A re-excitation mechanism for strychnine-induced doublets in molluscan neurons

The effects of strychnine on Aplysia R2 neurons were evaluated using simultaneous intracellular recordings of the soma and axon potentials. 1 mM strychnine produced a slight enlargement of the somatic spike and a large increase of the axon spike duration. Following direct stimulation, the soma displayed depolarizing afterpotentials ( DAPs ) which might trigger extra-spikes, both produced electronically by long-lasting axon spikes. Cobalt suppressed both the axon spike lengthening and the somatic extra-spikes or DAPs , and induced large depolarizing shifts in the soma. The region of largest spike lengthening (proximal axon) had a large density of Ca channels. The different effects of strychnine on the soma and on the axon were assumed to result from a selective blockage of the V-dependent K channels which would predominate in the axon whereas Ca-activated K channels would predominate in the soma.     

Prolonged modification of action potential shape by synaptic inputs in molluscan neurones

1. Somatic action potentials of Lymnaea neurons are modified by excitatory or inhibitory synaptic inputs and have been studied using phase-plane techniques and an action potential duration monitor. 2. Excitatory synaptic inputs increase the rate of neuronal discharge, cause action potential broadening, a decrease in the maximum rate of depolarization (Vd) and a decrease in the maximum rate of repolarization (Vr). 3. Inhibitory synaptic inputs decrease the discharge rate and cause narrowing of action potentials, an increase in Vd and an increase in Vr. 4. The effects reported above outlast the original synaptic inputs by many seconds and, if the somatic action potentials are similar to those in the axon terminals, they may have far-reaching effects on transmitter release.     

Methods for studying store-operated calcium entry

Activation of surface membrane receptors coupled to phospholipase C results in the generation of cytoplasmic Ca2+ signals comprised of both intracellular Ca2+ release, and enhanced entry of Ca2+ across the plasma membrane. A primary mechanism for this Ca2+ entry process is attributed to store-operated Ca2+ entry, a process that is activated by depletion of Ca2+ ions from an intracellular store by inositol 1,4,5-trisphosphate. Our understanding of the mechanisms underlying both Ca2+ release and store-operated Ca2+ entry have evolved from experimental approaches that include the use of fluorescent Ca2+ indicators and electrophysiological techniques. Pharmacological manipulation of this Ca2+ signaling process has been somewhat limited; but recent identification of key molecular players, STIM and Orai family proteins, has provided new approaches. Here we describe practical methods involving fluorescent Ca2+ indicators and electrophysiological approaches for dissecting the observed intracellular Ca2+ signal to reveal characteristics of store-operated Ca2+ entry, highlighting the advantages, and limitations, of these approaches.     

Locust nymphal neurones in culture: a new technique for studying the physiology and pharmacology of insect central neurones

A new technique for studying the physiology and pharmacology of locust central neurones is described. Somata isolated from neurones in the meso and metathoracic ganglia of third instar locusts (Schistocerca gregaria) were maintained for up to 4 weeks in co-culture (monolayer) with embryonic locust neurones. Most of the cultured cells became multipolar but a few were monopolar like their in vivo counterparts. They had diameters of 40-80 microns and "clean" (glial free) surface membranes. Cells 6-14 days in vitro were depolarized by acetylcholine and usually hyperpolarized by gamma-aminobutyrate, taurine and glycine. L-Glutamate and L-aspartate were inactive but further pharmacological studies are required to confirm this. Cultured larval neurones should provide excellent opportunities to study the molecular basis for drug-receptor interactions and voltage-sensitive membrane channels using the patch clamp technique.     

Sensing of extracellular calcium by neurones

Transient changes in the intracellular concentration of Ca2+ provide a major signal for the regulation of many ion channels and enzymes in central neurones. In contrast, changes in extracellular Ca2+ are thought to play little or no signaling role. However, concentrations of extracellular calcium in the central nervous system do change dramatically during intense physiological and pathological stimulation, and recent studies have identified a number of membrane proteins that can sense and respond to changes in extracellular Ca2+. These include the recently cloned Ca(2+)-sensing receptor, hemi-gap-junction channels, and a potential Ca(2+)-sensing cation channel. Lowering extracellular Ca2+ strongly depolarizes and excites cultured hippocampal neurones. The excitation can be detected with decreases from physiological concentrations of as little as 100 microM. The depolarization results from activation of a nonselective cation current, which is sensitive to block by divalent and polyvalent cations. In outside-out patches, lowering Ca2+ induces a single-channel current with a conductance of 36 pS. Activation of this cation channel, in response to decreases in extracellular Ca2+, likely plays a key role in a positive feedback system of excessive neuronal depolarization, which accompanies intense excitatory activity in the hippocampus.     

