Presynaptic Calcium Concentration Research Articles

Calcium channels control tDCS-induced spontaneous vesicle release from axon terminals

BackgroundTranscranial direct current stimulation (tDCS) is a subthreshold neurostimulation therapeutic method that ameliorate neuropsychiatric impairments. The most sensitive subcellular compartment for tDCS are the axons that polarize. However, how these relatively small polarizations significantly alter synaptic dynamics is still unknown.Objective/Hypothesis: We hypothesized that tDCS-induced axonal polarization modulates calcium channel activity at the presynaptic compartment, thus playing a crucial role in synaptic vesicle release. MethodsFor this aim, we examined how different DCS conditions and orientations affect the spontaneous excitatory post synaptic currents (sEPSCs) recorded from hippocampal CA1 pyramidal neurons. Since P/Q-type calcium-channels are the main presynaptic voltage-dependent calcium-channels in the hippocampus, we further examined the DCS effects while applying a P/Q-type calcium channels blocker, ω-agatoxin. Additionally, to explain the DCS-induced calcium channel-regulated vesicle release dynamics, we developed a simplified model to complement our experimental results. ResultsWe demonstrated that anodal-DCS application in a dorso-ventral orientation, similar to that of in-vivo experiments, enhanced the sEPSCs frequency, while cathodal-DCS was ineffective. Moreover, DCS application in parallel to the Schaffer collaterals (medio-lateral orientation), showed both anodal and cathodal significant effects. Furthermore, the ω-agatoxin application occluded the DCS-induced modulation of sEPSC frequencies in any orientation. The model showed the interaction between DCS-induced membrane polarization, calcium channel activation and presynaptic vesicle release. ConclusionUsing experiments and modeling we show that DCS induces a small variation in terminal membrane potential sufficient to activate P/Q type voltage-gated calcium channels, and that this is sufficient to modify presynaptic calcium concentration, subsequently altering spontaneous vesicle release.

Imaging and Analysis of Presynaptic Calcium Influx in Cultured Neurons Using synGCaMP6f.

Presynaptic Ca2+ influx through voltage-gated calcium channels (VGCCs) is a key step in synaptic transmission that links action potential (AP)-derived depolarization to vesicle release. However, investigation of presynaptic Ca2+ influx by patch clamp recordings is difficult due to the small size of the majority of synaptic boutons along thin axons that hamper clamp control. Genetically encoded calcium indicators (GECIs) in combination with live cell imaging provide an alternative method to study Ca2+ transients in individual presynaptic terminals. The indicator GCaMP6f was developed for fast speed and high sensitivity in detecting Ca2+ transients even in subcellular compartments. We fused GCaMP6f to synaptophysin (synGCaMP6f) to enrich the calcium indicator in presynaptic boutons of transfected primary hippocampal neurons to study presynaptic Ca2+ changes in response to individual APs or short bursts. Changes in fluorescence intensity were evaluated by normalization to control level or, alternatively, by normalization to maximal fluorescence using the calcium ionophore ionomycin. Measurements revealed robust Ca2+ transients with amplitudes that depend on parameters like the number of APs, stimulation frequency or external calcium concentration. Our findings indicate an appropriate sensitivity of synGCaMP6f for studying total presynaptic Ca2+ transients induced by single APs or short bursts that showed little rundown of the response after repeated bursts. Moreover, these recordings are fast enough to even study short-term plasticity like paired pulse facilitation (PPF) and frequency dependence of Ca2+ transients. In addition, synGCaMP6f could be used to dissect the contribution of different subtypes of VGCCs to presynaptic Ca2+ influx. Our results demonstrate that synGCaMP6f allows the reliable analysis of changes in presynaptic calcium concentration at many individual synaptic boutons in parallel and provides the possibility to study the regulation of this important step in synaptic transmission.

Monitoring single-synapse glutamate release and presynaptic calcium concentration in organised brain tissue

Brain function relies in large part on Ca2+-dependent release of the excitatory neurotransmitter glutamate from neuronal axons. Establishing the causal relationship between presynaptic Ca2+ dynamics and probabilistic glutamate release is therefore a fundamental quest across neurosciences. Its progress, however, has hitherto depended primarily on the exploration of either cultured nerve cells or giant central synapses accessible to direct experimental probing in situ. Here we show that combining patch-clamp with time-resolved imaging of Ca2+ −sensitive fluorescence lifetime of Oregon Green BAPTA-1 (Tornado-FLIM) enables readout of single spike-evoked presynaptic Ca2+ concentration dynamics, with nanomolar sensitivity, in individual neuronal axons in acute brain slices. In parallel, intensity Tornado imaging of a locally expressed extracellular optical glutamate sensor iGluSnFr provides direct monitoring of single-quantum, single-synapse glutamate releases in situ. These two methods pave the way for simultaneous registration of presynaptic Ca2+ dynamics and transmitter release in an intact brain at the level of individual synapses.

