Activity-Dependent Depression of GABAergic IPSCs in Cultured Hippocampal Neurons

Kimmo Jensen,1 John D. C. Lambert,1 and Morten Skovgaard Jensen2

 1Department of Physiology and  2Department of Neurobiology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Jensen, Kimmo, John D. C. Lambert, and Morten Skovgaard Jensen. Activity-Dependent Depression of GABAergic IPSCs in Cultured Hippocampal Neurons. J. Neurophysiol. 82: 42-49, 1999. Short-term depression of monosynaptic GABAergic inhibitory postsynaptic currents (IPSCs) evoked between pairs of cultured rat hippocampal neurons was investigated using dual whole cell patch-clamp recordings. Paired stimuli applied to the GABAergic neuron resulted in paired-pulse depression (PPD) of the second IPSC (IPSC2) at interpulse intervals from 25 to 2,000 ms. CGP 55845A, but not CGP 35348, reduced PPD marginally. Brief paired-pulse applications of exogenous GABA indicated that postsynaptic factors made only minimal contribution to PPD of IPSCs. IPSC1 and PPD was reduced on lowering [Ca2+]o and enhanced on increasing [Ca2+]o. The potassium-channel blocker 4-aminopyridine (4-AP), which increases presynaptic Ca2+ influx, enhanced IPSC1 and PPD. Chelation of residual Ca2+ in the GABAergic boutons with EGTA-AM enhanced PPD. Stimulation of the presynaptic neuron at frequencies (f) ranging from 2.5 to 80 Hz resulted in tetanic depression of IPSCs, which declined rapidly and reached a plateau depending on f and [Ca2+]o. CGP 55845A decreased tetanic depression in the first part of the train, but this could be overcome with continued stimulation. We show that GABAergic IPSCs are robustly depressed by paired-pulse stimulation in cultured hippocampal neurons. The depression of IPSCs is mainly independent of presynaptic GABAB receptors and could be caused by depletion of releasable vesicles. Depleted synapses recover with a slow time course, depending on factors that regulate [Ca2+]i in the GABAergic boutons.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

GABA is the major inhibitory neurotransmitter in the CNS, where it acts on GABAA and GABAB receptors. GABA is released in response to an increase in Ca2+ influx through N- and P/Q-type Ca2+ channels located near the secretory apparatus (Ohno-Shosaku et al. 1994a). The release of neurotransmitter at single synapses depends on their history of activation, because different patterns of stimulation can evoke frequency-dependent facilitation or depression (McCarren and Alger 1985). In experiments with hippocampal slices, inhibitory postsynaptic potentials (IPSPs) evoked at fast rates are usually depressed for several seconds due to feedback of GABA onto presynaptic GABAB receptors (Nathan and Lambert 1991; Roepstorff and Lambert 1994), which depresses GABA release by a G protein coupled mechanism (Thompson et al. 1993). However, a fraction of inhibitory postsynaptic current (IPSC) paired-pulse depression (PPD) is insensitive to GABAB receptor antagonists (Lambert and Wilson 1994). Glutamatergic synapses are also subject to PPD, which seems to depend on mechanisms associated with the basal secretory apparatus at the active zones (Dobrunz et al. 1997). It is therefore likely that excitatory and inhibitory synapses share basal secretory mechanisms, as suggested by Lambert and Wilson (1994). These mechanisms can be studied in detail in cultured hippocampal neurons because GABAergic IPSCs show a substantial PPD, which is insensitive to GABAB receptor antagonists (Wilcox and Dichter 1994; Yoon and Rothman 1991), although functional GABAB receptors are present on the terminals (Harrison 1990).

We have made paired whole cell patch-clamp recordings from cultured hippocampal neurons and stimulated the presynaptic GABAergic neuron to evoke monosynaptic IPSCs, which were used to study the frequency-dependent depression of GABAergic IPSCs. Our results indicate that depletion of synaptic vesicles is responsible for depression of repetitively evoked IPSCs, although "spill-over" of GABA onto presynaptic GABAB autoreceptors does occur to a small extent. We also examined whether an increased level of internal Ca2+ contributes to refilling of depleted active zones. The latter process would be important for sustained release of transmitter during prolonged high-frequency activity at inhibitory GABAergic synapses.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Hippocampal culture preparation

Pregnant Sprague-Dawley rats were anesthetized by pentobarbital sodium (50 mg/kg ip) at gestational day 17-18. Fetuses were removed and decapitated, and the hippocampi were dissected free. The tissue was triturated mechanically in a HEPES-buffered dissection medium and plated on poly-D-lysine-coated coverslips in 35-mm Petri dishes. Plating medium consisted of minimal essential medium with Earle's salts and Glutamax-1 (glutamine) supplemented with horse serum (HS, 10%), fetal calf serum (FCS, 10%), penicillin (50 IU ml-1), and streptomycin (50 µg/ml). Cultures were grown in 5% CO2 and 10% O2 at 37°C. Plating medium was fully replaced by 2 ml feeding medium after 1 day in vitro, and thereafter 1 ml was exchanged twice weekly. Feeding medium had the same composition as plating medium except that FCS was omitted and HS was reduced to 5%. The mitosis inhibitors 5'-Fluoro-2'-Deoxyuridine (FUDR, 15 µg/ml) and uridine (35 µg/ml) were added after 3-4 days to inhibit glial overgrowth.

