Role of residual calcium in synaptic depression and posttetanic potentiation: fast and slow calcium signaling in nerve terminals.

Trains of action potentials evoked rises in presynaptic Ca2+ concentration ([Ca2+]i) at the squid giant synapse. These increases in [Ca2+]i were spatially nonuniform during the trains, but rapidly equilibrated after the trains and slowly declined over hundreds of seconds. The trains also elicited synaptic depression and augmentation, both of which developed during stimulation and declined within a few seconds afterward. Microinjection of the Ca2+ buffer EGTA into presynaptic terminals had no effect on transmitter release or synaptic depression. However, EGTA injection effectively blocked both the persistent Ca2+ signals and augmentation. These results suggest that transmitter release is triggered by a large, brief, and sharply localized rise in [Ca2+]i, while augmentation is produced by a smaller, slower, and more diffuse rise in [Ca2+]i.     

Evidence for a presynaptic action of chlordimeform at the insect neuromuscular junction

The mechanism of action of chlordimeform on the mealworm nerve-muscle preparation was studied with microelectrodes. Chlordimeform affected neither the mean amplitude nor the frequency of spontaneous miniature excitatory postsynaptic potentials. Extracellular focal recordings show that in the presence of 0.8 mM chlordimeform the presynaptic spike is almost unchanged, but the quantal content for evoked transmitter release is reduced. It is suggested that chlordimeform decreases the influx of calcium at the presynaptic terminal during the active phase of the nerve terminal action potential, thereby inhibiting evoked transmitter release.     

Presynaptic strontium dynamics and synaptic transmission

Strontium can replace calcium in triggering neurotransmitter release, although peak release is reduced and the duration of release is prolonged. Strontium has therefore become useful in probing release, but its mechanism of action is not well understood. Here we study the action of strontium at the granule cell to Purkinje cell synapse in mouse cerebellar slices. Presynaptic residual strontium levels were monitored with fluorescent indicators, which all responded to strontium (fura-2, calcium orange, fura-2FF, magnesium green, and mag-fura-5). When calcium was replaced by equimolar concentrations of strontium in the external bath, strontium and calcium both entered presynaptic terminals. Contaminating calcium was eliminated by including EGTA in the extracellular bath, or by loading parallel fibers with EGTA, enabling the actions of strontium to be studied in isolation. After a single stimulus, strontium reached higher peak free levels than did calcium (approximately 1.7 times greater), and decayed more slowly (half-decay time 189 ms for strontium and 32 ms for calcium). These differences in calcium and strontium dynamics are likely a consequence of greater strontium permeability through calcium channels, lower affinity of the endogenous buffer for strontium, and less efficient extrusion of strontium. Measurements of presynaptic divalent levels help to explain properties of release evoked by strontium. Parallel fiber synaptic currents triggered by strontium are smaller in amplitude and longer in duration than those triggered by calcium. In both calcium and strontium, release consists of two components, one more steeply dependent on divalent levels than the other. Strontium drives both components less effectively than does calcium, suggesting that the affinities of the sensors involved in both phases of release are lower for strontium than for calcium. Thus, the larger and slower strontium transients account for the prominent slow component of release triggered by strontium.     

Diffusion barriers limit the effect of mobile calcium buffers on exocytosis of large dense cored vesicles

Fast exocytosis in melanotropic cells, activated by calcium entry through voltage-gated calcium channels, is very sensitive to mobile calcium buffers (complete block at 800 microM ethylene glycol bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA)). This indicates that calcium diffuses a substantial distance from the channel to the vesicle. Surprisingly, 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), having a similar KD for calcium as EGTA but a approximately 100 times faster binding rate, blocked exocytosis only twice as effectively as EGTA. Using computer simulations, we demonstrate that this result cannot be explained by free diffusion and buffer binding rates. We hypothesized that local saturation of calcium buffers is involved. A diffusion barrier for both calcium and buffer molecules, located 50-300 nm from the membrane and reducing diffusion 1000 to 10,000 times, generated similar calcium concentrations for specific concentrations of EGTA and BAPTA. With such barriers, calcium rise phase kinetics upon short step depolarizations (2-20 ms) were faster for EGTA than for BAPTA, implying that short depolarizations should allow exocytosis with 50 microM EGTA but not with 25 microM BAPTA. This prediction was confirmed experimentally with capacitance measurements. Coupling exocytosis to calcium dynamics in the model, we found that a barrier with a approximately 3000 times reduced diffusion at approximately 130 nm beneath the membrane best explains the experimentally observed effects of EGTA and BAPTA on block and kinetics of release.     

