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Spontaneous [Ca2+]i fluctuations in rat chromaffin cells do not require inositol 1,4,5-trisphosphate elevations but are generated by a caffeine- and ryanodine-sensitive intracellular Ca2+ store.
No full textA considerable fraction (65%) of single rat chromaffin cells loaded with the fluorescent [Ca2+]i indicator fura-2 exhibited spontaneous rhythmic fluctuations with an average period of approximately 100 s. Parallel patch clamp experiments as well as fura-2 experiments carried out in Ca2(+)-free and other modified media in the presence of Ca2+ and Na+ channel blockers indicated an origin from intracellular stores. Appropriate concentrations of agonists (bradykinin and histamine) for receptors (B2 and H1) that trigger generation of inositol 1,4,5-trisphosphate induced increased fluctuation frequency, recruitment of silent cells, and large [Ca2+]i changes at high doses. These effects were blocked by cell pretreatment with neomycin, a drug that inhibits inositol 1,4,5-trisphosphate generation. In contrast, spontaneous fluctuations and the effects of another drug, caffeine, which also induced increased frequency and recruitment, were unaffected by neomycin. Ryanodine caused first a prolongation and then (approximately 10 min) a block of both spontaneous fluctuations and caffeine effects, where the single transients after bradykinin and histamine were maintained. Caffeine and ryanodine are known to affect selectively the process of calcium-induced Ca2+ release; this is the first demonstration of [Ca2+]i fluctuation activity arising from Ca2(+)-induced Ca2+ release in nonmuscle cells with no strict requirement for inositol 1,4,5-trisphosphate involvement
Static and dynamic properties of synaptic transmission at the cyto-neural junction of frog labyrinth posterior canal.
Full text linkThe properties of synaptic transmission have been studied at the cyto-neural junction of the frog labyrinth posterior canal by examining excitatory postsynaptic potential (EPSP) activity recorded intraaxonally from the afferent nerve after abolishing spike firing by tetrodotoxin. The waveform, amplitude, and rate of occurrence of the EPSPs have been evaluated by means of a procedure of fluctuation analysis devised to continuously monitor these parameters, at rest as well as during stimulation of the semicircular canal by sinusoidal rotation at 0.1 Hz, with peak accelerations ranging from 8 to 87 deg.s-2. Responses to excitatory and inhibitory accelerations were quantified in terms of maximum and minimum EPSP rates, respectively, as well as total numbers of EPSPs occurring during the excitatory and inhibitory half cycles. Excitatory responses were systematically larger than inhibitory ones (asymmetry). Excitatory responses were linearly related either to peak acceleration or to its logarithm, and the same occurred for inhibitory responses. In all units examined, the asymmetry of the response yielded nonlinear two-sided input-output intensity functions. Silencing of EPSPs during inhibition (rectification) was never observed. Comparison of activity during the first cycle of rotation with the average response over several cycles indicated that variable degrees of adaptation (up to 48%) characterize the excitatory response, whereas no consistent adaptation was observed in the inhibitory response. All fibers appeared to give responses nearly in phase with angular velocity, at 0.1 Hz, although the peak rates generally anticipated by a few degrees the peak angular velocity. From the data presented it appears that asymmetry, adaptation, and at least part of the phase lead in afferent nerve response are of presynaptic origin, whereas rectification and possible further phase lead arise at the encoder. To confirm these conclusions a simultaneous though limited study of spike firing and EPSP activity has been attempted in a few fibers
Pre-steady-state and transport-associated currents in the GABA cotransporter rGAT1 are simply related
No full textLa trasmissione sinaptica afferente nel labirinto di rana
No full textL'informazione sensoriale che proviene dai canali semicircolari dipende da una serie di processi in cascata. Un contributo importante alla conoscenza della funzione vestibolare è stato ottenuto studiando il meccanismo di liberazione del mediatore alla giunzione citoneurale. Lo studio è stato condotto mediante l'analisi degli EPSPs registrati a riposo e durante la rotazione sinusoidale da singole fibre afferenti del canale posteriore in prossimità della sinapsi, dove gli EPSPs si osservano unitamente ai potenziali d'azione. Ciò consente l'analisi dei soli EPSPs se i potenziali d'azione sono aboliti con l'impiego di tetrodotossina; d'altro canto, la coesistenza in condizioni normali di entrambi gli eventi permette di definire la funzione di trasferimento dell'encoder. La