Electrophysiology

Coverslips with the cultured cells were placed in a chamber mounted on an inverted Nikon Diaphot 200 microscope and perfused (1 ml/min) with an extracellular (control) medium containing (in mM) 140 NaCl, 3.5 KCl, 2.5 CaCl2, 2.5 MgCl2, 10 glucose, and 10 HEPES; pH 7.35 with NaOH (22°C), osmolality 305 mosm/kg (Wescor 5500 osmometer). Patch-clamp electrodes (3-6 MOmega ) were fabricated from borosilicate glass (1.2 mm OD) on a Flaming/Brown P-97 puller (Sutter Instruments). The presynaptic electrode contained (in mM) 140 KOH, 11 EGTA, 1 CaCl2, 2 MgCl2, 15 NaCl, 10 HEPES, 0.10 leupeptin, 2 MgATP; pH adjusted to 7.3 with methanesulfonic acid, 290 mosm/kg. To increase the driving-force for Cl- and to block Na+ and K+ currents, the postsynaptic electrode contained (in mM) 120 CsCl, 10 TEACl, 5 QX-314, 11 EGTA, 1 CaCl2, 1 MgSO4, 0.10 leupeptin, 4 MgATP, pH adjusted to 7.3 with CsOH, 290 mosm/kg. Whole cell recordings were made at a holding potential (Vhold) of -70 mV using Axopatch 200 and 200A amplifiers in voltage clamp. Excitatory synaptic responses were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 µM) and DL-2-amino-5-phosphonopentanoic acid (DL-AP5; 50 µM). GABAergic neurons were identified by the presence of autaptic IPSCs following a 3-ms depolarizing pulse to 0 mV.

Stimulation pulses (3 ms at 0 mV) were delivered by a pulse-generator (Master 8, AMPI) to the presynaptic GABAergic neuron. Paired-pulse stimulation was delivered at interpulse intervals (IPI) ranging from 25 ms to 1 s at a rate of 0.1 Hz and at IPIs of 2-4 s at 0.067 Hz. Stimulus trains consisting of 80 pulses were given at 2.5-80 Hz. Whole cell currents were low-pass filtered at 10 kHz, monitored on a pen recorder (Servogor 220), digitized using an AD converter (Instrutech VR-100 B), and stored simultaneously on a VTR and a Pentium PC equipped with Clampex (pClamp v. 6.0. software, Axon Instruments).

Drug application

Active substances were dissolved as stock solutions at 1,000 times the final concentration, diluted in extracellular medium just before use and perfused through the bath (exchange time 2-3 min). All changes in [Ca2+]o were compensated by changes in [Mg2+]o to keep the total extracellular divalent cation concentration constant at 5 mM. For experiments in which a rapid change of the extracellular medium was required, the neurons were continuously superfused from a three-barrel gravity-feed pipette (tip opening ~100 µm). Brief (10 ms duration) applications of GABA (1 mM) were made by applying pressure to a patch electrode with an opening of 4-5 µm placed close to the neuron. Response kinetics were improved by rapid removal of the applied GABA through a larger (100-µm opening) suction pipette placed nearby. In these experiments, TTX (0.2 µM) was added to the bathing solution to inhibit spontaneous synaptic currents.

Drugs and chemicals were purchased from Sigma except CNQX and DL-AP5 (Tocris Cookson) and EGTA-AM (Molecular Probes). CGP 35348 was a gift from Ciba-Geigy, and CGP 55845A was provided by Novartis. Culturing media were purchased from GIBCO, except for FUDR, uridine, and poly-D-lysine (Sigma).