Preparation of solutions with free calcium concentration in the nanomolar range using 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid

There are many uses for solutions with a known free calcium concentration ([Ca2+]free) in the nanomolar range. Most frequently ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) has been used as a buffer for the control of [Ca2+]free; however, under a variety of conditions the use of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) for this purpose would be advantageous. The theory and calculations necessary to make solutions with known [Ca2+]free appropriate for given conditions of pH, ionic strength, and temperature for use with EGTA or BAPTA are reviewed. Practical considerations and methods for making such solutions are detailed. The advantages and disadvantages associated with the use of each of the two chelators are discussed. As one example of the application of solutions with free calcium in the nanomolar range, the dissociation constant of the fluorescent indicator fura-2 for calcium has been determined in a physiologic buffer at 22 and 37 degrees C. For practical reasons, the use of BAPTA is advantageous when solutions with different known [Ca2+]free must be used on a daily basis.     

Retrograde inhibition of presynaptic calcium influx by endogenous cannabinoids at excitatory synapses onto Purkinje cells

Brief depolarization of cerebellar Purkinje cells was found to inhibit parallel fiber and climbing fiber EPSCs for tens of seconds. This depolarization-induced suppression of excitation (DSE) is accompanied by altered paired-pulse plasticity, suggesting a presynaptic locus. Fluorometric imaging revealed that postsynaptic depolarization also reduces presynaptic calcium influx. The inhibition of both presynaptic calcium influx and EPSCs is eliminated by buffering postsynaptic calcium with BAPTA. The cannabinoid CB1 receptor antagonist AM251 prevents DSE, and the agonist WIN 55,212-2 occludes DSE. These findings suggest that Purkinje cells release endogenous cannabinoids in response to elevated calcium, thereby inhibiting presynaptic calcium entry and suppressing transmitter release. DSE may provide a way for cells to use their firing rate to dynamically regulate synaptic inputs. Together with previous studies, these findings suggest a widespread role for endogenous cannabinoids in retrograde synaptic inhibition.     

The maintenance of LTP at hippocampal mossy fiber synapses is independent of sustained presynaptic calcium.

We have examined the role of presynaptic residual calcium in maintaining long-term changes in synaptic efficacy observed at mossy fiber synapses between hippocampal dentate granule cells and CA3 pyramidal cells. Calcium concentrations in individual mossy fiber terminals in hippocampal slice were optically measured with the calcium indicator fura-2 while stimulating the mossy fiber pathway and recording excitatory postsynaptic potentials extracellularly. Short-term synaptic enhancement was accompanied by increased presynaptic residual calcium concentration. A 2-fold enhancement of transmitter release was accompanied by a 10-30 nM increase in residual calcium. Following induction of mossy fiber LTP, transiently elevated presynaptic calcium decayed to prestimulus levels, whereas enhancement of synaptic transmission persisted. Our results demonstrate that, despite an apparent strong sensitivity of synaptic enhancement to presynaptic residual calcium levels, sustained increases in presynaptic residual calcium levels are not responsible for the maintained synaptic enhancement observed during mossy fiber LTP.     

Presynaptic calcium concentration microdomains and transmitter release.

n-Aequorin J, a luminescent protein which responds to calcium concentration changes in the order of several hundred micromoles, was injected into the preterminal fiber in the squid giant synapse. The activation of the presynaptic terminal leading to release of transmitter was accompanied by light emission at well-defined sites at the active zone in the presynaptic terminal. Location of these light emission sites was very much the same from one stimulus to the next, indicating that light emission was triggered by the inward calcium current occurring at specific and invariant locations. The distribution, size and number of these QEDs (quantum emission domains) coincides well with the location and number of active zones in the presynaptic terminal. The results imply that transmitter release is triggered by very well-localized calcium concentration changes that may be as high as several hundred micromoles.     

