The oscillatory responses of skate electroreceptors to small voltage stimuli

Tonic nerve activity in skate electroreceptors is thought to result from spontaneous activity of the lumenal membranes of the receptor cells which is modulated by applied stimuli. When physiological conditions are simulated in vitro, the receptor epithelium produces a current which flows inward across the lumenal surface. This epithelial current exhibits small spontaneous sinusoidal fluctuations about the mean that are associated with corresponding but delayed fluctuations in postsynaptic response. Small voltage stimuli produce damped oscillations in the epithelial current similar in time-course to the spontaneous fluctuations. For lumen-negative, excitatory stimuli, these responses are predominantly an increase over the mean inward current. For inhibitory stimuli they are predominantly a decrease. Increased inward current across the lumenal membranes of the receptor cells increases depolarization of the presynaptic membranes in the basal faces leading to increased release of transmitter and an excitatory postsynaptic response. Decreased inward current decreases depolarization of the presynaptic membranes leading to a reduction in transmitter release and an inhibitory postsynaptic response. Clear changes in postsynaptic response are detectable during stimuli as small as 5 microV with saturation occurring at +/- 400 microV. The evoked oscillations in epithelial current are damped and the postsynaptic responses decline during maintained stimuli with large off-responses occurring at stimulus termination. The initial peak of the off-response is similar to the response produced by onset of an oppositely directed stimulus. These observations substantiate the role of receptor cell excitability in the detection of small voltage changes.     

Effects of aspartic acid on skate electroreceptors

The effects of L-aspartic acid (L-ASP) on spontaneous and evoked activity in afferent nerve fibers were investigated by perfusing the basal membrane of sea skate electroreceptors (the ampullae of Lorenzini) with this substance. It was found that perfusion with physiological saline containing L-ASP exerted a primarily excitatory effect on afferent activity (threshold concentration: 10^–7 M). When synaptic transmission was blocked by magnesium ions, activity was restored in the afferent fibers if L-ASP was added to the solution and spike activity persisted for longer; this would imply the presence of desensitizing processes in the postsynaptic receptors of the ampullae. Finding would lead to the conclusion that L-ASP and L-glutamate fulfill a set of criteria for likely neurotransmitters in the ampullae of Lorenzini.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 19, No. 1, pp. 61–67, January–February, 1987.     

Calcium-activated conductance in skate electroreceptors: current clamp experiments

When current clamped, skate electroreceptor epithelium produces large action potentials in response to stimuli that depolarize the lumenal faces of the receptor cells. With increasing stimulus strength these action potentials become prolonged. When the peak voltage exceeds about 140 mV the repolarizing phase is blocked until the end of the stimulus. Perfusion experiments show that the rising phase of the action potential results from an increase in calcium permeability in the lumenal membranes. Perfusion of the lumen with cobalt or with a zero calcium solution containing EGTA blocks the action potential. Perfusion of the lumen with a solution containing 10 mM Ca and 20 mM EGTA initially slows the repolarizing process at all voltages and lowers the potential at which it is blocked. With prolonged perfusion, repolarization is blocked at all voltages. When excitability is abolished by perfusion with cobalt, or with a zero calcium solution containing EGTA, no delayed rectification occurs. We suggest that repolarization during the action potential depends on an influx of calcium into the cytoplasm, and that the rate of repolarization depends on the magnitude of the inward calcium current. Increasingly large stimuli reduce the rate of repolarization by reducing the driving force for calcium, and then block repolarization by causing the lumenal membrane potential to exceed ECa. Changes in extracellular calcium affect repolarization in a manner consistent with the resulting change in ECa.     