Analysis

The traces shown in the figures are averages of eight consecutive responses, unless otherwise indicated. Analysis of IPSCs and responses to GABA was performed in pClamp (Axon Instruments). IPSCs were inspected visually and rejected if spontaneous activity disturbed the measurements. PPD is expressed as the percentage of the net amplitude of the second IPSC (IPSC2) compared with the first (IPSC1). In the paired-pulse experiments, decays of IPSCs and responses to exogenous GABA were fitted by a monoexponential function using the Clampfit routine and inspected visually. Data were imported into a spreadsheet (Excel v. 7.0a), where statistical calculations were performed. All data are presented as means ± SE with n indicating the number of pairs of neurons tested. Changes were considered to be significant at P values < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Monosynaptic GABAAergic IPSCs

Monosynaptic GABAAergic IPSCs were investigated in pairs of rat hippocampal neurons, which were continuously perfused with CNQX (10 µM) and DL-AP5 (50 µM) to block glutamatergic excitation. The presynaptic GABAergic neuron was stimulated by stepping from -70 to 0 mV for 3 ms, which evoked a short-latency (1-3 ms) IPSC in most nearby neurons. The IPSC was blocked by bicuculline (10 µM) and had a reversal potential near ECl (n = 5), indicating that it is mediated by GABAA receptors. In spite of the inclusion of MgATP in the pipette solutions, minor rundown of IPSC amplitudes was usually seen. Rundown has previously been calculated to be ~10% after the first 20 min of recording (Jensen et al. 1999b).

PPD of IPSCs

To study use-dependent depression of the IPSCs, pairs of stimuli at an IPI of 150 ms were delivered at 0.1 Hz to the presynaptic GABAergic neuron. Single traces showed that the amplitude of IPSC2 varied considerably more than the amplitude of IPSC1 (Fig. 1A), and on rare occasions it was larger than IPSC2 (paired-pulse facilitation, not shown). Normally, eight consecutive pairs of IPSCs were averaged and used as the basis for further analysis. Averaged traces disclosed that the amplitude of IPSC2 was always depressed with respect to the first, commonly termed PPD. The time course of PPD was investigated by varying IPI between 25 and 4,000 ms (Fig. 1B). At short IPIs, IPSCs showed temporal summation with IPSC2 riding on the tail of IPSC1. IPSC2 was then measured as the net response after subtraction of the tail of IPSC1. When results from such experiments were pooled from seven pairs of neurons, significant PPD was seen at IPIs from 25 to 2,000 ms (Fig. 1C). Maximal depression occurred at the shortest IPI of 25 ms and was by 55.2 ± 7.4% (mean ± SE, P < 0.01).



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 1. Paired-pulse depression (PPD) of monosynaptic IPSCs in cultured hippocampal neurons. A: dual whole cell patch-clamp recordings were obtained from cultured hippocampal neurons coupled by GABAergic synapses. Eight consecutive postsynaptic responses (bottom trace) elicited by double stimulation of the presynaptic GABAergic neuron (top trace) are superimposed [interpulse interval (IPI) 150 ms, stimulation frequency 0.1 Hz]. The 2nd inhibitory postsynaptic current (IPSC2) was depressed compared with IPSC1 and showed a greater variability than IPSC1. B: traces show IPSCs evoked at the indicated IPIs. The 2 IPSCs showed temporal summation at IPIs of up to 250 ms. In this neuron, PPD of the 2nd IPSC was seen up to 4 s. C: graph showing the amplitude of IPSC2 normalized to IPSC1 as a function of IPI (log scale). For IPIs showing temporal summation, IPSC2 was measured as the net response after subtraction of the tail of IPSC1. Maximal PPD of 55.2 ± 7.4% was seen at the shortest IPI of 25 ms, whereas significant PPD of IPSC2 was seen at IPIs up to 2 s (** P < 0.01, paired t-tests).

The decay of the IPSCs could be fitted by a monoexponential function with a time constant, tau decay, of 41.9 ± 5.3 ms for IPSC1. tau decay for IPSC2 at an IPI of 250 ms (where the 2 IPSCs were completely separated) was 39.3 ± 4.5 ms, which was not significantly different from tau decay of IPSC1 (n = 7; P > 0.05). This is in contrast to IPSCs in intact neuronal preparations in which tau decay of IPSC2 is modulated by paired-pulse stimulation (Roepstorff and Lambert 1994).