Can presynaptic depolarization release transmitter without calcium influx?

Recent experimental evidence suggesting that presynaptic depolarization can evoke transmitter release without calcium influx has been re-examined. The presynaptic terminal of the squid giant synapse can be depolarized by variable amounts while recording presynaptic calcium current under voltage clamp and postsynaptic responses. Small depolarizations open few calcium channels with large single channel currents. Large depolarizations approaching the calcium equilibrium potential open many channels with small single channel currents. When responses to small and large depolarizations eliciting similar total macroscopic calcium currents are compared, the large pulses evoke more transmitter release. This apparent voltage-dependence of transmitter release may be explained by the greater overlap of calcium concentration domains surrounding single open calcium channels when many closely apposed channels open at large depolarizations. This channel domain overlap leads to higher calcium concentrations at transmitter release sites and more release for large depolarizations than for small depolarizations which open few widely dispersed channels. At neuromuscular junctions, a subthreshold depolarizing pulse to motor nerve terminals may release over a thousand times as much transmitter if it follows a brief train of presynaptic action potentials than if it occurs in isolation. This huge synaptic facilitation has been taken as indicative of a direct effect of voltage which is manifest only when prior activity raises presynaptic resting calcium levels. This large facilitation is actually due to a post-tetanic supernormal excitability in motor nerve terminals, causing the previously subthreshold test pulse to become suprathreshold and elicit a presynaptic action potential. When motor nerve terminals are depolarized by two pulses, as the first pulse increases above a certain level it evokes more transmitter release but less facilitation of the response to the second pulse.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optical measurement of presynaptic calcium currents.

Measurements of presynaptic calcium currents are vital to understanding the control of transmitter release. However, most presynaptic boutons in the vertebrate central nervous system are too small to allow electrical recordings of presynaptic calcium currents (I(Ca)pre). We therefore tested the possibility of measuring I(Ca)pre optically in boutons loaded with calcium-sensitive fluorophores. From a theoretical treatment of a system containing an endogenous buffer and an indicator, we determined the conditions necessary for the derivative of the stimulus-evoked change in indicator fluorescence to report I(Ca)pre accurately. Matching the calcium dissociation rates of the endogenous buffer and indicator allows the most precise optical measurements of I(Ca)pre. We tested our ability to measure I(Ca)pre in granule cells in rat cerebellar slices. The derivatives of stimulus-evoked fluorescence transients from slices loaded with the low-affinity calcium indicators magnesium green and mag-fura-5 had the same time courses and were unaffected by changes in calcium influx or indicator concentration. Thus both of these indicators were well suited to measuring I(Ca)pre. In contrast, the high-affinity indicator fura-2 distorted I(Ca)pre. The optically determined I(Ca)pre was well approximated by a Gaussian with a half-width of 650 micros at 24 degrees C and 340 micros at 34 degrees C.     

New fluorescent calcium indicators designed for cytosolic retention or measuring calcium near membranes.

A new family of fluorescent calcium indicators has been developed based on a new analog of BAPTA called FF6. This new BAPTA analog serves as a versatile synthetic intermediate for developing Ca2+ indicators targeted to specific intracellular environments. Two of these new Ca2+ indicators, fura-PE3 and fura-FFP18, are described in this report. Fura-PE3 is a zwitterionic indicator that resists the rapid leakage and compartmentalization seen with fura-2 and other polycarboxylate calcium indicators. In contrast to results obtained with fura-2, cells loaded with PE3 remain brightly loaded and responsive to changes in concentration of cytosolic free calcium for hours. Fura-FFP18 is an amphipathic indicator that to binds to liposomes and to cell membranes. Studies to be detailed later indicate that FFP18 functions as a near-membrane Ca2+ indicator and that calcium levels near the plasma membrane rise faster and higher than in the cytosol.     