Calcium-activated conductance in skate electroreceptors: voltage clamp experiments

Voltage clamp experiments allow further characterization of the calcium-dependent repolarizing process in skate electroreceptor epithelium. Four current components are described: a prolonged capacity current, a leakage current, an early active current which flows inward across the lumenal membranes of the receptor cells, and a late current which flows outward. The leakage and capacity currents are linear and may be substracted from the total current, giving net active currents. The early active current is carried by calcium and does not undergo inactivation for at least several seconds. When large stimuli exceed the reversal potential for the early calcium current, the late current is suppressed. Reduction of the ionized calcium concentration in the lumen lowers the reversal potential for the early current and the suppression potential for the late current by the same amount. We conclude that the late current is initiated by a calcium influx into the cytoplasm. During pulses of moderate duration, activation of the late current does not begin until a fixed amount of calcium has entered the receptor cells. The required amount of calcium is reduced if a recent calcium influx has occurred. We suggest that the calcium-activated outward current is mediated by a distinct macromolecule that is insensitive to voltage. Such macromolecules are likely to have an important role in the regulation of electrical activity in excitable cells.     

Effects of kainic acid on synaptic transmission in skate electroreceptors (ampullae of Lorenzini)

The effects of kainic acid on synaptic transmission in electroreceptors were investigated in the skate using techniques of uninterrupted superfusion of the synaptic area with a solution containing this substance and then recording the spike activity of single nerve fibers of the ampullae of Lorenzini. Kainic acid at threshold concentrations of the order of 10^–9 M effectively changed spontaneous and evoked activity of the receptors. Level of background activity served as an indication of the effects taking place. During blockage of synaptic transmission produced by magnesium ions the addition of kainic acid to the magnesium-saturated solution restored both spontaneous and evoked activity. It was deduced that the action of kainic acid on skate electroreceptors is of a primarily presynaptic nature.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 18, No. 2, pp. 147–153, March–April, 1986.     

Membrane current responses of skate photoreceptors

Light-evoked membrane currents were recorded with suction electrodes from the outer segments of individual photoreceptors enzymatically dissociated from the skate retina. The intensity-response relation of dark-adapted cells closely followed a Michaelis function for which a half-saturating response was elicited by a flash intensity that produced about 36 photoisomerizations. Dim-light responses, as well as the early rising phase of the responses to a wide range of flash intensities, could be described by a reaction scheme that involved a series of four first-order delay stages. The number of delay stages required to model the rising phase of the photocurrents did not change in light adaptation. However, background illumination that reduced sensitivity by 1.5 log units, or a bleaching exposure that resulted in a nearly equivalent desensitization, shortened significantly the time scale of the responses. In both instances there were two- to threefold increases in the rate constants of the transitional delays, and almost complete suppression of the tail current that characterized the response of the dark-adapted cell. These findings suggest that although light adaptation alters the gain and kinetics of the transduction mechanism, the nature of the intervening processes is the same in dark- and light-adapted photoreceptors. Moreover, the results show clearly that there is no need to postulate the existence of a second class of cone-like rods to account for the remarkable ability of skate photoreceptors to respond to incremental stimuli presented on "saturating" background fields or after exposure to an intense bleaching light.     

Osmolyte channel regulation by ionic strength in skate RBC

The aim of this study was to determine whether the opening of the osmolyte channel in skate red blood cells (RBC) is regulated by intracellular electrolyte concentration and conductivity. Consistent with previous studies, experiments with hyperosmotic preincubation before cell swelling or swelling with an isosmotic electrolyte (e.g., ammonium chloride) showed that an increase in ionic strength inhibits the opening of the taurine channel. However, a decrease in intracellular ionic strength did not always stimulate taurine efflux to the same degree. Whereas hyposmotic swelling caused a large increase in taurine efflux, swelling induced by treatment with isosmotic nonelectrolytes produced much smaller stimulation. Results with assays for band 3 phosphorylating enzymes were consistent with those from the taurine efflux studies; stimulation of enzyme activity was lower in cells that were swollen with isosmotic nonelectrolyte media than in cells swollen in hyposmotic media. These results indicate that a decrease in ionic strength is not the only signal for the opening of the taurine channel in skate RBC. Ionic strength does affect channel activity, but there must also be some other regulator.     