Effects of changing [Ca2+]o on IPSCs and PPD

The probability of transmitter release at cultured hippocampal GABAergic synapses is steeply dependent on [Ca2+]o (Ohno-Shosaku et al. 1994b). In 30 experiments performed on 14 pairs of neurons, we examined the effect of changing [Ca2+]o in the range of 0.8-4.0 mM on pairs of IPSCs elicited at an IPI of 150 ms (Fig. 2A). We measured IPSC1 relative to IPSC1 in 2.5 mM Ca2+ and plotted this against PPD (Fig. 2C; triangle ). Linear regression showed that PPD was correlated to the size of IPSC1 (P < 0.05), and for every 100% increase in IPSC1, PPD increased by 33%.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 2. Changing release probability modulates PPD. A: representative traces showing the effect of altering [Ca2+]o on IPSCs evoked by paired presynaptic stimulation (IPI 150 ms). In control medium, PPD was 65%. Perfusion of 0.8 mM Ca2+ depressed IPSC1 more than IPSC2, resulting in PPD being reduced to 22%. In 4.0 mM Ca2+, IPSC1 was enhanced by 29%, whereas IPSC2 was again reduced. This resulted in an increase of PPD to 79%. The effects of changing [Ca2+]o were fully reversible on washing. B: traces showing the effect of 4-aminopyridine (4-AP; 50 µM) on IPSCs evoked by paired presynaptic stimulation (IPI 150 ms). PPD was 24% in control medium. 4-AP enhanced IPSC1 by 40%, whereas the net IPSC2 was decreased by 9%, resulting in a PPD of 51%. The effect of 4-AP reversed on washing and a small rundown of both IPSCs was seen. C: graph showing the relationship between PPD (IPI 150 ms) and the relative amplitude of IPSC1. The results are pooled from experiments with changed [Ca2+]o (black-triangle, 17 trials in 13 pairs of neurons) and 4-AP (triangle , 18 trials on 11 pairs of neurons). IPSC1 in test solution was normalized to IPSC1 in 2.5 mM Ca2+. Ca2+ was varied between 0.8 and 4.0 mM, whereas 4-AP ranged from 20 to 100 µM. Linear regression of the initial part of the curve showed that PPD increased by 33% for every 100% increase in IPSC1. D: depression of IPSC2 plotted against IPI in the presence of 100 µM 4-AP for a single neuron. The data in control medium (black-down-triangle ) are from Fig. 1C. IPSC2 was nearly abolished by 4-AP at short IPIs. Although PPD was greater in the presence of 4-AP, the time course of the depression was similar to the control.

Effect of 4-aminopyridine (4-AP) on IPSCs and PPD

4-AP inhibits A-type K+ channels and delays repolarisation after APs. This prolongs presynaptic Ca2+ influx and enhances transmitter release (Buckle and Haas 1982). During paired stimulation, 4-AP (20-100 µM) enhanced IPSC1 and PPD (Fig. 2B). From the plot of PPD as a function of IPSC1 (Fig. 2C; black-triangle), it is seen that PPD did not increase as steeply with IPSC1 as was the case in the experiments in which [Ca2+]o was increased. We also wanted to examine recovery from PPD after depletion of the entire readily releasable pool of vesicles. We found that this could be accomplished by perfusing 100 µM 4-AP, which enhanced PPD to nearly 100% at the shortest IPIs. Results from a single pair of neurons are shown in Fig. 2D. The time course of PPD in the presence of 4-AP was similar to the control (t1/2 ~500 ms, and full recovery following PPD ~3 s in both cases).

PPD is reduced by a GABAB receptor antagonist

It has been reported that PPD of IPSCs in cultured hippocampal neurons is completely insensitive to the GABAB receptor antagonists 2-OH saclofen (Yoon and Rothman 1991) and CGP 35348 (Wilcox and Dichter 1994). Synaptically released GABA is, therefore, not thought to activate presynaptic GABAB receptors in this preparation. Accordingly, we found that bath perfusion of CGP 35348 (100 µM) had no effect on either IPSC1 or PPD, which was 36.0 ± 6.3% in control compared with 35.1 ± 5.1% in CGP 35348 (P > 0.05, n = 6, paired t-test). The GABAB agonist, baclofen (10 µM) depressed IPSCs by 41.8 ± 6.9% (n = 7, not shown), and this effect could be blocked by CGP 35348 (n = 3), demonstrating that this antagonist was indeed able to block actions at presynaptic GABAB receptors.

In contrast to CGP 35348, the newer potent blocker CGP 55845A (1-5 µM) (Davies et al. 1993) caused a significant reduction of PPD (Fig. 3B) from 39.4 ± 7.9% to 31.8 ± 10.4% (P < 0.05, n = 6, paired t-test). Full reversal of the effect on PPD was obtained after wash out of the drug. Because rundown was usually seen, the effect of CGP 55845A on a single IPSC was difficult to evaluate. However, when CGP 55845A was applied through a perfusion pipette that allowed medium exchange within a few seconds (see METHODS), single IPSCs were slightly enhanced (by 7% superimposed on a rundown of 2%, n = 2). This could indicate that presynaptic GABAB receptors are tonically activated in control conditions.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 3. CGP 55845A reduces PPD. A: IPSCs elicited by paired presynaptic stimulation with an IPI of 150 ms. In control solution, PPD was 39%. CGP 55845A (2 µM) reduced PPD to 34%. The effects of CGP 55845A were superimposed on a small rundown of the responses, which accounts for the apparent incomplete reversal of IPSC1 on washing. Shown below are IPSCs scaled to the same IPSC1 amplitude and superimposed to illustrate the reduction of PPD by CGP 55845A. B: CGP 55845A (1-5 µM) reduced PPD significantly from 39.4 to 31.8% (n = 6). PPD was not affected by the rundown of the IPSCs and reversed to 37.6% on washing.