Rhythmic Activity of Murine Neuromuscular Junctions upon Activation of Release of Stored Calcium in the Presence of a Calcium Buffer

Using a two-electrode voltage-clamp technique, we recorded end-plate currents (EPCs) in neuromuscular synaptic junctions of the murine diaphragm upon rhythmic stimulation of the n. phrenicus with frequencies of 7, 20, 50, 70, and 100 sec⁻¹. Parameters of EPC series were analyzed against the background of the action of a mobilizer of intracellular calcium, ryanodine (0.5 μM), after the loading of terminals by 1.2 mM BAPTA (calcium buffer with rapid dynamics of binding of calcium), and upon the action of ryanodine in the presence of BAPTA. Under the action of ryanodine, the amplitude and quantum content of EPC within the plateau phase increased by 100 to 150% (P < 0.05). Loading with BAPTA evoked sharp decreases in the quantum content of unitary EPCs, the intensity of the initial facilitation, and the level of the EPC plateau in series within the entire range of stimulation frequencies used. Against the background of the action of BAPTA, the facilitatory effect of ryanodine increased; inhibitory effects of BAPTA with respect to the amplitude of unitary EPC and the level of the initial facilitation were completely compensated, whereas the level of EPC at the plateau stage increased to levels exceeding the control values by 50 to 70%. The ability of ryanodine to facilitate the transmitter (acetylcholine) release, which was enhanced in the presence of BAPTA, was completely neutralized by a blocker of L-type calcium channels, verapamil (5 μM). In the absence of BAPTA, verapamil did not influence the effects of ryanodine. We hypothesize that in the presence of BAPTA calcium channels of L type whose activity is resistive to the buffer action of BAPTA are disinhibited. The calcium current through L-type channels, perhaps, is capable of stimulating calcium release from the stores of nerve terminals and, as a consequence, of intensifying the facilitatory effect of ryanodine on the release of acetylcholine. After verapamil-induced blockade of this current, BAPTA demonstrates the ability to prevent the facilitatory effect of ryanodine on the transmitter release. Neirofiziologiya/Neurophysiology, Vol. 37, No. 4, pp. 330–338, July–August, 2005.     

Single synaptic events evoke NMDA receptor-mediated release of calcium from internal stores in hippocampal dendritic spines

We have used confocal microscopy to monitor synaptically evoked Ca2+ transients in the dendritic spines of hippocampal pyramidal cells. Individual spines respond to single afferent stimuli (<0.1 Hz) with Ca2+ transients or failures, reflecting the probability of transmitter release at the activated synapse. Both AMPA and NMDA glutamate receptor antagonists block the synaptically evoked Ca2+ transients; the block by AMPA antagonists is relieved by low Mg2+. The Ca2+ transients are mainly due to the release of calcium from internal stores, since they are abolished by antagonists of calcium-induced calcium release (CICR); CICR antagonists, however, do not depress spine Ca2+ transients generated by backpropagating action potentials. These results have implications for synaptic plasticity, since they show that synaptic stimulation can activate NMDA receptors, evoking substantial Ca2+ release from the internal stores in spines without inducing long-term potentiation (LTP) or depression (LTD).     

Hyperosmotic media inhibit voltage-dependent calcium influx and peptide release in Aplysia neurons

Summary The bag cell neurons of Aplysia provide a model system in which to investigate the effects of hyperosmolality on the electrical and secretory properties of neurons. Brief stimulation of these neurons triggers an afterdischarge of action potentials that lasts approximately 20–30 min, during which time they release several neuroactive peptides. We have found that pre-incubation of intact clusters of bag cell neurons in hyperosmotic media prior to stimulation prevents the initiation of afterdischarges. Furthermore, an increase in osmolality of the external medium during an ongoing afterdischarge causes its premature termination. Hyperosmotic media attenuate the release of peptide evoked by both electrically stimulated afterdischarges and potassium-induced depolarization. The ability of high potassium to depolarize the bag cell neurons is, however, not impaired. Exposure of isolated bag cell neurons to hyperosmotic media also inhibits the amplitude of action potentials evoked by depolarizing current injection and attenuates the voltage-dependent calcium current. In isolated bag cell neurons loaded with the calcium indicator dye, fura-2, hyperosmotic media reduced the rise in intracellular calcium levels that normally occurs in response to depolarization. Our results suggest that the effects of hyperosmotic media on peptide secretion in bag cell neurons can largely be attributed to their effects on calcium entry.This work was supported by NIH Grant NS-18492 to L.K. Kaczmarek.     

