were analyzed in further detail. The synaptic signal was filtered at 1 kHz (8-Pole Bessel) and digitized (12 bits) at 20-10 kHz. All events detected via internal-triggering
68
were visually selected. Frequent negative slopes during the °rising' phase were attributed to closely clustered multiple events; these were discarded from further analysis. Amplitude and rise-time (10-90% of peak amplitude) properties of more than 20,000 MPSCs from 13 of the 20-odd neurons exhibiting spontaneous synaptic activity were analyzed with the SCAN program, courtesy of Dr.
A
J. Dempster. All of the synaptic events reversed polarity at ~0 mV (Eel) and at - 8 0 mV had an averaged decay fitted by a single exponential with a time constant ->20 ms [3]. Either 50/aM bicuculline or 10/aM picrotoxin blocked all of this spontaneous synaptic activity. We conclude that all of this activity was mediated by GABA. Amplitude vs. rise-time plots revealed that
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69 spontaneous MPSCs group as two classes, which could be either quite distinct (Fig. 1A) or overlapping (Fig. 2B) regardless of the holding potential. One class of tetrodotoxin (TTX)-resistant MPSCs exhibited amplitude distributions typically more skewed than bell-shaped (Figs. 1El and 2B1). Their rise-time distribution was also skewed (Fig. IF1). Some skew-MPSC rise-time distributions showed clearly recognizable peaks involving up to 50% of the counts (Fig. 1F1). The mode of skew-MPSCs was usually about 120 pS; but in some cases the resolution of the smallest MPSCs (