Effects of some agonists of excitatory amino acids on synaptic transmission in the skate electroreceptors (ampullae of Lorenzini)

The effects on synaptic transmission of blockers of amino acids at postsynaptic receptors were investigated by means of external perfusion of the basal membrane of electroreceptor cells in ampullae of Lorenzini and by recording of spike activity from individual nerve fibers in the skateRaja clavata: glutamic acid diethyl ester (DEE-GLU), glutamic acid dimethyl ester (DME-GLU), D--aminoadipic acid (DAA), kynurenic acid (KYN), and cis-2,3-piperidine dicarboxylic acid (PDA). The most effective blocker was found to be DEE-GLU, producing reversible blockade of postsynaptic amino acid response. It was found that DAA did not change synaptic transmission in the electroreceptors, while PDA largely affected response produced by applying L-aspartic acid (L-ASP); KYN affected response produced by L-glutamic acid (L-GLU). It is deduced that L-GLU and L-ASP are the most effective agonists at the afferent synapse of ampullae of Lorenzini and that their excitatory effect is produced by activating quisqualate receptors.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 20, No. 4, July–August, 1988, pp. 457–463.     

On oscillatory responses of the Limulus retina

Published observations of the dynamic properties of lateral and self-inhibition in the Limulus retina lead to a non-linear integral equation for the response of ommatidia located near the center of a uniformly illuminated region. Coleman and Renninger (1976, 1978) showed that when the excitation is constant in time and the sum of the inhibitory coefficients for the illuminated region exceeds a critical value, the integral equation has a stable periodic solution describing a sustained, spatially synchronized, oscillatory response in which bursts of activity alternate with silent periods. Such spatially synchronized bursting has been observed in the Limulus retina in situ by Barlow and Fraioli (1978), using the preparation of Barlow and Kaplan (1971). Employing experimental data on the temporal dependence of lateral and self-inhibition, which were then available only for the excised eye, Coleman and Renninger calculated a value of 0.34 s for the period p of the bursting response, which is significantly above the range, 0.11–0.20 s, of values of p observed for the Limulus eye in situ. Brodie et al. (1978) have recently published measurements of the temporal dependence of lateral and self-inhibition for the in situ preparation. Here we show that when the kernel functions in Coleman and Renninger's integral equation are chosen in accord with these new data, the periodic solutions of the equation have a period of approximately 0.13s, which is in the range (0.11–0.20 s) required for agreement with experiment. Other properties of the periodic solutions, i.e., their general form and the threshold levels of inhibition required for their existence, are also in accord with published observations of the behavior of the retina in situ.     

The ionic basis of electrical activity in embryonic cardiac muscle

The intracellular sodium concentration reported for young, embryonic chick hearts is extremely high and decreases progressively throughout the embryonic period, reaching a value of 43 mM immediately before hatching. This observation suggested that the ionic basis for excitation in embryonic chick heart may differ from that responsible for electrical activity of the adult organ. This hypothesis was tested by recording transmembrane resting and action potentials on hearts isolated from 6-day and 19-day chick embryos and varying the extracellular sodium and potassium concentrations. The results show that for both young and old embryonic cardiac cells the resting potential depends primarily on the extracellular potassium concentration and the amplitude and rate of rise of the action potential depend primarily on the extracellular sodium concentration.     

The ionic basis of the resting potential of molluscan unstriated muscle

Summary The membrane potential (Vm) of unstriated, non-spiking fibres from the buccal retractor muscle of the opisthobranch molluscPhiline aperta is primarily determined by the distribution of the potassium ion across the membrane. In salines where potassium is varied and chloride remains constant or nearly so, the membrane potential varied with log external K⁺ with a slope of 50.6 (±2.3) mV per decade. In chloride-free salines the slope was 48.5 mV per decade. The experiments were conducted at temperatures of 18–20° C.A ten-fold reduction in external chloride concentration depolarised the fibres by around 10 mV, indicating that chloride permeability makes some contribution to Vm. In salines where [K]₀·[Cl]₀ is constant the Nernst slope was 55.8 mV per decade compared with the theoretical value of 58 mV.The experimental data suggest that the internal potassium concentration of the fibres is 247±31 mM and pNa/pK is 0.01, giving a predicted value of Vm in sea water of –72 mV. The membrane potential of 90 fibres measured in sea water was –74.2±1.3 mV. The membrane contains an electrogenic sodium pump which contributes 4–5 mV to the membrane potential.     

