Neuromuscular synapse: stochastic properties of spontaneous release of transmitter.

The spontaneous quantal release of transmitter from the motor nerve endings is a random process which follows the Poisson theorem; the liberation of each quantum is independent of the release of previous quanta. Increase in the extracellular calcium concentration produces a statistical interdependence in the release of the spontaneously appearing packages.

Neuromuscular transmission: inhibition by manganese ions.

Manzganiese ions. are potent blocking argenits of synaptic transmission at the neuromuscular junction in the frog. The main site of action is the presynaptic nerve terminal, where the ions decr-ease the amount of transmitter liberated by a never impulse. The inihibition produced by manganese is reversible.

Adrenocorticotropic hormone causes long-lasting potentiation of transmitter release from frog motor nerve terminals.

Exposure of frog neuromuscular preparations to adrenocorticotropic hormone for several minutes increased both nerve-evoked and spontaneous transmitter release for several hours. No changes in postsynaptic sensitivity to transmitter were detected. The long-lasting potentiation shows little sensitivity to changes in extracellular calcium concentration and seems to be entirely presynaptic in origin.

GABA- and glutamate-mediated network activity in the hippocampus of neonatal and juvenile rats revealed by fast calcium imaging.

In the rat hippocampus, during the first postnatal week, network activity is characterized by GABA-driven giant depolarizing potentials (GDPs) associated with calcium signals that are readily blocked when the GABAA antagonist bicuculline is applied to the bath. Towards the end of the first postnatal week, in concomitance with the shift of GABA responses from the depolarizing to the hyperpolarizing direction, functional glutamatergic connections start appearing. At this developmental stage, application of bicuculline blocks GABAA-mediated inhibition and induces the appearance of interictal epileptiform discharges. In the present experiments, we have used a high spatio-temporal resolution imaging system to compare, on a time scale of tens of ms, the onset and propagation of fast calcium transients generated within a GABAergic or glutamatergic network. We found that, during the first postnatal week, calcium signals associated to evoked GDPs arise from the activation of a local circuitry of neurons spanning the stratum radiatum and the pyramidal layer. Similar activation patterns were elicited by focal application of GABA in the presence of kynurenic acid, a broad spectrum ionotropic glutamatergic antagonist, and were blocked by bicuculline. During the second postnatal week, in the presence of bicuculline, calcium signals associated with interictal discharges evoked by stimulation of glutamatergic fibres propagated along the well-defined three-synaptic pathway from the dentate gyrus to the CA1 hippocampal area.

Intracellular and extracellular calcium ions in transmitter release at the neuromuscular synapse.

The theme of this presentation has been to show that the control of transmitter release at the neuromuscular synapse is achieved by extracellular and intracellular calcium. For the fast information transfer represented by the end-plate potential, the electrochemical gradient for calcium across the presynaptic membrane and the associated calcium conductance seem to play the primary role. For slower processes such as tetanic and posttetanic potentiation, the combined effect of both sources for calcium determine the amount of transmitter liberated.

Voltage dependent switch in the activity mode of the K+ channel in presynaptic nerve terminals.

The bursting K+ channel is the most common channel in fused Torpedo presynaptic nerve terminals. It possesses the property of 'statistical memory', demonstrated by non-random probability of channel opening. We examined the voltage dependence of the statistical memory and report that removal of channel inactivation by hyperpolarization abolishes it. Addition of the potassium channel blocker 4-aminopyridine to the bath solution led to disappearance of statistical memory, while raising extracellular potassium concentration had the opposite effect. Another common channel at Torpedo nerve terminals which is a non-selective channel did not exhibit statistical memory. We conclude that statistical memory is a channel-specific phenomenon and speculate regarding its possible role in cellular and network properties of the nervous system.

Visualization of synaptic structure and function with confocal microscopy: calcium fluctuations and oscillations.

