Nonstationary fluctuation analysis and direct resolution of single channel currents at postsynaptic sites.

In order to measure unitary properties of receptor channels at the postsynaptic site, the noise within the decay phases of inhibitory postsynaptic currents (IPSCs) and of N-methyl-D-aspartate (NMDA)-dependent excitatory postsynaptic currents (EPSCs) in rat hippocampal neurons was studied by nonstationary fluctuation analysis. Least squares scaling of the mean current was used to circumvent the wide variation in amplitude of postsynaptic currents. The variance of fluctuations around the expected current was analyzed to calculate single channel conductance, and fluctuation kinetics were studied with power spectra. The single channel conductance underlying the IPSC was measured as 14 pS, whereas that underlying the EPSC was 42 pS. Openings of the EPSC channel could also be resolved directly in low-noise whole-cell recordings, allowing verification of the accuracy of the fluctuation analysis. The results are the first measurements of the properties of single postsynaptic channels activated during synaptic currents, and suggest that the technique can be widely applicable in investigations of synaptic mechanism and plasticity.     

Temperature compensation of postsynaptic currents from the extraocular muscle of temperate teleost fishes

Miniature end plate currents were recorded from white inferior oblique extraocular muscle fibres of one temperate marine teleost (Aldrichetta forsteri, Family Mugilidae) and two temperate freshwater teleosts (Galaxias fasciatus, Family Galaxiidae and Oncorhynchus mykiss, Family Salmonidae). Miniature end plate currents were digitised and averaged over a temperature range of 5-25 degrees C. For each species, decay of miniature end plate currents was exponential and exhibited a strong temperature dependence. Lower temperatures resulted in prolonged decay phases, which decreased exponentially as a function of absolute temperature. Although values of the exponential time constant tau (tau) obtained for each species at 5 degrees C, 15 degrees C and 25 degrees C were significantly different, at any given temperature, there were no significant differences between tau values for the three species, despite differences in phylogeny (different families) and habitat (marine versus freshwater). At their normal temperature of 15 degrees C, mean values of tau for the three species ranged from 840 micros to 940 micros, and apparent activation energies ranged from -41 kJ mol(-1) deg(-1) to 50 kJ mol(-1) deg(-1). These observations confirm earlier reports that teleost miniature end plate currents are consistently shorter than those of other vertebrates.     

Effect of sodium nitroprusside on the mediator release and functional properties of the postsynaptic membrane at the neuromuscular junction]

The effect of nitric oxide donor sodium nitroprusside on the end-plate currents was studied under two-electrode voltage-clamp condition at frog neuro-muscular junction. Sodium nitroprusside (10(-4) M) reduced to the half the amplitude of end-plate currents while did not change miniature end-plate currents indicating the presynaptic nature of end-plate depression. In keeping with such suggestion sodium nitroprusside essentially (to 33%) suppressed the frequency of miniature end-plate currents but did not affect the decay time constant and voltage-dependence of miniature end-plate decay. In contrast to another presynaptic inhibitors sodium nitroprusside rather reduced than increased the presynaptic facilitation and did not change postsynaptic potentials. Thus, nitric oxide is the powerful inhibitor of both evoked and spontaneous transmitter release and did not change postsynaptic potential.     

Comparison of excitatory currents activated by different transmitters on crustacean muscle. I. Acetylcholine-activated channels

The properties of acetylcholine-activated excitatory currents on the gm1 muscle of three marine decapod crustaceans, the spiny lobsters Panulirus argus and interruptus, and the crab Cancer borealis, were examined using either noise analysis, analysis of synaptic current decays, or analysis of the voltage dependence of ionophoretically activated cholinergic conductance increases. The apparent mean channel open time (tau n) obtained from noise analysis at -80 mV and 12 degrees C was approximately 13 ms; tau n was prolonged e-fold for about every 100-mV hyperpolarization in membrane potential; tau n was prolonged e- fold for every 10 degrees C decrease in temperature. Gamma, the single- channel conductance, at 12 degrees C was approximately 18 pS and was not affected by voltage; gamma was increased approximately 2.5-fold for every 10 degrees C increase in temperature. Synaptic currents decayed with a single exponential time course, and at -80 mV and 12 degrees C, the time constant of decay of synaptic currents, tau ejc, was approximately 14-15 ms and was prolonged e-fold about every 140-mV hyperpolarization; tau ejc was prolonged about e-fold for every 10 degrees C decrease in temperature. The voltage dependence of the amplitude of steady-state cholinergic currents suggests that the total conductance increase produced by cholinergic agonists is increased with hyperpolarization. Compared with glutamate channels found on similar decapod muscles (see the following article), the acetylcholine channels stay open longer, conduct ions more slowly, and are more sensitive to changes in the membrane potential.     