IPSCs can be mimicked by exogenous applications of GABA

Investigations of the reversal potentials for pairs of IPSCs showed that EIPSC did not change between IPSC1 and IPSC2 (n = 2, not shown). To examine whether desensitization of postsynaptic GABAA receptors plays a role in PPD, these were activated directly by applying brief (10 ms) pulses of GABA (1 mM) to 25 single neurons using a focal perfusion system (see METHODS). The responses to GABA qualitatively mimicked GABAergic IPSCs, although the response to GABA (Fig. 4Ab) had slower kinetics (tau decay 82.7 ± 7.3 ms, n = 25) than IPSCs (41.9 ± 5.3 ms). Paired application of GABA was made in 71 trials at different IPIs. The absolute amplitudes of the two responses to GABA were similar, whereas the net amplitude of the second response (GABA2) depended on whether a tail of the first response was still present (Fig. 4B). PPD of the net amplitude of GABA2 was therefore mainly a result of temporal summation. This is as illustrated in Fig. 4Ca, where the decay of GABA1 (dotted line) is shown in relation to PPD of the GABA responses. The curve for PPD deviates from the decay of GABA1 in the interval 200-1,000 ms, indicating that there is a slight decrease in postsynaptic responsiveness during GABA2. The GABAergic IPSCs showed profound PPD at intervals up to 3 s, irrespective of the decay of IPSC1 (Fig. 4Cb), indicating that PPD of IPSCs was not affected by temporal summation. Taken together, these data suggest that PPD mainly involves a presynaptic mechanism.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 4. Responses evoked by exogenous GABA show less paired-pulse depression than IPSCs. Aa: pair of monosynaptic IPSCs evoked by presynaptic stimulation at an IPI of 150 ms displayed PPD of 47%. Ab: GABAA receptor-mediated whole cell currents in another neuron elicited by brief applications of 1 mM GABA through a local perfusion system (solid bars; pulse duration 10 ms; IPI 150 ms, average of 4 responses). Responses evoked by exogenous GABA mimicked the IPSCs qualitatively, although they had a slower decay kinetics (t1/2 39 ms for the 1st GABAA response compared with 28 ms for IPSC1 in Aa). The 2nd GABAA response (GABA2) reached the same amplitude as the 1st. B: responses from another neuron elicited by applications of GABA at the indicated IPIs. Note that the absolute peak values of the 2 responses were similar, irrespective of IPI. Ca: summary of results from 71 trials in 25 neurons of paired GABA applications at different IPIs. PPD of GABA responses was calculated as the net amplitude of GABA2 compared with GABA1 and plotted against IPI (triangle ; as in Fig. 1C). Dotted line (+) represents the decay of GABA1 calculated from the average tau decay (82.7 ms) of single GABA responses. This shows that the net PPD of GABA responses is mainly caused by temporal summation, with a slight decrease in postsynaptic responsiveness at IPIs of 200-1,000 ms. Cb: PPD of IPSCs (taken from Fig. 1C) and the decay of IPSC1 (calculated as above). IPSCs show profound PPD that is independent of the decay of IPSC1.

EGTA-AM increases PPD

It has recently been reported that the rate of recovery from PPD at excitatory glutamatergic synapses depends on residual Ca2+ in the boutons, which is thought to stimulate refilling of depleted vesicles at the active zones (Dittman and Regehr 1998; Stevens and Wesseling 1998; Wang and Kaczmarek 1998). It has not been established whether a similar mechanism is present at inhibitory GABAergic synapses. To investigate this, we made recordings from presynaptic neurons in the perforated-patch configuration to preserve the internal environment. We evoked pairs of IPSCs at an IPI of 500 ms (each 15 s) and monitored PPD (Fig. 5). After stable control recordings had been obtained, perfusion of the membrane-permeable Ca2+ chelator, EGTA-AM (100 µM), was started. PPD increased over the course the next minute and stabilized at a higher level (Fig. 5B), whereas no effect was seen on IPSC1. In four pairs of neurons, PPD increased from 26.6 ± 4.1% in control to 42.9 ± 1.7% in EGTA-AM (P < 0.05, Fig. 5C). These results indicate that chelation of Ca2+ in the boutons by exogenously applied Ca2+ buffers depresses IPSC2.



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 5. EGTA-AM enhances PPD. A: perforated-patch recording was made from a presynaptic GABAergic neuron, and this was stimulated at an IPI of 500 ms to evoke pairs of IPSCs. PPD was 26% under control conditions. After 5 min extracellular application of EGTA-AM (100 µM), PPD had increased to 38%, whereas there was no effect on IPSC1. Responses are scaled to the same IPSC1 and superimposed to the right. B: graph from another pair of neurons showing the effect of perfusing EGTA-AM on PPD measured from single pairs of responses. PPD was on average 21% at the start of the experiment. Two minutes after start of perfusion with EGTA-AM, PPD had increased to a mean value of 48%. C: similar experiments were performed in a total of 4 pairs of neurons. PPD increased from 26.6 ± 4.1 in control to 42.9 ± 1.7% in EGTA-AM.