Inactivation of calcium currents in the molluscan neuron somatic membrane

The time course of weakening of inward calcium currents (inactivation) during prolonged (of the order of 1 sec) depolarizing shifts of membrane potential was studied in isolated dialyzed neurons of snailHelix pomatia. This decay of the current recorded in this way can be approximated by two exponential functions with time constants of 20–70 and 250–350 msec, respectively. With an increase in pH of the intracellular solution to 8.5 the fast component of the decay disappeared completely; the kinetics of the slow component in this case was very slightly retarded. It is concluded that the fast component of decay of the recorded current does not reflect a change in the calcium current but is due to parallel activation of the nonspecific outward current; the slow component, however, is true in activation of the calcium current. The rate of inactivation of this current was shown to be determined by its maximal value and not by the level of the depolarizing potential shift and it depends on the conditions of accumulation of calcium ions near the inner surface of the membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 14, No. 5, pp. 525–531, September–October, 1982.     

Dependence upon external calcium for contractile activity in two molluscan proboscis muscles.

1. Both the radular sac and odontophore retractor muscles of Buccinum undatum depend upon [Ca]0 to raise the [Ca]i concentration of the contractile system to activation level. 2. The K-induced responses of the muscles depend mainly upon [Ca]0 for activator Ca while the ACh responses depend upon [Ca]0 to raise stored intracellular Ca to activation levels. 3. In the radular sac muscle, it is probable that the inward current is carried by Na+ or is Na(+)-dependent and this current may release [Ca]i for contraction since the muscle became spontaneously active during ACh- and K-contractures in Ca-free seawater containing 2 mM EGTA as a calcium chelator. 4. It is proposed that since calcium antagonists are more inhibitory on ACh responses than on K-contractures, ACh releases the activator calcium for the contractile system through a slow-type Ca channel while high K releases Ca through a fast-type calcium channel in these muscles.     

Analysis by simulation of the transient rectification in molluscan neurons

Central neurons ofLymnaea stagnalis exhibit, by the dne of a hyperpolarizing current pulse, a breaking-off of the rise of the membrane potential to the resting level. By using an electrical model of the membrane, this effect is accounted for by the activation of the fast outward current.     

Computer simulation study of the relationship between the profile of excitatory postsynaptic potential and stimulus-correlated motoneuron firing

This paper shows the results of computer simulation of changes in motoneuron (MN) firing evoked by a repetitively applied synaptic volley that consists of a single excitatory postsynaptic potential (EPSP). Spike trains produced by the threshold-crossing MN model were analyzed as experimental results. Various output functions were applied for analysis; the most useful was a peristimulus time histogram, a special modification of a raster plot and a peristimulus time frequencygram (PSTF). It has been shown that all functions complement each other in distinguishing between the genuine results evoked by the excitatory volley and the secondary results of the EPSP-evoked synchronization. The EPSP rising edge was best reproduced by the PSTF. However, whereas the EPSP rise time could be estimated quite accurately, especially for high EPSP amplitudes at high MN firing rates, the EPSP amplitude estimate was also influenced by factors unrelated to the synaptic volley, such as the afterhyperpolarization duration of the MN or the amplitude of synaptic noise, which cannot be directly assessed in human experiments. Thus, the attempts to scale any estimate of the EPSP amplitude in millivolts appear to be useless. The decaying phase of the EPSP cannot be reproduced accurately by any of the functions. For the short EPSPs, it is extinguished by the generation of an action potential and a subsequent decrease in the MN excitability. For longer EPSPs, it is inseparable from the secondary effects of synchronization. Thus, the methods aimed at extracting information about long-lasting and complex postsynaptic potentials from stimulus-correlated MN firing, should be refined, and the theoretical considerations checked in computer simulations.     

Enzymatic regulation of calcium current in dialyzed and intact molluscan neurons

Isolated neurons of Helix aspersa were dialyzed and voltage clamped under conditions that isolate the Ca current. The rapid time-dependent run-down, or washout, of Ca current could be slowed by addition of 1 mM EGTA to the dialysis solution. A more effective means of slowing washout was the use of agents that promote protein phosphorylation, such as cAMP, Mg-ATP and the catalytic subunit (CS) of cAMP-dependent protein kinase, along with leupeptin, a tripeptide inhibitor of proteases. In the presence of these agents, no internal EGTA was required to prevent Ca current washout. Thus, during dialysis with 100 microM leupeptin, 7 mM Mg-ATP and 20 micrograms/ml CS, the Ca current remained stable for up to several hours. The rate of Ca-dependent inactivation of the current that occurs during a depolarizing step showed only a small decline during prolonged dialysis. Under these conditions, introduction of 10 microM calmodulin plus 40 micrograms/ml calcineurin, a Ca-calmodulin-dependent phosphatase, caused a significant increase in the rate of Ca current inactivation during a depolarizing step. This increase in rate of inactivation, as well as the original inactivation, was eliminated by introduction of EGTA or replacement of external Ca with Ba, results that are consistent with the ion dependency for activation of calcineurin. When internal ATP was replaced with ATP-gamma-S, a hydrolysis-resistant analogue, the rate of Ca current inactivation slowed, providing further evidence that inactivation involves a dephosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

T-type calcium channel in mammalian CNS neurones.