frequenza degli EPSPs è elevata a riposo (>100 EPSPs/s) ed aumenta in modo significativo con la stimolazione eccitatoria. Ciò produce un notevole grado di fusione dei singoli eventi che rende impossibile una valutazione diretta del loro numero. Per ovviare a questo inconveniente abbiamo trattato il segnale elettrico che si origina dalla sommazione degli EPSPs come "rumore". L'analisi è stata condotta usando una procedura statistica basata sull'estensione del teorema di Campbell ai momenti di ordine superiore della distribuzione del rumore. Il metodo è in grado di fornire una valutazione puntuale della frequenza e dell'ampiezza degli EPSPs a riposo e durante la rotazione. Abbiamo dimostrato che, a parità di accelerazione angolare, la risposta eccitatoria è sistematicamente maggiore di quella inibitoria. Questa asimmetria della risposta dà luogo ad una funzione input-output non-lineare in tutte le unità esaminate. Il confronto dell'attività durante il primo ciclo di stimolazione con quella media dei cicli successivi ha fornito una valutazione quantitativa dell'adattamento della scarica. Abbiamo dimostrato che asimmetria e adattamento della risposta sono di origine presinaptica (Rossi et al., J. Gen. Physiol., 94, 303, 1989). L'analisi simultanea delle proprietà di EPSPs e potenziali d'azione afferenti indica che: 1) la rettificazione (azzeramento della scarica degli spikes per accelerazioni inibitorie del canale) è una proprietà dell'encoder in quanto, a parità di stimolo, la frequenza degli EPSPs non rettifica; 2) la frequenza degli spikes durante la fase eccitatoria della risposta aumenta più rapidamente di quella degli EPSPs; 3) la frequenza degli spikes dopo il picco eccitatorio diminuisce più rapidamente di quella degli EPSPs. La comparsa di potenziali d'azione è legata alla probabilità che il potenziale postsinaptico fluttuante raggiunga la soglia di scarica della fibra. I risultati ottenuti dimostrano che nel labirinto di rana questa probabilità non aumenta linearmente con la frequenza degli EPSPs; ne consegue che la relazione EPSPs-spikes non è lineare. In particolare i risultati suggeriscono che alcune distorsioni dinamiche siano effettivamente presenti nella relazione input-output dell'encoder, dovute probabilmente a conduttanze specifiche voltaggio-dipendenti dell'elemento postsinaptico. Per descrivere la funzione di trasferimento l'encoder viene assimilato a due elementi in serie: il primo presenta caratteristiche statiche ed un guadagno non lineare; il secondo è un filtro a guadagno lineare responsabile delle distorsioni dinamiche. La funzione di trasferimento dell'encoder viene calcolata con le tecniche classiche dell'analisi spettrale o attraverso regressione lineare dell'output sull'input
Pre- and postsynaptic effects of temperature at the posterior canal cytoneural junction in the isolated frog labyrinth
No full text--
THE FREQUENCY DEPENDENCE OF TRANSMITTER RELEASE, IN RESPONSE TO SINUSOIDAL ROTATION, AT THE CYTONEURAL JUNCTION IN FROG LABYRINTH
No full textClassically, the response of the semicircular canal to rotational stimulation has been studied in terms of the spike firing pattern of the primary afferents. However this is the final result of a series of signal processing steps. These involve the hydrodynamic properties of the cupula-endolymph system, the processes of mechano-electrical transduction, regulation of membrane potential and transmitter release by the hair cell, and the properties of the spike encoder which translates the time varying postsynaptic potential into action potentials. The regulation of transmitter release by the hair cell has been the subject of few studies. We have previousiy demonstrated that quantitative measurements of quantal release (rates of occurrence of excitatory postsynaptic potentials, EPSPs) can be obtained by means of noise analysis during intense mechanical stimulation of the canal. Transmitter release was studied at the frog posterior canal cytoneural junction, in response to sinusoidal rotation with varying intensity at fixed frequency (0.1 Hz), and it was concluded that asymmetry, adaptation and at least part of the phase lead which are generally observed in the firing pattern of the primary afferents are of presynaptic origin, as they are observed in the pattern of transmitter release as well. Conversely, rectification (silencing of spikes during inhibitory rotation) and a possible further phase lead appeared to arise at the encoder.
In this study the characterization of presynaptic activity has been completed by measuring the response to stimuli of relatively constant intensity (peak acceleration: 15-18 deg-s-2) but at different frequencies (0.02 to 0.5 Hz) and to rotations at varying frequency (0.02-0.5 Hz) and acceleration (2.5-63 deg-s-2). Thus, the frequency response of transmitter release at the cytoneural junction has been defined.
