Synaptic transmission in slices of rat hippocampus using a modified voltage clamp technique

A small modification to a voltage-clamp set-up for studying isolated neurons, and the use of simple hippocampal slices allowed stable recording of excitatory postsynaptic currents (EPSCs) that were evoked by stimulating the Shaffer's collaterals of individual CA1 pyramidal neurons. With the developed method EPSCs and focal extracellular potentials could be recorded simultaneously. It was confirmed that the EPSC consists of two components that are mediated via N-methyl-D-aspartate (NMDA)- and non-NMDA-receptors. The effects of different blockers of these receptors on the postsynaptic current were investigated, as were the effects of adenosine, which, depending on its concentration, could either depress or potentiate the synaptic transmission.A. A. Bogomolets Institute of Physiology, Ukrainian Academy of Sciences, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 6, pp. 731–738, November–December, 1991.     

An electrically coupled network of skeletal muscle in zebrafish distributes synaptic current

Fast and slow skeletal muscle types are readily distinguished in larval zebrafish on the basis of differences in location and orientation. Additionally, both muscle types are compact, rendering them amenable to in vivo patch clamp study of synaptic function. Slow muscle mediates rhythmic swimming, but it does so purely through synaptic drive, as these cells are unable to generate action potentials. Our patch clamp recordings from muscle pairs of zebrafish reveal a network of electrical coupling in slow muscle that allows sharing of synaptic current within and between segmental boundaries of the tail. The synaptic current exhibits slow kinetics (tau(decay) approximately 4 ms), which further facilitates passage through the low pass filter, a consequence of the electrically coupled network. In contrast to slow muscle, fast skeletal muscle generates action potentials to mediate the initial rapid component of the escape response. The combination of very weak electrical coupling and synaptic kinetics (tau(decay) <1 ms) too fast for the network low pass filter minimizes intercellular sharing of synaptic current in fast muscle. These differences between muscle types provide insights into the physiological role(s) of electrical coupling in skeletal muscle. First, intrasegmental coupling among slow muscle cells allows effective transfer of synaptic currents within tail segments, thereby minimizing differences in synaptic depolarization. Second, a fixed intersegmental delay in synaptic current transit, resulting from the low pass filter properties of the slow muscle network, helps coordinate the rostral-caudal wave of contraction.     

AII (Rod) amacrine cells form a network of electrically coupled interneurons in the mammalian retina

AII (rod) amacrine cells in the mammalian retina are reciprocally connected via gap junctions, but there is no physiological evidence that demonstrates a proposed function as electrical synapses. In whole-cell recordings from pairs of AII amacrine cells in a slice preparation of the rat retina, bidirectional, nonrectifying electrical coupling was observed in all pairs with overlapping dendritic trees (average conductance approximately 700 pS). Coupling displayed characteristics of a low-pass filter, with no evidence for amplification of spike-evoked electrical postsynaptic potentials by active conductances. Coincidence detection, as well as precise temporal synchronization of subthreshold membrane potential oscillations and TTX-sensitive spiking, was commonly observed. These results indicate a unique mode of operation and integrative capability of the network of AII amacrine cells.     

The component community structure of larval trematodes in the pulmonate snail Helisoma anceps

Factors that affected the component community structure of larval trematodes in the pulmonate snail Helisoma anceps in Charlie's Pond, North Carolina, were studied over a 15-mo period using a multiple mark-recapture protocol. Patent infections of 8 species were observed in 1,485 of 4,899 snails examined. Reproductive activity, population size, and survival rate of the snail population were estimated to evaluate the extent of resource availability for the parasites. Antagonistic interactions between trematode species that occurred at the infracommunity level had a neglible effect on the composition and structure of the component community. The patterns observed at this level were related to temporal heterogeneity in the abundance of infective stages (mostly miracidia), differential responses of trematode species to the diverse and constantly changing distribution of snail size and abundance, differential mortality of snails infected with certain trematode species, constant recruitment of 1 trematode species over time, and the existence of predictable disturbances such as the complete mortality of the host population and recruitment of a replacement population during a 6-8 wk period. The last factor operated as a reset mechanism for this snail-trematode system once each year. A model of patch dynamics, with snails as patch resources, best explains the organization and dynamics of this system.     

Action potential shape change in an electrically coupled network during propagation: a computer simulation

We applied compartmental computer modeling to test a model of spike shape change in the jellyfish, Polyorchis penicillatus, to determine whether adaptive spike shortening can be attributed to the inactivation properties of a potassium channel. We modeled the jellyfish outer nerve-ring as a continuous linear segment, using ion channel and membrane properties derived in earlier studies. The model supported action potentials that shortened as they propagated away from the site of initiation and this was found to be largely independent of potassium channel inactivation. Spike broadening near the site of initiation was found to be due to a depolarization plateau that collapsed as two spikes spread from the point of initiation. The lifetime of this plateau was found to depend critically on the inward current flux and the space constant of the membrane. These data suggest that the spike shape changes may be due not only to potassium channel inactivation, but also to the passive properties of the membrane.     