Single calcium channels on a cholinergic presynaptic nerve terminal.

The calyx-type synapse of the chick ciliary ganglion was used to examine single calcium channels in a vertebrate cholinergic presynaptic nerve terminal by means of the cell-attached, patch-clamp technique. Calcium channels were recorded on the internal, transmitter-release face of the nerve terminal, but were not detected on the external face. These channels were recruited at -30 mV, with maximum activation at about +30 mV, and were sometimes clustered at high densities. Single-channel conductance estimates with voltage-pulse or -ramp techniques gave values of 11-14 pS with 110 mM barium, which is in the intermediate, N-type range for calcium channels on a control neuron. This nerve terminal calcium channel, termed the NPT-type, may link action potentials to transmitter release at many vertebrate fast-transmitting synapses.     

Presynaptic facilitation at the crayfish neuromuscular junction. Role of calcium-activated potassium conductance

Membrane potential was recorded intracellularly near presynaptic terminals of the excitor axon of the crayfish opener neuromuscular junction (NMJ), while transmitter release was recorded postsynaptically. This study focused on the effects of a presynaptic calcium-activated potassium conductance, gK(Ca), on the transmitter release evoked by single and paired depolarizing current pulses. Blocking gK(Ca) by adding tetraethylammonium ion (TEA; 5-20 mM) to a solution containing tetrodotoxin and aminopyridines caused the relation between presynaptic potential and transmitter release to steepen and shift to less depolarized potentials. When two depolarizing current pulses were applied at 20-ms intervals with gK(Ca) not blocked, the presynaptic voltage change to the second (test) pulse was inversely related to the amplitude of the first (conditioning) pulse. This effect of the conditioning prepulse on the response to the test pulse was eliminated by 20 mM TEA and by solutions containing 0 mM Ca2+/1 mM EGTA, suggesting that the reduction in the amplitude of the test pulse was due to activation of gK(Ca) by calcium remaining from the conditioning pulse. In the absence of TEA, facilitation of transmitter release evoked by a test pulse increased as the conditioning pulse grew from -40 to -20 mV, but then decreased with further increase in the conditioning depolarization. A similar nonmonotonic relationship between facilitation and the amplitude of the conditioning depolarization was reported in previous studies using extracellular recording, and interpreted as supporting an additional voltage-dependent step in the activation of transmitter release. We suggest that this result was due instead to activation of a gK(Ca) by the conditioning depolarization, since facilitation of transmitter release increased monotonically with the amplitude of the conditioning depolarization, and the early time course of the decay of facilitation was prolonged when gK(Ca) was blocked. The different time courses for decay of the presynaptic potential (20 ms) and facilitation (greater than 50 ms) suggest either that residual free calcium does not account for facilitation at the crayfish NMJ or that the transmitter release mechanism has a markedly higher affinity or stoichiometry for internal free calcium than does gK(Ca). Finally, our data suggest that the calcium channels responsible for transmitter release at the crayfish NMJ are not of the L, N, or T type.     

Presynaptic actions of botulinal neurotoxins at vertebrate neuromuscular junctions