Tetanic depression of IPSCs depends on frequency and [Ca2+]o

Because a substantial depression of IPSC2 was observed with paired stimulation, it was expected that IPSCs would be further reduced in response to prolonged train stimulation. We applied train stimuli consisting of 80 pulses at frequencies (f) ranging from 2.5 to 80 Hz (37 trains in 20 pairs of neurons). At all frequencies, a gradual decrement of net IPSC amplitudes (tetanic depression) was observed, which reached a plateau after about five pulses (Fig. 6B). The constants for the initial decrease in IPSC (tau depr) did not change in any consistent pattern with f (Table 1, middle column). On the other hand, when the first IPSC was compared with the net amplitudes of the last 10 IPSCs in the train, the plateau phase during tetanic depression depended strongly on f (Table 1, right column).



View larger version (41K):
[in this window]
[in a new window]
 
Fig. 6. Tetanic depression of IPSCs. A: single traces from 4 different neurons showing tetanic depression of IPSCs evoked by stimulation with 80 pulses at 5, 20, 40, and 80 Hz, respectively. IPSCs showed temporal summation at 20, 40, and 80 Hz, and at 20 and 80 Hz a gradual increase in the plateau current occurred toward the end of the train. Tetanic depression of IPSCs was assessed by measuring the average net amplitude at the end of the train (IPSC71 to IPSC80), and was 42% at 5 Hz, 67% at 20 Hz, 89% at 40 Hz, and 91% at 80 Hz (vertical scale bars: 5 Hz, 2,000 pA; 20 Hz, 200 pA; 40 and 80 Hz, 1,000 pA). B: tetanic depression of net IPSCs as a function of stimulus number at the frequencies shown. Thirty-seven trains consisting of 80 pulses were applied to 20 pairs of neurons. For each frequency the net IPSC amplitudes were measured, calculated as a percentage with respect to the 1st IPSC and pooled. Only results from the 1st and last 10 pulses are shown. Net IPSCs reached a relatively stable amplitude after 5-6 pulses, irrespective of stimulus frequency. At 2.5, 10, and 20 Hz IPSCs had increased slightly compared with the plateau earlier in the train, whereas a decrease was seen at 40 and 80 Hz. Results for 5 Hz lay between 2.5 and 10 Hz and are omitted for clarity. C: graph showing tetanic depression of net IPSCs at 40 Hz in different [Ca2+]o. Results are presented as in B and show that tetanic depression increases with [Ca2+]o.


                              
View this table:
[in this window]
[in a new window]
 
Table 1. The dependence of the rate and extent of tetanic depression depends on stimulating frequency

Tetanic depression also depended on extracellular Ca2+ as shown when this was decreased from 2.5 to 1.2 mM or increased to 4.0 mM (Fig. 6C). tau depr was 1.55 stimuli in 1.2 mM Ca2+, 0.90 stimuli in 2.5 mM Ca2+, and 0.41 stimuli in 4.0 mM Ca2+. In 1.2 mM [Ca2+]o the last 10 IPSCs were depressed by 76.3 ± 3.1%, in 2.5 mM Ca2+ by 82.1 ± 1.4% and in 4.0 mM Ca2+ by 89.9 ± 1.3%.

Effects of CGP 55845A on tetanic depression

To see whether increasing the level of extracellular GABA could cause a greater activation of GABAB autoreceptors, we stimulated the presynaptic neuron at 80 Hz for 1 s and tested the effect of CGP 55845A on the response (Fig. 7, n = 8). CGP 55845A reduced tetanic depression during the early part of the train but, surprisingly, not later in the train (Fig. 7). To quantify this differential effect, IPSC areas were measured with respect to the baseline during an early (at 150-300 ms) and a later (at 850-1,000 ms) period of the train (stippled thin lines in Fig. 7B). Areas were normalized to the area of a single pretetanic IPSC in each solution. Accordingly, a value of 1.0 means that the IPSC area was the same as the area of a single pretetanic IPSC. In CGP 55845A, the early area was 2.28 ± 0.3, which was 16% larger than in control solution (1.96 ± 0.2, P < 0.05, Fig. 7C). There was no significant difference between the late areas, which were 2.45 ± 0.3 in CGP 55845A compared with 2.28 ± 0.3 (P > 0.05). This indicates that the effect of GABAB autoreceptor activation is overcome by continued tetanic stimulation.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 7. CGP 55845A partially reverses tetanic depression of IPSCs. A: single traces showing tetanic depression in control solution (top traces) and in the presence of CGP 55845A (5 µM). Trains of IPSCs were evoked at 80 Hz for 1 s in each solution. A single pretetanic IPSC is shown to the left, which was unaffected by CGP 55845A. After start of tetanization (100-200 ms), CGP 55845A discretely reduced tetanic depression, while there was no obvious difference late in the train. B: single traces from another cell showing trains of 80 Hz for 1 s in control solution and in CGP 55845A. Traces are scaled to the same amplitude of the 1st IPSC in the train, and superimposed. CGP 55845A partially reversed tetanic depression early in the train, while the traces converged later in the train. C: early (150-300 ms) and late (850-1,000 ms) areas were measured with respect to the baseline and normalized to the area of a preceding single IPSC. In CGP 55845A the early area was 2.28 ± 0.3, which is 15% larger than in control solution (1.96 ± 0.2, * P < 0.05, n = 8). No differences were observed between the late areas.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Presynaptic GABAB receptors in cultured neurons