Effects of Propofol on Glutamate-Induced Calcium Mobilization in Presynaptic Boutons of Rat Hippocampal Neurons

Recent reports have suggested that various general anesthetics affect presynaptic processes in the central nervous system. However, characterizations of the influence of intravenous anesthetics on neurotransmitter release from presynaptic nerve terminals (boutons) are insufficient. Because the presynaptic calcium concentration ([Ca2+]pre) regulates neurotransmitter release, we investigate the effects of the intravenous anesthetic propofol on neurotransmitter release by measuring [Ca2+]pre in the presynaptic boutons of individual dissociated hippocampal neurons. Brain slices were prepared from Sprague–Dawley rats (10 - 14 days of age). The hippocampal CA1 area was isolated with a fire-polished glass pipette, which vibrated horizontally to dissociate hippocampal CA1 neurons along with their attached presynaptic boutons. Presynaptic boutons were visualized under a confocal laser scanning microscope after staining with FM1-43 dye, and [Ca2+]pre was measured using fluo-3 AM dye. Glutamate (3 – 100 μM) administration increased [Ca2+]pre in Ca2+- containing external solution in a concentration-dependent manner. Propofol (3 – 30 μM) dose-dependently suppressed this glutamate (30 μM)-induced increase in [Ca2+]pre in boutons attached to dendrites, but not to the soma or base of the dendritic tree. The large majority of excitatory synapses on CA1 neurons are located on dendritic spines; therefore, propofol may affect glutamate-induced Ca2+ mobilization in excitatory, but not inhibitory, presynaptic boutons. Propofol may possibly have some effect on glutamate-regulated neurotransmitter release from excitatory presynaptic nerve terminals through inhibiting the increase in [Ca2+]pre induced by glutamate.

Sod1 gene ablation in adult mice leads to physiological changes at the neuromuscular junction similar to changes that occur in old wild-type mice

Reactive oxygen species (ROS) are believed to be important mediators of muscle atrophy and weakness in aging and many degenerative conditions. However, the mechanisms and physiological processes specifically affected by elevated ROS in neuromuscular units that contribute to muscle weakness during aging are not well defined. Here we investigate the effects of chronic oxidative stress on neurotransmission and excitation–contraction (EC) coupling mechanisms in the levator auris longus (LAL) muscle from young (4–8 months) and old (22–28 months) wild-type mice and young adult Cu-Zn superoxide dismutase 1 knockout (Sod1−/−) mice. The frequency of spontaneous neurotransmitter release and the amplitude of evoked neurotransmitter release in young Sod1−/− and old wild-type LAL neuromuscular junctions were significantly reduced from the young wild-type values, and those declines were mirrored by decreases in synaptic vesicle pool size. Presynaptic cytosolic calcium concentration and mitochondrial calcium uptake amplitudes showed substantial increases in stimulated young Sod1−/− and old axon terminals. Surprisingly, LAL muscle fibers from old mice showed a greater excitability than fibers from either young wild-type or young Sod1−/− LAL. Both evoked excitatory junction potential (EJP) and spontaneous mini EJP amplitudes were considerably higher in LAL muscles from old mice than in fibers from young Sod1−/− LAL muscle. Despite a greater excitability, sarcoplasmic calcium influx in both old wild-type and young Sod1−/− LAL muscle fibers was significantly less. Sarcoplasmic reticulum calcium levels were also reduced in both old wild-type and young Sod1−/− mice, but the difference was not statistically significant in muscle fibers from old wild-type mice. The protein ratio of triad calcium channels RyR1/DHPR was not different in all groups. However, fibers from both young Sod1−/− and old mice had substantially elevated levels of protein carbonylation and S-nitrosylation modifications. Overall, our results suggest that young Sod1−/− recapitulate many neuromuscular and muscle fiber changes seen in old mice. We also conclude that muscle weakness in old mice might in part be driven by ROS-mediated EC uncoupling, while both EC uncoupling and reduced neurotransmitter release contribute to muscle weakness in Sod1−/− mice.

Calcium channels for endocytosis.

Calcium channels for endocytosis.