1. In the present paper we review some presynaptic aspects of the mode of action of botulinal toxins (BoTxs) at vertebrate neuromuscular junctions with emphasis on studies carried out in our laboratories using electrophysiological and morphological techniques. 2. Spontaneous quantal transmitter release recorded as miniature end-plate potentials is drastically affected by BoTxs. The low probability of release at poisoned terminals can be enhanced by carbonyl cyanide m-chlorophenylhydrazone (CCCP), Cd2+ and La3+. However, CCCP and La3+ which drastically deplete clear synaptic vesicles from unpoisoned terminals failed to markedly affect the density of synaptic vesicles at poisoned terminals. It is concluded that poisoned terminals have a reduced sensitivity to the release-promoting action of Ca2+, Cd2+ and La3+. 3. When comparing the effect of the various BoTxs on nerve-impulse evoked transmitter release it appears that increasing phasic Ca2+ entry into the terminals enhances evoked synchronized quantal release only from terminals poisoned with serotypes A and E. In contrast, enhanced Ca2+ entry into terminals poisoned with serotypes B, D and F induced a period of high frequency asynchronous release suggesting that these BoTxs may affect a presynaptic step beyond the influx of Ca2+, that may be involved in the synchronization of transmitter quanta. These data suggest that the actions of BoTxs involve several steps of the acetylcholine release process. 4. The analysis of presynaptic currents which depend on both Ca2+ entry and intraterminal background Ca2+ levels strongly suggests that neither Ca2+ entry nor intraterminal Ca2+ levels are altered by BoTxs. Furthermore, poisoned terminals are no more efficient than unpoisoned ones in dealing with Ca2+ overloads. 5. Finally, the morphological examination of junctions paralysed by BoTx-A indicates that the toxin triggers a particularly important overgrowth of the nerve terminals and suggests that the in vivo functional recovery may occur from an extension of the original nerve terminal arborization and the concomitant remodelling of postsynaptic structures.     

Calcium-activated potassium conductance in presynaptic terminals at the crayfish neuromuscular junction

Membrane potential changes that typically evoke transmitter release were studied by recording intracellularly from the excitor axon near presynaptic terminals of the crayfish opener neuromuscular junction. Depolarization of the presynaptic terminal with intracellular current pulses activated a conductance that caused a decrease in depolarization during the constant current pulse. This conductance was identified as a calcium-activated potassium conductance, gK(Ca), by its disappearance in a zero-calcium/EGTA medium and its block by cadmium, barium, tetraethylammonium ions, and charybdotoxin. In addition to gK(Ca), a delayed rectifier potassium conductance (gK) is present in or near the presynaptic terminal. Both these potassium conductances are involved in the repolarization of the membrane during a presynaptic action potential.     

Okadaic acid suppresses calcium regulation of mitosis onset in sea urchin embryos.

We show that a phosphatase inhibitor, okadaic acid, induces premature and persistent mitosis during the first cell cycle in sea urchin embryos. Okadaic acid-induced mitosis requires protein synthesis, suggesting that it activates the protein synthesis-requiring mitotic H1 kinase. By microinjecting the calcium chelators BAPTA and EGTA and by measuring Cai using fura-2, an indicator dye, we show that okadaic acid-induced mitosis is independent of the calcium signal that usually triggers mitosis onset in sea urchin embryos. Disabling the calmodulin kinase II that is thought to respond to the mitotic Cai signal using a peptide inhibitor fails to prevent mitosis in response to okadaic acid. These data suggest that okadaic acid bypasses calcium regulation of mitosis by inducing constitutive phosphorylation of a site on the H1 kinase that is normally under the control of the calmodulin-regulated kinase.     

Mechanism of long-term potentiation of transmitter release induced by adrenaline in bullfrog sympathetic ganglia

A mechanism of the long-term potentiation of transmitter release induced by adrenaline (ALTP) was studied by recording intracellularly the fast excitatory postsynaptic potentials (fast EPSPs). The ALTP was produced during the blockade of K+ channels at the presynaptic terminals by tetraethylammonium (TEA). The synaptic delay, possibly reflecting a relative change in the duration of an action potential at the presynaptic terminal, was not changed during the course of the ALTP. By contrast, it was significantly lengthened by TEA and other K+ channel inhibitors (4-aminopyridine and Cs+) that markedly enhanced the evoked release of transmitter. The magnitude of facilitation of the fast EPSP, induced by a conditional stimulus to the preganglionic nerve, was decreased during the generation of the ALTP, but was unchanged during the potentiation of transmitter release caused by TEA. These results, together with theoretical considerations applying the residual Ca2+ hypothesis to the facilitation, suggest that the enhancement of transmitter release during the ALTP is not caused by an increased Ca2+ influx during a presynaptic impulse owing to the blockade of K+ channel or the modulation of Ca2+ channel, but presumably is induced by a rise in the basal level of free Ca2+ in the presynaptic terminal.