Variability of excitatory currents due to single-channel noise, receptor number and morphological heterogeneity

Patch clamp recordings of excitatory postsynaptic currents (EPSCs) in central neurons reveal large fluctuations in amplitudes and decay times of AMPA-receptor-mediated EPSCs. By using Monte Carlo simulations of synaptic transmission in brainstem interneurons, we tested several hypothesis that could account for the observed variability. The coefficient of variation (CV) of 0.5 for miniature amplitudes cannot be explained by fluctuations in vesicle content or receptor distribution, but is traced to variations in receptor number, which is estimated as 77+/-39 receptors per bouton. As the variability of rise times reflects fluctuations in size of the post-synaptic density and heterogeneity of the receptor distribution, the relatively small CV=0.37 of experimentally determined values points to a homogeneous arrangement of receptors. Within our model the large variability of decay times (CV=0.49) can only be explained by fluctuations in the transmitter time course (mean residence times of 0.4+/-0.13 ms), presumably resulting from heterogeneities in synaptic morphology. Hence, our simulations indicate that different noise sources control the variability of amplitudes, rise and decay times. In particular, the distribution of decay times yields information about the synaptic transmission process, which cannot be obtained from other observables.     

Synapse-specific contribution of the variation of transmitter concentration to the decay of inhibitory postsynaptic currents

Synaptic transmission is characterized by a remarkable trial-to-trial variability in the postsynaptic response, influencing the way in which information is processed in neuronal networks. This variability may originate from the probabilistic nature of quantal transmitter release, from the stochastic behavior of the receptors, or from the fluctuation of the transmitter concentration in the cleft. We combined nonstationary noise analysis and modeling techniques to estimate the contribution of transmitter fluctuation to miniature inhibitory postsynaptic current (mIPSC) variability. A substantial variability (approximately 30%) in mIPSC decay was found in all cell types studied (neocortical layer2/3 pyramidal cells, granule cells of the olfactory bulb, and interneurons of the cerebellar molecular layer). This large variability was not solely the consequence of the expression of multiple types of GABA(A) receptors, as a similar mIPSC decay variability was observed in cerebellar interneurons that express only a single type (alpha(1)beta(2)gamma(2)) of GABA(A) receptor. At large synapses on these cells, all variance in mIPSC decay could be accounted for by the stochastic behavior of approximately 36 pS channels, consistent with the conductance of alpha(1)beta(2)gamma(2) GABA(A) receptors at physiological temperatures. In contrast, at small synapses, a significant amount of variability in the synaptic cleft GABA transient had to be present to account for the additional variance in IPSC decay over that produced by stochastic channel openings. Thus, our results suggest a synapse-specific contribution of the variation of the spatiotemporal profile of GABA to the decay of IPSCs.     

Effects of Etomidate on GABAergic and Glutamatergic Transmission in Rat Thalamocortical Slices

Although accumulative evidence indicates that the thalamocortical system is an important target for general anesthetics, the underlying mechanisms of anesthetic action on thalamocortical neurotransmission are not fully understood. The aim of the study is to explore the action of etomidate on glutamatergic and GABAergic transmission in rat thalamocortical slices by using whole cell patch-clamp recording. We found that etomidate mainly prolonged the decay time of spontaneous GABAergic inhibitory postsynaptic currents (sIPSCs), without changing the frequency. Furthermore, etomidate not only prolonged the decay time of miniature inhibitory postsynaptic currents (mIPSCs) but also increased the amplitude. On the other hand, etomidate significantly decreased the frequency of spontaneous glutamatergic excitatory postsynaptic currents (sEPSCs), without altering the amplitude or decay time in the absence of bicuculline. When GABA_A receptors were blocked using bicuculline, the effects of etomidate on sEPSCs were mostly eliminated. These results suggest that etomidate enhances GABAergic transmission mainly through postsynaptic mechanism in thalamocortical neuronal network. Etomidate attenuates glutamatergic transmission predominantly through presynaptic action and requires presynaptic GABA_A receptors involvement.     