A comparison of calcium homeostasis in isolated and attached growth cones of the snail Helisoma

This study examines the capability of growth cones from identified neurons of the snail Helisoma trivolvis to perform calcium homeostasis. Calcium influx into the cytoplasm was eliminated or increased experimentally to alter [Ca]i, and the compensatory response of the growth cone was measured with the fluorescent calcium indicator Fura-2. Growth cones compensated for both increases and decreases in calcium influx by restoring [Ca]i towards basal levels under both types of challenges. The intrinsic ability of growth cones to control [Ca]i was examined in physically isolated growth cones. Isolated growth cones demonstrated essentially identical calcium homeostatic properties to their intact counterparts, indicating that mechanisms governing calcium homeostasis exist intrinsically in the growth cone. Such independence may add significantly to the growth cone's potential to locally interpret and respond to stimuli encountered en route to its appropriate target.     

Continuous registration of membrane input resistances of small plant cells using a double-pulse current clamp technique for single-electrode impalements : comparison with the conventional two-electrode method

To measure the cell input resistance in Elodea leaf cells, a new single-microelectrode method was explored by comparing the results with conventional two-microelectrode experiments. The new method takes advantage of the difference in the frequency response curves between electrode and cell impedances. By application of electrical stimuli, which contain specific frequency bands, the different impedances can be analyzed separately. To get a distinct separation in the frequency response of cell and electrode, respectively, the electrode capacitance has to be compensated during the impalement. Different time constants of the cell membrane can be accounted for by adjustment of the stimulus length. It is shown that both the single- and the double-electrode method yield the same results, even if the cell input resistances change considerably during the course of the experiment. This demonstrates the usefulness of the new single-electrode method for continuous measurements of cell membrane resistances, especially in cells so small that the double-electrode method is no longer applicable.     

Studies of the mechanism of the electrical polyspermy block using voltage clamp during cross-species fertilization

Prevention of polyspermic fertilization in sea urchins (Jaffe, 1976, Nature (Lond.). 261:68-71) and the worm Urechis (Gould-Somero, Jaffe, and Holland, 1979, J. Cell Biol. 82:426-440) involves an electrically mediated fast block. The fertilizing sperm causes a positive shift in the egg's membrane potential; this fertilization potential prevents additional sperm entries. Since in Urechis the egg membrane potential required to prevent fertilization is more positive than in the sea urchin, we tested whether in a cross-species fertilization the blocking voltage is determined by the species of the egg or by the species of the sperm. With some sea urchin (Strongylocentrotus purpuratus) females, greater than or equal to 90% of the eggs were fertilized by Urechis sperm; a fertilization potential occurred, the fertilization envelope elevated, and sometimes decondensing Urechis sperm nuclei were found in the egg cytoplasm. After insemination of sea urchin eggs with Urechis sperm during voltage clamp at +50 mV, fertilization (fertilization envelope elevation) occurred in only nine of twenty trials, whereas, at +20 mV, fertilization occurred in ten of ten trials. With the same concentration of sea urchin sperm, fertilization of sea urchin eggs occurred, in only two of ten trials at +20 mV. These results indicate that the blocking voltage for fertilization in these crosses is determined by the sperm species, consistent with the hypothesis that the fertilization potential may block the translocation within the egg membrane of a positively charged component of the sperm.     

Stimulation-induced changes in filopodial dynamics determine the action radius of growth cones in the snail Helisoma trivolvis

Filopodia on neuronal growth cones constantly extend and retract, thereby functioning as both sensory probes and structural devices during neuronal pathfinding. To better understand filopodial dynamics and their regulation by encounters with molecules in the environment, we investigated filopodial dynamics of identified B5 neurons from the buccal ganglion of the snail Helisoma trivolvis before and after treatment with nitric oxide (NO). We have previously demonstrated that treatment with several NO-donors caused a transient, cGMP-mediated elevation in [Ca(2+)](i), which was causally related to an increase in filopodial length and a reduction in the number of filopodia on growth cones. We demonstrate here that these effects were the result of distinct changes in filopodial dynamics. The NO-donor SIN-1 induced a general increase in filopodial motility. Filopodial elongation after treatment with SIN-1 resulted from a significant increase in the rate at which filopodia extended, as well as a significant increase in the time filopodia spent elongating. The reduction in filopodial number was caused by a significant decrease in the frequency with which new filopodia were inserted into the growth cone. With the exception of the back where filopodia appeared less motile, filopodial dynamics appeared to be mostly independent of the location on the growth cone. These results suggest that NO can regulate filopodial dynamics on migrating growth cones and might function as a messenger to adjust the action radius of a growth cone during pathfinding.     