Concurrent imaging of synaptic vesicle recycling and calcium dynamics

Synaptic transmission involves the calcium dependent release of neurotransmitter from synaptic vesicles. Genetically encoded optical probes emitting different wavelengths of fluorescent light in response to neuronal activity offer a powerful approach to understand the spatial and temporal relationship of calcium dynamics to the release of neurotransmitter in defined neuronal populations. To simultaneously image synaptic vesicle recycling and changes in cytosolic calcium, we developed a red-shifted reporter of vesicle recycling based on a vesicular glutamate transporter, VGLUT1-mOrange2 (VGLUT1-mOr2), and a presynaptically localized green calcium indicator, synaptophysin-GCaMP3 (SyGCaMP3) with a large dynamic range. The fluorescence of VGLUT1-mOr2 is quenched by the low pH of synaptic vesicles. Exocytosis upon electrical stimulation exposes the luminal mOr2 to the neutral extracellular pH and relieves fluorescence quenching. Reacidification of the vesicle upon endocytosis again reduces fluorescence intensity. Changes in fluorescence intensity thus monitor synaptic vesicle exo- and endocytosis, as demonstrated previously for the green VGLUT1-pHluorin. To monitor changes in calcium, we fused the synaptic vesicle protein synaptophysin to the recently improved calcium indicator GCaMP3. SyGCaMP3 is targeted to presynaptic varicosities, and exhibits changes in fluorescence in response to electrical stimulation consistent with changes in calcium concentration. Using real time imaging of both reporters expressed in the same synapses, we determine the time course of changes in VGLUT1 recycling in relation to changes in presynaptic calcium concentration. Inhibition of P/Q- and N-type calcium channels reduces calcium levels, as well as the rate of synaptic vesicle exocytosis and the fraction of vesicles released.

Dynasore, an inhibitor of dynamin, increases the probability of transmitter release

Dynasore was recently developed as a small molecule, selective non-competitive inhibitor of the protein dynamin. This inhibitor has been shown to block dynamin-dependent endocytosis and is now used commonly to study vesicular recycling at synapses. We have measured the effects of dynasore on spontaneous and evoked transmitter release at the frog neuromuscular junction and shown that, in addition to inhibiting endocytosis, dynasore increases the probability of transmitter release. Furthermore, we have shown that dynasore exposure leads to an increase in resting intra-terminal calcium, but this effect does not completely account for the dynasore-mediated increase in the probability of transmitter release. Therefore, in interpreting effects of the dynamin inhibitor dynasore at synapses, one must be alert to potential increases in presynaptic calcium concentration and transmitter release probability.

Increased asynchronous release and aberrant calcium channel activation in amyloid precursor protein deficient neuromuscular synapses

Despite the critical roles of the amyloid precursor protein (APP) in Alzheimer’s disease pathogenesis, its physiological function remains poorly established. Our previous studies implicated a structural and functional activity of the APP family of proteins in the developing neuromuscular junction (NMJ). Here we performed comprehensive analyses of neurotransmission in mature neuromuscular synapse of APP deficient mice. We found that APP deletion led to reduced paired-pulse facilitation and increased depression of synaptic transmission with repetitive stimulation. Readily releasable pool size and total releasable vesicles were not affected, but probability of release was significantly increased. Strikingly, the amount of asynchronous release, a measure sensitive to presynaptic calcium concentration, was dramatically increased, and pharmacological studies revealed that it was attributed to aberrant activation of N- and L-type Ca 2+ channels. We propose that APP modulates synaptic transmission at the NMJ by ensuring proper Ca 2+ channel function.

Post‐tetanic potentiation in the rat calyx of Held synapse

We studied synaptic plasticity in the calyx of Held synapse, an axosomatic synapse in the auditory brainstem, by making whole-cell patch clamp recordings of the principal cells innervated by the calyces in a slice preparation of 7- to 10-day-old rats. A 5 min 20 Hz stimulus train increased the amplitude of excitatory postsynaptic currents (EPSCs) on average more than twofold. The amplitude of the synaptic currents took several minutes to return to control values. The post-tetanic potentiation (PTP) was accompanied by a clear increase in the frequency, but not the amplitude, of spontaneous EPSCs, which returned to baseline more rapidly than the potentiation of evoked release. The size of the readily releasable pool of vesicles was increased by about 30%. In experiments in which presynaptic measurements of the intracellular calcium concentration were combined with postsynaptic voltage clamp recordings, PTP was accompanied by an increase in the presynaptic calcium concentration to about 210 nM. The decay of the PTP matched the decay of this increase. When the decay of the calcium transient was shortened by dialysing the terminal with EGTA, the PTP decay sped up in parallel. Our experiments suggest that PTP at the calyx of Held synapse is due to a long-lasting increase in the presynaptic calcium concentration following a tetanus, which results in an increase in the release probability of the vesicles of the readily releasable pool. Although part of the PTP can be explained by a direct activation of the calcium sensor for phasic release, other mechanisms are likely to contribute as well.