Postsynaptic target specificity of neurotrophin-induced presynaptic potentiation

The role of the target cell in neurotrophin-induced modifications of glutamatergic synaptic transmission was examined in cultured hippocampal neurons. Brain-derived neurotrophic factor (BDNF) induced rapid and persistent potentiation of evoked glutamate release when the postsynaptic neuron was glutamatergic, or excitatory (E-->E), but not when it was GABAergic, or inhibitory (E-->1). This target-specific action of BDNF was also found at divergent outputs of a single presynaptic neuron innervating both glutamatergic and GABAergic neurons, suggesting that individual terminals can be independently modified. Surprisingly, BDNF increased the frequency of miniature postsynaptic currents at both E-->E and E-->I, although it had no effect on evoked currents at E-->I. Finally, potentiation by neurotrophin-3 (NT-3) was also target specific. The selective effect at E-->E suggests that retrograde signaling by the postsynaptic target cell endows a localized presynaptic action of neurotrophins.     

Effects of voltage and temperature changes on postsynaptic potentiation at the frog neuromuscular junction

The difference in the decay time constants of multiquantal endplate currents (EPC) produced by presenting paired stimuli 100 msec apart was measured during experiments on transversely cut neuromuscular preparations of the frog sartorius muscle. When acetylcholinesterase was inhibited by 3×10^–6 M prostigmine, decay time of the 2nd EPC (₂) was 39±8% longer than that of the first (₁) due to postsynaptic potentiation. It was found that degree of potentiation was not affected by membrane potential level within the –30 to –120 mV range. Several effects were produced by a drop in temperature: an increase in EPC decay time constant and in that of miniature endplate currents (MEPC) in particular, a slight drop in MEPC amplitude, and a reduction in EPC quantal content. By comparing paired EPC of equal quantal content at different temperatures it was found that potentiation was more pronounced at 12°C than at 22°C and the temperature coefficient Q₁₀ at which ₂ exceeds ₁ was 2.0±0.2 (n=7). The processes determining postsynaptic potential are clearly not voltage-dependent but have a complex dependence on temperature. Quantal content of EPC falls with reduced temperature, thereby restraining potentiation, while helping to retain residual transmitter activity.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Kurashov Medical Institute, Kazan'. Translated from Neirofiziologiya, Vol. 18, No. 4, pp. 512–518, July–August, 1986.     

Multiple kinetic states underlying macroscopic M-currents in bullfrog sympathetic neurons.

M-current is a time- and voltage-dependent potassium current which is suppressible by muscarinic receptor activation. We have used curve fitting and noise analysis to determine if macroscopic M-currents deviate from a previously predicted simple two-state kinetic scheme. The M-current was best described by three kinetically distinct components: 'fast' (tau 0), 'intermediate' (tau 1) and 'slow' (tau 2) time constants. The 'fast' (tau 0) and 'intermediate' (tau 1) components were identified from the spectra of M-current noise at potentials positive to the cells' resting membrane potential. The 'intermediate' (tau 1) and 'slow' (tau 2) components were seen by curve fitting M-current deactivation currents. The 'intermediate' (tau 1) time constant was voltage dependent (decreasing e-fold in 23 mV), but voltage dependence of the 'fast' (tau 0) and 'slow' (tau 2) components was not obvious. All kinetic components were sensitive to muscarine, with the 'intermediate' (tau 1) and 'slow' (tau 2) being equally so. These data suggest that all components may derive from the same channel population, and that the M-channel may have at least four kinetic states.     

L-type Ca2+ channels access multiple open states to produce two components of Bay K 8644-dependent current in GH3 cells