Activity-dependent depression of IPSPs is a prominent feature of GABAergic synaptic transmission in the mammalian CNS (Thompson et al. 1993). Experiments with hippocampal and cortical brain slices have shown that PPD of IPSPs is partly caused by activation of presynaptic GABAB autoreceptors, leading to a reduction in GABA release (Davies et al. 1990; Deisz and Prince 1989; Mott et al. 1993; Nathan and Lambert 1991). In contrast to structurally intact preparations, investigations of PPD in cultured hippocampal neurons have shown that PPD of IPSCs is independent of presynaptic GABAB receptor activation (Wilcox and Dichter 1994), despite the fact that functional GABAB receptors are present on the presynaptic terminals and can be selectively activated by baclofen (Harrison 1990). However, we show here that the potent GABAB antagonist, CGP 55845A, reduces PPD by ~20%, illustrating that presynaptic GABAB receptors on cultured neurons are activated to a small extent by synaptically released GABA. A submaximal concentration of baclofen depressed IPSCs by >40%, suggesting a larger depressant potential for the presynaptic GABAB receptors. That synaptically released GABA could not activate the GABAB receptors fully would indicate that these are located at some distance from the GABA release site. Application of CGP 55845A also caused a small enhancement of single IPSCs. If this reflects tonic activation of the high affinity presynaptic GABAB receptors, the concentration of ambient extracellular GABA would probably be in the submicromolar range (Dittman and Regehr 1997).

Postsynaptic factors in PPD

Although PPD is probably expressed at a presynaptic locus (Wilcox and Dichter 1994), postsynaptic factors such as a shift in ECl (Thompson and Gähwiler 1989) or desensitization of GABAA receptors (Frosch et al. 1992; Oh and Dichter 1992) could contribute to PPD. We can, however, rule out that a shift in ECl contributes to the depression of GABAAergic IPSCs because the reversal potentials for IPSC1 and IPSC2 were similar. To investigate whether desensitization is involved in the depression of IPSCs, we made paired applications of exogenous GABA to voltage-clamped neurons in the whole cell mode. The concentration of GABA in the synaptic cleft has been estimated to be ~500 µM, which saturates the postsynaptic receptors (Maconochie et al. 1994). The brief application of GABA (1 mM) used in our experiments is therefore likely to mimic simultaneous release at all GABAergic synapses on the entire neuron. Our results show that the postsynaptic responsiveness was unchanged following the first application of GABA at all IPIs, thereby illustrating that the receptors were not desensitized after the first pulse. This would indicate that desensitization does not contribute to PPD of the IPSCs.

Presynaptic mechanism for PPD

Because activation of presynaptic GABAB autoreceptors plays only a minor role in PPD, the most likely mechanism is depletion of releasable vesicles at the active zones. Changing [Ca2+]o or applying 4-AP are established ways of modulating probability of release at individual release sites by altering presynaptic Ca2+ influx (Klapstein and Colmers 1992; Ohno-Shosaku et al. 1994b). The results show that PPD was positively related to the probability of release (Fig. 2C). Because this relationship was investigated at an IPI of 150 ms, PPD asymptoted toward 70%, which is the maximal obtainable PPD (see Fig. 2D). In the presence of 100 µM 4-AP, PPD at the shortest IPIs was close to 100%, and the probability of release was maximized. Although the readily releasable pool was maximally depleted by the first stimulation, the rate of recovery from PPD was nevertheless the same as the control.

Our results indicate that GABAergic boutons are refractory following exocytosis, as has previously been suggested for excitatory synapses in culture (Debanne et al. 1996). Depleted active zones are replenished at a slow rate as found at glutamatergic synapses, where refilling takes several seconds (Dobrunz and Stevens 1997; Stevens and Tsujimoto 1995). Disregarding the marginal contribution from GABAB receptors, the plot of PPD as a function of IPI (Fig. 2D) would reflect replenishment of depleted vesicles at boutons that had released on pulse1. Furthermore, there is increasing evidence against the involvement of other possible sites in PPD such as axonal branch block (MacKenzie et al. 1996) or inactivation of presynaptic voltage-dependent Ca2+ channels (VDCCs) (Dobrunz and Stevens 1997; Mintz et al. 1995). The latter could, however, play a role at IPIs of <20 ms (Dobrunz et al. 1997).