Inter-bouton variability of synaptic strength correlates with heterogeneity of presynaptic Ca(2+) signals.

The elevation of presynaptic calcium concentration is a crucial step in excitation-secretion coupling. However, the amplitudes of action-potential-induced presynaptic calcium transients can display high variability among different terminals. The aim of this study was to clarify whether, at individual boutons, synaptic strength correlates with the average amplitude of presynaptic calcium transients. Low-density collicular cultures were loaded with the calcium indicator Oregon Green bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) 1. Action potentials were blocked with tetrodotoxin. Presynaptic terminals were identified with FM4-64, a use-dependent vesicle marker. Presynaptic calcium influx was elicited by a focal electrical stimulation of single boutons. Whole cell patch-clamp and calcium imaging techniques were used to record GABAergic evoked inhibitory postsynaptic currents (eIPSCs) and presynaptic fluorescence changes in the stimulated terminal. To make the eIPSCs from different boutons comparable, they were normalized to the mean value of miniature IPSCs (mIPSCs) of the postsynaptic cell. Records from 47 boutons showed that eIPSCs varied between 0.5 and 3.0 and presynaptic calcium transients varied between 0.1 and 1.3. However, there was a strong correlation between the mean amplitudes of eIPSCs and presynaptic calcium responses. The eIPSC-[Ca(2+)](pre) relationship allows to use the amplitudes of presynaptic calcium transients as an indicator of release efficacy and, in a set of contacts made by one axon, to predict the relative impact of individual terminals.

Temporal Synaptic Tagging by I h Activation and Actin : Involvement in Long-Term Facilitation and cAMP-Induced Synaptic Enhancement

Presynaptic I h channels become activated during a tetanus through membrane hyperpolarization resulting from Na + accumulation and electrogenic Na +/K + exchange. I h activation is obligatory for inducing long-term facilitation (LTF), a long-lasting synaptic strengthening. cAMP-induced synaptic enhancement also requires I h activation, and both processes are sensitive to actin depolymerization. Other mechanisms are responsible for expression of the responses. Once initiated, continued response to cAMP is I h and actin independent. Moreover, LTF-induced activation of I h renders subsequent cAMP enhancement insensitive to both I h blockers and actin depolymerization. This actin-stabilized “temporal synaptic tagging” set by I h activation is prolonged when I h is activated concurrent with an elevation in presynaptic calcium concentration ([Ca 2+] i), permitting the further strengthening of synapses given appropriate additional stimuli.

Giant miniature EPSCs at the hippocampal mossy fiber to CA3 pyramidal cell synapse are monoquantal.

The mechanisms generating giant miniature excitatory postsynaptic currents (mEPSCs) were investigated at the hippocampal mossy fiber (MF) to CA3 pyramidal cell synapse in vitro. These giant mEPSCs have peak amplitudes as large as 1,700 pA (13.6 nS) with a mean maximal mEPSC amplitude of 366 +/- 20 pA (mean +/- SD; 5 nS; n = 25 cells). This is compared with maximal mEPSC amplitudes of <100 pA typically observed at other cortical synapses. We tested the hypothesis that giant mEPSCs are due to synchronized release of multiple vesicles across the release sites of single MF boutons by directly inducing vesicular release using secretagogues. If giant mEPSCs result from simultaneous multivesicular release, then secretagogues should increase the frequency of small mEPSCs selectively. We found that hypertonic sucrose and spermine increased the frequency of both small and giant mEPSCs. The peptide toxin secretagogues alpha-latrotoxin and pardaxin failed to increase the frequency of giant mEPSCs, but the possible lack of tissue penetration of the toxins make these results equivocal. Because a multiquantal release mechanism is likely to be mediated by a spontaneous increase in presynaptic calcium concentration, a monoquantal mechanism is further supported by results that giant mEPSCs were not affected by manipulations of extracellular or intracellular calcium concentrations. In addition, reducing the temperature of the bath to 15 degrees C failed to desynchronize the rising phases of giant mEPSCs. Together these data suggest that the giant mEPSCs are generated via a monovesicular mechanism. Three-dimensional analysis through serial electron microscopy of the MF boutons revealed large clear vesicles (50 to 160 nm diam) docked presynaptically at the MF synapse in sufficient numbers to account for the amplitude and frequency of giant mEPSCs recorded electrophysiologically. It is concluded that release of the contents of a single large clear vesicle generates giant mEPSCs at the MF to CA3 pyramidal cell synapse.