This article summarizes the basic principles of confocal microscopy and how they can be employed to visualize synaptic structure and function. Optical 'sectioning' of living cells allows the examination of a large number of biological processes at different subcellular localities. Different fluorescent markers enable the study of processes in the extracellular, intracellular and membrane domains of the nerve cell. The excellent spatial resolution of confocal microscopy permits to study the changes in intracellular calcium concentration in single synaptic boutons, without a substantial interference from supporting cells. Intracellular calcium concentration shows coordinated fluctuations in space and periodic oscillations. Periodic oscillations can serve as time keeping devices in nerve terminals. Oscillations were previously observed also in the process of transmitter release. We speculate therefore that these calcium oscillations may be of significance, if the quantal transmitter release is governed by a sequence of calcium dependent steps, which have a different affinity for calcium.

Vasopressin produces long-lasting increase in transmitter release.

Arginine-vasopressin has a widespread distribution in the nervous system, and has been implicated in cellular and behavioral functions. Its effect on the neuromuscular synapse reveals that it produces long-lasting augmentation in synaptic transmission due to an increase in spontaneous and nerve stimulation evoked quantal transmitter release. No significant postsynaptic effect was detected.

Presynaptic action of the neurosteroid pregnenolone sulfate on inhibitory transmitter release in cultured hippocampal neurons.

The effects of the neurosteroid pregnenolone sulfate (PS) were studied in 3- to 9-week-old hippocampal cultures from neonatal rats. Cells were voltage clamped using CsCl filled electrodes, while action potentials and excitatory glutamatergic currents were abolished by superfusing with a combination of tetrodotoxin, 6-cyano-7-nitroquinoxaline (CNQX) and 2-amino-5-phosphonopentanoic acid (AP-5). Under these conditions spontaneous GABAergic inhibitory postsynaptic currents (sIPSCs) were seen as inward currents at a holding potential of -70 mV. Their amplitude distributions were skewed without clearly detectable peaks. PS at 1-50 microM concentrations decreased the frequency of sIPSCs, with 1 microM being the most effective concentration. The effect appeared after 10-15 min of steroid application and the magnitude of the reduction increased during the early wash period. No recovery of sIPSC frequency was found after 30 min of washing with steroid-free medium. sIPSC amplitudes were not significantly changed at the time the effect of PS on sIPSC frequency was observed. The slow onset of this effect and its duration suggest a novel presynaptic action of the neurosteroid PS on GABAergic inhibition in the mammalian brain.

The action of the sodium ionophore, monensin, or transmitter release at the frog neuromuscular junction.

The action of the sodium ionophore, monensin, on spontaneous and evoked transmitter release at the frog neuromuscular junction was studied. Ringer's solutions with low calcium concentrations (0.4 mM or 0 Ca 1 mM EGTA) were used to bathe the preparation. Following addition of monensin in the bathing solution: (1) substantial increases in miniature end-plate potential frequency occurred; (2) tetanic nerve stimulation caused increases in potentiation of approximately 10 times over control values; (3) there was a dual action on the amplitude of the end-plate potential. We conclude that sodium ions take part in the regulation of transmitter release at the neuromuscular junction.

Noradrenaline augments tetanic potentiation of transmitter release by a calcium dependent process.

Noradrenaline (25 microM-50 microM) causes an increase in tetanic potentiation and in the augmentation phase of posttetanic potentiation of miniature and plate potential frequency. These effects were observed at both the frog and the rat neuromuscular junctions. The action of noradrenaline on quantal transmitter release depends on the presence of calcium ions in the extracellular medium.

Apamin-sensitive nitric oxide- and ATP-mediated motor effects on the guinea pig small intestine.