Regulation and restoration of motoneuronal synaptic transmission during neuromuscular regeneration in the pulmonate snail Helisoma trivolvis

Regeneration of motor systems involves reestablishment of central control networks, reinnervation of muscle targets by motoneurons, and reconnection of neuromodulatory circuits. Still, how these processes are integrated as motor function is restored during regeneration remains ill defined. Here, we examined the mechanisms underlying motoneuronal regeneration of neuromuscular synapses related to feeding movements in the pulmonate snail Helisoma trivolvis. Neurons B19 and B110, although activated during different phases of the feeding pattern, innervate similar sets of muscles. However, the percentage of muscle fibers innervated, the efficacy of excitatory junction potentials, and the strength of muscle contractions were different for each cell's specific connections. After peripheral nerve crush, a sequence of transient electrical and chemical connections formed centrally within the buccal ganglia. Neuromuscular synapse regeneration involved a three-phase process: the emergence of spontaneous synaptic transmission (P1), the acquisition of evoked potentials of weak efficacy (P2), and the establishment of functional reinnervation (P3). Differential synaptic efficacy at muscle contacts was recapitulated in cell culture. Differences in motoneuronal presynaptic properties (i.e., quantal content) were the basis of disparate neuromuscular synapse function, suggesting a role for retrograde target influences. We propose a homeostatic model of molluscan motor system regeneration. This model has three restoration events: (1) transient central synaptogenesis during axonal outgrowth, (2) intermotoneuronal inhibitory synaptogenesis during initial neuromuscular synapse formation, and (3) target-dependent regulation of neuromuscular junction formation.     

Method of isolating FMRF-amide-like substances in the snail, using affinity chromatography coupled with HPLC

An extraction procedure of FMRFa-like substances from brain of Helix aspersa was developed. It consists of using affinity chromatography coupled with reverse phase HPLC. Three synthetic peptides (FMRFa, pQDPFLRFa, Met-enkephalin) were used to evaluate the specificity and yield of the affinity column. Its efficiency was tested by use of snail brain extracts. The results showed that this method is efficient and reproducible.     

Measurement of GABA-evoked conductance changes of lobster muscle fibres by a three-microelectrode voltage clamp technique

The effective membrane conductance and capacity of lobster muscle fibres was measured by a three-intracellular-microelectrode voltage clamp technique. Conductance values agreed well with those determined under current clamp, by means of the 'short' cable equations. Reversible increases in conductance evoked by gamma-aminobutyric acid (GABA) were reflected by differences (delta V) in electrotonic potential amplitude recorded at the centre, and midway between the centre and fibre end respectively. GABA dose--conductance curves derived from cable theory or from delta V measurements were virtually identical. The effective capacity (ceff), determined from the area beneath the 'on' delta V capacity transient, yielded values of the membrane time constant consistently lower than those obtained by the graphical method of E. Stefani & A.B. Steinbach (J. Physiol., London. 203, 383-401 (1969)); one possible explanation for this discrepancy is discussed. In the presence of GABA, the effective capacity was reduced in a dose-related manner. The results were interpreted in terms of an equivalent circuit in which surface membrane was arranged in parallel with cleft-tubular membrane of finite conductance, charged through an access resistance. GABA was though to be decreasing ceff by selectively increasing the conductance of the cleft-tubular membranes.     

Mapping conformational changes of a type IIb Na+/Pi cotransporter by voltage clamp fluorometry

The fluorescence of a fluorophore depends on its environment, and if attached to a protein it may report on conformational changes. We have combined two-electrode voltage clamp with simultaneous fluorescence measurements to detect conformational changes in a type IIb Na(+)/P(i) cotransporter expressed in Xenopus oocytes. Four novel Cys, labeled with a fluorescent probe, yielded voltage- and substrate-dependent changes in fluorescence (F). Neither Cys substitution nor labeling significantly altered the mutant electrogenic properties. Different F responses to voltage and substrate were recorded at the four sites. S155C, located in an intracellular re-entrant loop in the first half of the protein, and E451C, located in an extracellular re-entrant loop in the second half of the protein, both showed Na(+), Li(+), and P(i)-dependent F signals. S226C and Q319C, located at opposite ends of a large extracellular loop in the middle of the protein, mainly responded to changes in Na(+) and Li(+). Hyperpolarization increased F for S155C and S226C but decreased F for Q319C and E451C. The labeling and F response of S155C, confirmed that the intracellular loop containing Ser-155 is re-entrant as it is accessible from the extracellular milieu. The behavior of S155C and E451C indicates a strong involvement of the two re-entrant loops in conformational changes during the transport cycle. Moreover, the data for S226C and Q319C suggest that also the large extracellular loop is associated with transport function. Finally, the reciprocal voltage dependences of the S155C-E451C and S226C-Q319C pairs suggest reciprocal conformational changes during the transport cycle for their respective local environments.     