Roles for Mitochondrial and Reverse Mode Na+/Ca2+Exchange and the Plasmalemma Ca2+ATPase in Post-Tetanic Potentiation at Crayfish Neuromuscular Junctions

We have explored the processes regulating presynaptic calcium concentration ([Ca(2+)](i)) in the generation of post-tetanic potentiation (PTP) at crayfish neuromuscular junctions, using spectrophotometric dyes to measure changes in [Ca(2+)](i) and [Na(+)](i) and effects of inhibitors of Ca(2+)-transport processes. The mitochondrial Na(+)/Ca(2+) exchange inhibitor CGP 37157 was without effect, whereas the reverse mode plasmalemmal Na(+)/Ca(2+) exchange inhibitor KB R7943 reduced PTP and Ca(2+) accumulation caused by increased [Na(+)](i). Exchange inhibitory peptide and C28R2 had opposite effects, consistent with their block of the plasma membrane Ca(2+) ATPase. All drugs except CGP 37157 reduced Ca(2+) accumulation caused by Na(+) accumulation, which occurred on block of the Na(+)/K(+) pump, acting in proportion to their effects on plasmalemmal Na(+)/Ca(2+) exchange. We find no role for mitochondrial Na(+)/Ca(2+) exchange in presynaptic Ca(2+) regulation. The plasma membrane Na(+)/Ca(2+) exchanger acts in reverse mode to admit Ca(2+) into nerve terminals during and for some minutes after tetanic stimulation, while at the same time the plasma membrane Ca(2+) ATPase operates as an important Ca(2+) removal process. The interplay of these two Ca(2+) transport processes with Na(+)-independent mitochondrial Ca(2+) fluxes and the plasmalemma Na(+)/K(+) pump determines the magnitude of tetanic [Ca(2+)](i) accumulation and potentiation of excitatory transmission, and the post-tetanic time courses of decay of elevated [Ca(2+)](i) and PTP.

Calcium sensitivity of glutamate release in a calyx-type terminal.

Synaptic efficacy critically depends on the presynaptic intracellular calcium concentration ([Ca2+]i). We measured the calcium sensitivity of glutamate release in a rat auditory brainstem synapse by laser photolysis of caged calcium. A rise in [Ca2+]i to 1 micromolar readily evoked release. An increase to >30 micromolar depleted the releasable vesicle pool in <0.5 millisecond. A comparison with action potential-evoked release suggested that a brief increase of [Ca2+]i to approximately 10 micromolar would be sufficient to reproduce the physiological release pattern. Thus, the calcium sensitivity of release at this synapse is high, and the distinction between phasic and delayed release is less pronounced than previously thought.

Cooperative Ca 2+ Removal from Presynaptic Terminals of the Spiny Lobster Neuromuscular Junction

Stimulation-induced changes in presynaptic free calcium concentration ([Ca 2+] i) were examined by fluorescent imaging at the spiny lobster excitor motor nerve terminals. The Ca 2+ removal process in the terminal was analyzed based on a single compartment model, under the assumption that the Ca 2+ removal rate from the terminal cytoplasm is proportional to nth power of [Ca 2+] i. During 100 nerve stimuli at 10–100 Hz, [Ca 2+] i reached a plateau that increased in a less-than-linear way with stimulation frequency, and the power index, n, was about 2. In the decay time course after stimulation, n changed with the number of stimuli from about 1.4 after 10 stimuli to about 2 after 100 stimuli. With the change of n from 1.4 to 2, the rate became larger at high [Ca 2+] i (>1.5 μM), but was smaller at low [Ca 2+] i (<1 μM). These results suggest that a cooperative Ca 2+ removal mechanism of n = 2, such as mitochondria, may play an important role in the terminal. This view is supported by the gradual increase in the [Ca 2+] i plateau during long-term stimulation at 20–50 Hz for 60 s and by the existence of a very slow [Ca 2+] i recovery process after this stimulation, both of which may be due to accumulation of Ca 2+ in the organelle.