The involvement of nitric oxide and ATP in both spontaneous and electrically-induced nonadrenergic noncholinergic (NANC) motor activity with special interest in the apamin-sensitive mechanisms was studied in a guinea pig ileum model. Depending on the concentration (0.1 or 1 micromol/l), apamin, a blocker of the calcium-activated potassium channels and antagonist of ATP action, induced either TTX (0.1 micromol/l)-resistant increase in tone or contractions. SNP, a nitric oxide donor, applied cumulatively (0.1-100 micromol/l) evoked a concentration-dependent relaxation, the EC50 value being 0.39 +/- 0.12 micromol/l. At concentrations of 0.1 or 1 micromol/l, apamin decreased the SNP effects and shifted the concentration-response curves for SNP to the right. The EC50 value for SNP in the presence of apamin at a concentration of 0.1 micromol/l increased to 59.34 +/- 36.53 micromol/l. ATP (1 or 50 micromol/l) induced TTX-resistant contractions. The effects of ATP were reduced by apamin (1 micromol/l). The contractile effect of ATP occurred in the presence of SNP. SNP provoked relaxation on the background of ATP. The NANC responses to electrical stimulation (0.8 ms, 40 V, 2 or 20 Hz, 20 s) consisted of an initial relaxation phase followed by a phase of contractions, twitch-like and tonic. L-NNA (0.5 mmol/l), an inhibitor of nitric oxide syntheses, abolished the relaxation phase. L-arginine (0.5 mmol/l) restored it. Apamin (0.1 or 1 micromol/l) completely eliminated the relaxation phase and concentration-dependently inhibited the tonic contraction of the phase of contractions. The present findings indicate that the apamin-sensitive nitric oxide-evoked relaxation could be realized by calcium-activated potassium channels and that the apamin-sensitive ATP-induced contraction is mediated via contraction-producing P2 purinoceptors.

Confocal microscopy of the lizard motor nerve terminals.

Confocal imaging was performed on the ceratomandibularis nerve muscle preparation of the lizard Anolis carolinensis, using 4-Di-2-ASP as a fluorescent probe. The imaging system consisted of a Sarastro Phoibos 1000 (Molecular Dynamics) scanning system and a Zeiss Universal microscope. The data were analyzed using the VANIS set of programs on a Silicon Graphics Personal Iris computer. A three dimensional reconstruction of the nerve terminals was performed using look-through and depth-coded projections. The volume of the nerve terminal was estimated in 15 neuromuscular junctions and found to be 84,835 microns 3 (+/- 8558 SEM). The largest diameter in each one of the 178 individual boutons was estimated from the look-through projections of 17 nerve terminals in 9 preparations. It was found to be 4.71 microns (+/- 0.08 SEM). The diameter perpendicular to the largest diameter in the same projection at 0 degrees was 3.3 microns (+/- 0.054 SEM). Thus it seems that the synaptic boutons of the ceratomandibularis are suitable for combined optical and electrophysiological recordings.

The methodology for studying coordinated calcium concentration changes in a pancreatic beta cell line.

Intracellular calcium concentration was imaged in beta Tc cells with the aid of Fluo-3 indicator and the Meridian ACAS 570 interactive laser cytometry. This cell line does not respond by an elevation in [Ca2+]in to increase in extracellular [glucose], but does respond to 10 microM forskolin. It was found that forskolin increases the mean [Ca2+]in and produces calcium spikes. Time series analysis was performed on individual pixels. Autocorrelation revealed that forskolin induces oscillation in [Ca2+]in. Cross-correlation analysis showed that all the intracellular pixels along the line scan are highly correlated, indicating that the increase in [Ca2+]in encompasses the entire cell.

Intracellular calcium dynamics--sparks of insight.

Calcium ions are of key importance in a large number of cellular functions. In the past decade a large variety of cells have been found to show localized increases in the intracellular calcium concentration named calcium sparks. In this brief review, the methodology of detecting calcium sparks by confocal microscopy is summarized. Some of the properties of calcium sparks in muscle (cardiac, skeletal and smooth muscles), neurons, nerve terminals and oocytes aredescribed. Speculations are put forward regarding their possible role in microcontrol of cell function.