Transmission in the squid giant synapse: a model based on voltage clamp studies

1. Voltage clamp studies were performed in squid giant synapse after blockage of the voltage-dependent sodium and potassium conductances. 2. Presynaptic depolarization under these conditions demonstrates the presence of voltage-dependent calcium conductance change for the duration of the voltage step, and a tail current at the break of the pulse. 2. This calcium current triggers a postsynaptic response which can be measured directly at the postsynaptic fiber. 4. These voltage clamp experiments have allowed the development of a mathematical model that describes the kinetics of the calcium current and the relationship between calcium current and transmitter release.     

Measurement of axonal membrane conductances and capacity by means of a varying potential control voltage clamp

Summary A new mode of voltage clamping in the squid giant axon is introduced and its advantages are analyzed, tested, and utilized to investigate membrane conductances and capacity. This method replaces the constant command potentials of the standard voltage clamp with potentials which vary with time. Some of the advantages in using the varying potential clamp are: (1) slowly varying potentials generate practically pureI _K ; (2) rapidly varying potentials generate practically pureI _Na; (3) triangular waves generate, under proper conditions, pure capacity currents and easy-to-analyze leakage currents; (4) the method gives direct, on-line display of sodium or potassium I–V characteristics within milliseconds; (5) it enables rapid and accurateE _Na andE _K determinations; and (6) it enables simple and accurate determination ofC _m. The method was utilized to study the effects of various ions on membrane conductances and the effects of ionic composition, ionic strength, and temperature on membrane capacity. Membrane capacity was found to be practically independent of frequency in the 200 to 2,000 Hz range. Replacement of external sodium by Ca⁺⁺, by impermeable Tris⁺, or even by dextrose or sucrose (low ionic-strength solutions) had negligible effects onC _m.C _m showed a small, positive temperature coefficient of 1.39% per °C in the 3 to 21°C range, and little change with temperature in the 20 to 40°C range. Above 40°C, bothC _m andg _L increased considerably with temperature.     

In-phase and antiphase self-oscillations in a model of two electrically coupled pacemakers

The dynamic behavior of a model of two electrically coupled oscillatory neurons was studied while the external polarizing current was varied. It was found that the system with weak coupling can demonstrate one of five stable oscillatory modes: (1) in-phase oscillations with zero phase shift; (2) antiphase oscillations with halfperiod phase shift; (3) oscillations with any fixed phase shift depending on the value of the external polarizing current; (4) both in-phase and antiphase oscillations for the same current value, where the oscillation type depends on the initial conditions; (5) both in-phase and quasiperiodic oscillations for the same current value. All of these modes were robust, and they persisted despite small variations of the oscillator parameters. We assume that similar regimes, for example antiphase oscillations, can be detected in neurophysiological experiments. Possible applications to central pattern generator models are discussed.     

Guild structure of larval trematodes in the snail Helisoma anceps: patterns and processes at the individual host level

Factors that influenced the infracommunity structure of trematodes parasitizing the pulmonate snail Helisoma anceps were studied over a 15-mo period; the guild included 8 species of parasites. Infracommunities were depauperate, with double patent infections observed in only 7 of 1,485 infected snails; a total of 4,899 was examined. Halipegus occidualis-Haematoloechus longiplexus was the most common dual infection. Both species share the same definitive host and, in both cases, eggs are the infective stage for the snail. Switches and losses of infections in individual snails were observed, suggesting the occurrence of dynamic interactions within the guild. A dominance hierarchy was constructed based on field observations and experimental infections. Echinostomatids were dominant; species without rediae in their life cycles were subordinates. Halipegus occidualis (which has rediae) was intermediate in dominance. Spatial and temporal heterogeneity in the distribution and abundance of trematode infective stages indicate that not all the snails have the same probability of becoming infected. Habitat structure, behavior of the definitive host, the nature of the infective stages, and snail population dynamics (mortality, recruitment, and size structure) generated spatial and temporal heterogeneity in this system. As a consequence, predictions of the probabilities of interspecific interactions based on an analysis of observed and expected frequencies of multiple infections could be inappropriate unless the potential sources of heterogeneity are considered.