1. In all neurones freshly isolated from various brain regions of newborn, adult and aged rats, the T-type Ca2+ currents were elicited by step depolarizations to potentials more positive than -60 mV from a holding potential of -100 mV, and reached a peak in the current-voltage relationship around -30 mV. 2. The activation and inactivation processes were highly potential-dependent, and the latter was fitted by a single exponential function. 3. It was concluded that mammalian brain neurones possess a definite class of T-type Ca2+ channel characterized by both current kinetics and ion selectivity for Ca2+, Ba2+ and Sr2+. However, the pharmacological nature of the T-type Ca2+ channel differed from that in other tissues such as cardiac and smooth muscle cells, peripheral neurones, and cultured cells.     

Model for calcium dependent oscillatory growth in pollen tubes

Experiments have shown that pollen tubes grow in an oscillatory mode, the mechanism of which is poorly understood. We propose a theoretical growth model of pollen tubes exhibiting such oscillatory behaviour. The pollen tube and the surrounding medium are represented by two immiscible fluids separated by an interface. The physical variables are pressure, surface tension, density and viscosity, which depend on relevant biological quantities, namely calcium concentration and thickness of the cell wall. The essential features generally believed to control oscillating growth are included in the model, namely a turgor pressure, a viscous cell wall which yields under pressure, stretch-activated calcium channels which transport calcium ions into the cytoplasm and an exocytosis rate dependent on the cytosolic calcium concentration in the apex of the cell. We find that a calcium dependent vesicle recycling mechanism is necessary to obtain an oscillating growth rate in our model. We study the variation in the frequency of the growth rate by changing the extracellular calcium concentration and the density of ion channels in the membrane. We compare the predictions of our model with experimental data on the frequency of oscillation versus growth speed, calcium concentration and density of calcium channels.     

Stabilizing role of calcium store-dependent plasma membrane calcium channels in action-potential firing and intracellular calcium oscillations

In many biological systems, cells display spontaneous calcium oscillations (CaOs) and repetitive action-potential firing. These phenomena have been described separately by models for intracellular inositol trisphosphate (IP3)-mediated CaOs and for plasma membrane excitability. In this study, we present an integrated model that combines an excitable membrane with an IP3-mediated intracellular calcium oscillator. The IP3 receptor is described as an endoplasmic reticulum (ER) calcium channel with open and close probabilities that depend on the cytoplasmic concentration of IP3 and Ca2+. We show that simply combining this ER model for intracellular CaOs with a model for membrane excitability of normal rat kidney (NRK) fibroblasts leads to instability of intracellular calcium dynamics. To ensure stable long-term periodic firing of action potentials and CaOs, it is essential to incorporate calcium transporters controlled by feedback of the ER store filling, for example, store-operated calcium channels in the plasma membrane. For low IP3 concentrations, our integrated NRK cell model is at rest at -70 mV. For higher IP3 concentrations, the CaOs become activated and trigger repetitive firing of action potentials. At high IP3 concentrations, the basal intracellular calcium concentration becomes elevated and the cell is depolarized near -20 mV. These predictions are in agreement with the different proliferative states of cultures of NRK fibroblasts. We postulate that the stabilizing role of calcium channels and/or other calcium transporters controlled by feedback from the ER store is essential for any cell in which calcium signaling by intracellular CaOs involves both ER and plasma membrane calcium fluxes.     

A simulation method for the firing sequences of motor units.

The firing sequences of motoneurons contain important information with regard to the underlying neural processes. Several methods have been proposed in the literature to simulate these sequences, however, one of the limitations is that they are not capable of simulating the complex neural dynamics of motor neurons, especially those of concurrently active ones, such as motor unit synchrony and motor unit common drive. In this paper, a novel model based on the Hodgkin-Huxley (HH) system is proposed, which has the ability to simulate the complex neurodynamics of the firing sequences of motor neurons. The model is presented at the cellular level and network level, and some simulation results from a simple 3-neuron network are presented to demonstrate its applications.     

Metabolic abnormalities in children of non-insulin dependent diabetics.

Non-insulin dependent diabetes appears to be an inherited condition. A study of young offspring of non-insulin dependent diabetics was conducted to determine whether metabolic abnormalities could be found at a young age before clinical diabetes developed. Thirteen patients with non-insulin dependent diabetes were selected who fulfilled the following criteria: they had a sibling who also had non-insulin dependent diabetes, their spouse was non-diabetic, and the offspring were aged between 12 and 45 years, not diabetic, and available for study. All 32 offspring had a 75 g oral glucose tolerance test, and results in 13 of them, one randomly selected from each family, were compared with 13 controls of similar age, sex, and weight. The offspring had significantly higher fasting concentrations of glucose, higher proportions of haemoglobin A1, and higher concentrations of insulin, C peptide, and glucagon. After glucose challenge the increases in both glucose and C peptide concentrations were significantly greater in the offspring. These differences were maintained in all 32 offspring when compared with 18 controls of similar age, sex, and weight; seven of the 32 offspring had impaired glucose tolerance. These results indicate that young offspring of selected non-insulin dependent diabetics can show extensive metabolic changes including impaired glucose tolerance. These changes are associated with hyperinsulinaemia and hyperglucagonaemia.