The biochemical basis of antimicrobial responses in Manduca sexta

Innate immunity is essential for the wellbeing of vertebrates and invertebrates. Key components of this defense system include pattern recognition receptors that bind to infectious agents, extra-and intra-cellular proteins that relay signals, as well as molecules and cells that eliminate pathogens. We have been studying the defense mechanisms in a biochemical model insect, Manduca sexta. In this insect, hemolin, peptidoglycan recognition proteins, β-1,3-glucan recognition proteins and C-type lectins detect microbial surface molecules and induce immune responses such as phagocytosis, nodulation, encapsulation, melanization and production of antimicrobial peptides. Some of these responses are mediated by extracellular serine proteinase pathways. The proteolytic activation of prophenoloxidase （proPO） yields active phenoloxidase （PO） which catalyzes the formation of quinones and melanin for wound healing and microbe killing. M. sexta hemolymph proteinase 14 （HP 14） precursor interacts with peptidoglycan or β-1,3-glucan, autoactivates, and leads to the activation of other HPs including HP21 and proPO-activating proteinases （PAPs）. PAP-1, -2 and -3 cut proPO to generate active PO in the presence of two serine proteinase homologs. Inhibition of the proteinases by serpins and association of the proteinase homologs with bacteria ensure a localized defense reaction. M. sexta HP1, HP6, HP8, HP17 and other proteinases may also participate in proPO activation or processing of spatzle and plasmatocyte spreading peptide.     

The genetic basis of flowering responses to seasonal cues

Plants respond to the changing seasons to initiate developmental programmes precisely at particular times of year. Flowering is the best characterized of these seasonal responses, and in temperate climates it often occurs in spring. Genetic approaches in Arabidopsis thaliana have shown how the underlying responses to changes in day length (photoperiod) or winter temperature (vernalization) are conferred and how these converge to create a robust seasonal response. Recent advances in plant genome analysis have demonstrated the diversity in these regulatory systems in many plant species, including several crops and perennials, such as poplar trees. Here, we report progress in defining the diverse genetic mechanisms that enable plants to recognize winter, spring and autumn to initiate flower development.     

The ionic basis of cardiac activity in the bivalve mollusc Perna perna

The ionic basis of cardiac activity and aspects of excitation-contraction (E-C) coupling were investigated in the isolated heart of the bivalve mollusc Perna perna, using the sucrose-gap technique. The role of the principal ions was established employing artificial seawater, in which specific ion concentrations were modified, and ion channel blockers. The mean membrane resting potential (MP) and the action potential (AP) were -33+/-0.7 mV (n=89) and 13+/-0.3 mV (n=71), respectively. The MP potential was primarily dependent on K(+) ions. Three types of cardiac APs were identified: fast, slow and spike-plateau potentials. Cardiac activity was maintained in Na(+)- or Ca(2+)-free salines but ceased when either Cd(2+) or EDTA was added to these salines. Other Ca(2+) channel blockers reduced the amplitude and increased duration of the cardiac APs. Tetrodotoxin (TTX) and procaine did not alter the AP. The data showed that the depolarizing phase of the AP was dependent on Ca(2+) influx while the plateau phase, when present, resulted from Na(+) influx that was modulated by Ca(2+). The mechanical responses were more sensitive to changes in extracellular Ca(2+) concentration than were the electrical responses.     

Functional properties of lamprey electroreceptors

Spike discharges in nerve fibers, evoked by stimulation of electroreceptors by an electric field directed along the body axis (square pulses of current and a sinusoidal current) were recorded in the lampreyLamperta fluviatilis (L.). Excitation of electroreceptors was shown to arise through the action of the cathode. Minimal values of electric field at which appreciable changes took place in spike activity were 10–60 µV/cm for different nerve fibers. The optimal frequency range of sinusoidal electrical stimulation was 0.05–0.5 Hz. It is suggested that electroreceptors of the Agnatha (caudata) and of gnathostomatous cartilaginous fish share a common origin.Murmansk Marine Biological Institute, Kola Branch, Academy of Sciences of the USSR, Dal'nie Zelentsy, Murmansk Region. Translated from Neirofizioloigya, Vol. 16, No. 1, pp. 105–110, January–February, 1984.