The waveform of EPSPs was evaluated by power spectral analysis; EPSP amplitude and rate of occurrence were determined by means of a procedure of fluctuation analysis devised to continuously monitor these parameters in a rapidly changing system, with a time resolution such that rotation frequencies up to 1Hz could be studied. Asymmetry in the response was generally observed at all rotation frequencies, i.e. the increase in EPSP rate in response to excitatory acceleration was systematically larger than the decrease produced by the corresponding inhibitory acceleration. At all frequencies of rotation the time course of EPSP rate of occurrence was well described by analytical *asymmetrical" sinusoid. For each unit and at each frequency of sinusoidal rotation it was possible to compute a “gain" of the response (peak-to-peak swing in EPSP rate per unit peak acceleration), a *phase" (lead with respect to angular acceleration or velocity) and an *asymmetry factor" (difference between excitatory and inhibitory responses, divided by peak-to-peak swing). In the experiments with fixed peak acceleration the gain-vs.-frequency relations were well described by one-pole low-pass fílter functions. The estimated corner frequencies ranged 0.04-0.24 Hz in the different units, phase leads being generally consistent with the functions fit to gain data. However, the confidence limits for these estimates are compatible with the idea that the frequency dependence may be common to all units and determined by the hydrodynamic properties of the cupula-endolymph system. Asymmetry in the response did not display any definite frequency-dependence. The same behaviour was observed in those units where peak accelerations were changed up to 25-fold, as expected from the linearity of the stimulus-response relations in the range of stimulation intensities here explored. From the frequency responses the static gain (zero frequency extrapolation) of each unit was defined. A decrease in the response to successive cycles of rotation (adaptation) was observed when the EPSP rate during the first cycle exceeded in each unit a value proportional to its own static gain. No well defined patterns or dominant frequencies of oscillation (tuning) could be identified in the fluctuations of EPSP rate, either at rest or in the residuals from fits during rotation. This study constitutes the first attempt at estimating the frequency response of neurotransmitter release by the hair cell. The results here reported indicate that most of the properties of spike firing at the cytoneural junction directly follow from the properties of neurotransmitter release by the presynaptic cell
FUNCTIONAL PROPERTIES OF THE POSTERIOR CANAL AFFERENT SYNAPSE IN THE ISOLATED FROG LABYRINTH
No full textInformation on the modalities of transmitter release at the cyto-neural junction was derived from the analysis of the EPSP discharge, intra-axonaily recorded from the posterior nerve either at rest or during sinusoidal canal rotation. The high EPSP emission rate (often >100 s-1 ) results in temporal summation and overlapping of the individual events. A noise analysis procedure, using higher moments of fluctuations in membrane potential, was developed to evaluate EPSP amplitude and frequency. EPSP response was quantified in terms of maximum and minimum peak rates as well as of total numbers of EPSPs normalized to the spontaneous activity. With respect to the resting level, the increase in EPSP rate produced by the excitatory phase of stimulation was more conspicuous than the decrease in EPSP frequency observed during the corresponding inhibitory phase of rotation. Both maximum and minimum EPSP rates were linearly related either with peak acceleration or with its logarithm. A consistent adaptation characterizes excitation. The results suggest that response asymmetry, adaptation and intensity function are intrinsic features of the synaptic transmission mechanism being then mirrored in the spike discharge pattern
PRE- AND POSTSYNAPTIC EFFECTS OF TEMPERATURE AT THE CYTONEURAL JUNCTION OF THE ISOLATED FROG LABYRINTH.
No full textResting mEPSPs were intracellularly recorded from the posterior nerve of frogs (Rana esculenta), which had been stored at room temperature (20-22° C), in the presence of TTX (10-6 M) to abolish action potentials. For each preparation continuous recordings were obtained from single units during a 10° C change of the bath temperature imposed by means of a Peltier device with a feedback controller. The range explored was from 8° to 30° C. mEPSP waveform was estimated at the beginning and at the end of the temperature step while mEPSP rate was checked also during the temperature change. In units where mEPSP rate was high (>100/s) single events extensively overlap preventing any direct evaluation of their numbers; therefore the estimate of their mean rate and size was obtained by means of two alternative noise analysis procedures in which the mEPSP waveform was calculated from the autoregressive (minimum phase) fit to the autocorrelation of the summated potentials. If mEPSP rate was less than 300/s the event waveform was used to build a Wiener filter to deconvolve the signal. Tests of Wiener filtering performance on simulated records indicated that this procedure gave unreliable results when mEPSP rate was higher than 300/s; under these conditions, mEPSP rate and amplitude were thus determined by means of an alternative statistical method based on the measure of variance and skewness of membrane potential fluctuations. mEPSP rate increased on increasing temperature and vice-versa, the Q10 ranging from 1.11 to 2.66. Statistical analysis in low- or high-frequency units (alter Wiener-filtering) performed before and at the end of the temperature change showed that: 1) mEPSP peak amplitude distributions were continuous, unimodal and well fitted by lognormal functions; 2) mEPSPS time interval distributions were always monoexponential. Peak amplitude, however, proved to decrease on increasing temperature leading to lognormal distributions markedly shifted to the left with respect to those obtained before the temperature change. Average mEPSP rate increased with increased temperature, this relation being approximately exponential. In the fibres where isolated events could be detected, the elementary waveform was evaluated by fitting the average of 20-50 unitary events with a modified gamma function. From this fit the following parameters were evaluated: 1) shape; 2) duration; 3) maximum amplitude; 4) time-to-peak. Not only peak amplitude, but also time-to-peak and event duration proved to decrease on increasing temperature. Results demonstrate a limited dependence of transmitter release on temperature, which does not affect the basic mechanism of secretion. More consistent are the postsynaptic effects, as suggested by the modifications in the event properties
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