Activity-dependent short-term enhancement of intercellular coupling

It was reported previously that repeated brief tetanization of the posterior eight nerve can produce long-term homosynaptic potentiations of the electrotonic and chemical components of the mixed EPSP evoked in the Mauthner cell lateral dendrite by a single stimulus to the nerve. We show here that the same stimulus paradigm can lead, alternatively, to short-term enhancements of both excitatory responses. These transient modifications last for approximately 3 min, with a time course similar to post-tetanic potentiation at chemical synapses. However, a different stimulus pattern that transiently increases the presynaptic calcium concentration, paired-nerve stimuli, does not have any significant effect on electrotonic transmission, whereas it facilitates the chemically mediated EPSP. On the other hand, induction of the short-lasting potentiation of coupling, which depended on the discontinuous or burst-like property of the tetanizing paradigm, required NMDA-receptor activation and was blocked by postsynaptic intradendritic injections of the calcium chelator bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid. The ineffectiveness of presynaptic calcium in potentiating electrotonic coupling likely reflects the involvement of a calcium-dependent regulatory protein in the postsynaptic cell and suggests that hemichannels on the two sides of a gap junction plaque can be modified independently. NMDA-mediated modulation of gap junctions could be widespread, because both types of channels coexist during development and in several mammalian adult central nervous system structures such as hippocampus.

Neuron-Muscle Contact Changes Presynaptic Resting Calcium Set-Point

The role of retrograde signaling between target cells and their presynaptic partners during early cell-cell interactions was examined in cell culture. Using time-lapse video microscopy and fura-2 calcium analysis, we followed contacts between presynaptic neurites of Helisoma motoneurons and muscle fibers dissociated from identified partners in chemical synaptogenesis. Cytosolic calcium rose dramatically at the region of cell-cell contact and subsequently increased throughout the entire neuron. Changes in the calcium set-point of presynaptic neurons were maintained even when connections with muscle targets were severed. Changes in presynaptic calcium concentration were not detected following contact with partners in electrical synapse formation. These data, taken together with the fact that calcium changes coincide with the time course of chemical synaptogenesis in these neuron-muscle cultures, suggest that calcium changes may be involved in early stages of synapse formation.

Synaptic transmission between pairs of retinal amacrine cells in culture

We have examined synaptic transmission between isolated pairs of chick GABAergic amacrine cells, maintained in sparse culture and identified by their binding of an amacrine cell-selective antibody. Using the perforated-patch method to whole-cell clamp both cells of a pair, postsynaptic currents were examined for step depolarizations of the "presynaptic" cell. Synaptic transmission, frequently reciprocal, was calcium dependent and reversibly blocked by bicuculline. Post-synaptic currents, excluding those due to ohmic electrical coupling, were elicited only for presynaptic voltage steps positive to about -40 mV and were always very noisy, suggesting that they were summed from relatively small numbers of quanta. Postsynaptic currents continued well after the termination of the 100 msec presynaptic voltage step when the step was to -10 mV, or positive to this value. This result is interpreted to imply that presynaptic calcium concentration remains elevated after the membrane is returned to its holding potential. When presynaptic voltages were kept low or else presynaptic voltage was uncontrolled, spontaneous quantal events mediated by GABAA receptors could often be seen. Quanta rose quickly (less than 4 msec) and decayed with a mean time constant of 19.3 msec. The amplitude distributions of quantal currents were positively skewed, sometimes showing rare quanta of exceptionally large amplitude. Peak conductance per quantum was about 300 pS, corresponding to the simultaneous opening of only 17 GABAA channels and corresponding to a net flux of only 32 x 10(3) Cl- ions per millivolt of driving force. Estimates of the maximum sustained release rate at individual release sites suggest an upper bound of between 19 and 42 quanta per second.

Presynaptic calcium transients evoked by paired-pulse stimulation in the hippocampal slice.

The fluorescent dye Calcium Green and optical recording techniques were used to record intracellular Ca2+ transients resulting from paired-pulse stimulation in stratum moleculare of area CA1 in guinea-pig hippocampal slices. Presumed presynaptic calcium transients were recorded while glutamatergic synaptic transmission was blocked by kynurenic acid. Peak responses to paired-pulses (10-160 ms interval) were higher than responses to single pulses of same stimulation strength (42-23% increase). The isolated response to the second pulse, however, was of smaller magnitude in comparison to the first one; the difference in magnitude depended on the interstimulus interval. Thus, the residual presynaptic free calcium concentration may be responsible for paired-pulse facilitation of synaptic transmission in hippocampus. At the same time, a use-dependent inactivation of presynaptic calcium channels may occur.