Recovery of synaptic depression

In three recent reports, it has been shown that recovery from PPD at excitatory glutamatergic synapses is accelerated by residual Ca2+ in the boutons (Dittman and Regehr 1998; Stevens and Wesseling 1998; Wang and Kaczmarek 1998). In the rat brain stem, refilling of the readily releasable pool could be slowed by reducing the spike frequency (Wang and Kaczmarek 1998). In the cerebellum, the membrane-permeable Ca2+ chelator, EGTA-AM, enhanced PPD and delayed recovery (Dittman and Regehr 1998). Accordingly, biochemical data show that synaptic vesicle dynamics are regulated by internal free Ca2+ in the range of 1 µM (reviewed by Burgoyne and Morgan 1995). Our results would indicate that a similar mechanism operates at inhibitory GABAergic synapses (Fig. 5). At a fixed IPI of 500 ms, perfusion of EGTA-AM enhanced PPD from 27 to 43%. This is interesting because GABAergic interneurons are believed to have a strong internal Ca2+ buffering capacity (Freund and Buzsáki 1996), which would not allow any significant accumulation of free Ca2+ in the boutons following a single action potential. Nevertheless, the results with EGTA-AM suggest that internal Ca2+ is indeed elevated after a single stimulus, and that Ca2+ might stimulate replenishment of the readily releasable pool. However, the precise mechanism responsible for this presynaptic regulation remains to be determined.

Tetanic depression of IPSCs

Given that depletion is mainly responsible for frequency-dependent synaptic depression, IPSCs would decline in response to longer stimulus trains until depletion was in equilibrium with the restorative processes (slow refilling), at which point the IPSCs would stabilize at a plateau level. It is apparent that both the initial decline and the initial plateau level depended on both the stimulating frequency and the probability of release (Fig. 6, B and C). However, although IPSCs were unable to follow the stimulation at the highest frequencies (80 Hz, Fig. 6, A and B), an appreciable GABAA current was maintained by asynchronous GABA release that continued 1-2 s after end of tetanization. This component of release has been analyzed in detail elsewhere (Jensen et al. 1999b) and can be blocked by EGTA-AM. It is therefore likely that asynchronous release is caused by an increase in [Ca2+]i in the boutons during the train stimulation (Jensen et al. 1999b). Bearing this in mind, it was interesting to find that CGP 55845A reduced tetanic depression early in the train, but that this effect declined with continued stimulation (Fig. 7B). GABAB-mediated inhibition of N- and P/Q-type VDCCs at the secretory apparatus (Ohno-Shosaku et al. 1994b) was overcome with continued stimulation, probably because transmitter release is maintained during the train by Ca2+ entering via L-type VDCCs (Jensen et al. 1999a). Because GABAB receptors do not desensitize (Misgeld et al. 1995), we have identified an alternative mechanism whereby presynaptic GABAB receptor function can be attenuated. This observation is important in the context of understanding the function of GABAB autoreceptors during high-frequency activity.

Conclusion

We have shown that GABAergic IPSCs in cultured hippocampal neurons show robust PPD that is mediated by a presynaptic mechanism. We suggest that PPD is caused by depletion of vesicles at the active zones, although spill-over of GABA onto presynaptic GABAB receptors is also present to a minor degree. Depleted vesicles are replenished at a slow rate that, however, depended on [Ca2+]i in the boutons. One could speculate that the loss of such Ca2+-mediated acceleration of vesicular recycling at GABAergic synapses in vivo could lead to an increased susceptibility for epileptic discharges. Finally, the effect of activation of GABAB autoreceptors is overcome with prolonged presynaptic activity. This observation is important in the context of understanding the actions of GABAB modulators as antiepileptic agents, and contributes to the understanding of how the action of high extracellular levels of GABA at presynaptic GABAB receptors is counteracted by accumulation of Ca2+ in the boutons during high-frequency activity.


    ACKNOWLEDGMENTS

We thank Ciba-Geigy for a gift of CGP 35348. Novartis supplied CGP 55845A, subject to the terms of their Statement of Investigator. We thank K. Kandborg for preparation of the cultures and S. Kristensen for technical assistance.

We are grateful to the Danish Medical Research Council and Aarhus Universitets Forsknings Fond for financial support.


    FOOTNOTES

Address for reprint requests: J.D.C. Lambert, Dept. of Physiology, University of Aarhus, Ole Worms Alle 160, DK-8000 Aarhus C, Denmark.

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 6 January 1999; accepted in final form 15 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society