To determine the number of L-channel populations responsible for producing the two components of whole-cell L-type Ca2+ channel current revealed by Bay K 8644 (Fass, D.M., and E.S. Levitan. 1996. J. Gen. Physiol. 108:1-11), L-type Ca2+ channel activity was recorded in cell- attached patches. Ensemble tail currents from most (six out of nine) single-channel patches had double-exponential time courses, with time constants that were similar to whole-cell tail current decay values. Also, in single-channel patches subjected to two different levels of depolarization, ensemble tail currents exactly reproduced the voltage dependence of activation of the two whole-cell components: The slow component is activated at more negative potentials than the fast component. In addition, deactivation of Bay K 8644-modified whole-cell L-current was slower after long (100-ms) depolarizations than after short (20-ms) depolarizations, and this phenomenon was also evident in ensemble tail currents from single L-channels. Thus, a single population of L-channels can produce the two components of macroscopic L-current deactivation. To determine how individual L-channels produce multiple macroscopic tail current components, we constructed ensemble tail currents from traces that contained a single opening upon repolarization and no reopenings. These ensemble tails were biexponential. This type of analysis also revealed that reopenings do not contribute to the slowing of tail current deactivation after long depolarizations. Thus, individual L-channels must have access to several open states to produce multiple macroscopic current components. We also obtained evidence that access to these open states can vary over time. Use of several open states may give L-channels the flexibility to participate in many cell functions.     

Miniature excitatory synaptic ion currents in the earthworm Lumbricus terrestris body wall muscles

The miniature excitatory postsynaptic currents (MEPCs) of the muscle cells of the earthworm Lumbricus terrestris were recorded by glass microelectrodes. In a single synaptic zone, three types of MEPC were recorded: a fast single-exponential type that decayed with tau =0.9 ms, a slow single-exponential with tau = 9.2 ms and a two-exponential MEPC with tau = 1.3 and 8.5 ms, respectively. The muscle cells of earthworms contain populations of yet-unidentified ionic channels that might be different from the common nicotinic and muscarinic groups of acetylcholine receptors, since these MEPCs are not sensitive to d-tubocurarine, atropine, benzohexonium or proserine. Alternatively, besides ACh receptors, the membrane may contain receptors for another yet-unidentified excitatory transmitter.     

Inhibitory synaptic currents in voltage-clamped locust muscle fibres desensitized to their excitatory transmitter

Inhibitory junctional currents (i.j.c.s) have been examined in locust muscle fibres to give properties of GABA-channels activated by the neurally released transmitter. A nerve-muscle preparation is described which has proved suitable for voltage-clamp analysis of inhibitory transmission. I.j.c.s were recorded from fibres in which excitatory synapses had been desensitized with glutamate, to abolish excitatory junctional currents. This procedure had no apparent effect on inhibitory channel properties. The time constant of decay of the i.j.c. was 7.7 +/- 0.3 ms, slightly exceeding the time constant of the membrane noise induced by externally applied GABA. Peak i.j.c. conductance decreased with hyperpolarization. I.j.c.s showed measurable fluctuations permitting an estimate of the mean size of the quantal events composing the i.j.c. Their mean size coincided with the spontaneously occurring miniature inhibitory junctional currents that could be directly recorded in some fibres. The inhibitory nerve-impulse released an average of 35 transmitter packets at sites distributed along the muscle fibre length. Since each m.i.j.c. produced a current of about 0.6 nA (at Vm = -80 mV, ECl = -40 mV) the single quantum of inhibitory transmitter opens 600-1000 postsynaptic chloride channels. This is roughly three to four times the number of channels opened by the excitatory transmitter packet at glutamate synapses in the same fibres.     

Postsynaptic currents in phasic and tonic muscle fibers of the rat extraocular muscle

Focal extracellular recordings were made of postjunctional currents produced at synapses of the inferior rectus eye muscle fibers by the spontaneous release of quanta of transmitter. These consisted of miniature endplate currents, or MEPC, in phasic fibers and miniature postjunctional currents, or MPJC, in tonic fibers. Open time of ionic channels (_chan) was also registered. In tonic fibers, MPJC lasted considerably longer than MEPC did in phasic fibers: rising time, decay time, and _chan in the former measured respectively 2.5, 4–5, and 2.2 times higher than in the latter. Acetylcholinesterace (AChE) inhibition produced a much greater (4.4-fold extension of current decay in phasic than in tonic fibers, where a 1.8-fold increase was seen, thereby reducing the gap between the decay time of currents in these fibers to a difference of 1.6 times. The more protracted decay of MPJC in tonic fibers compared with MEPC in phasic fibers is determined by the lower functional activity of AChE as well as the higher value of _chan. Duration of MEPC and magnitude of _chan in the "slow" phasic fibers of rat skeletal muscles fell well below the same parameters measured in the tonic fibers of the ocular muscle.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 19, No. 1, pp. 120–129, January–February, 1987.     

Effects of "non-quantal" acetylcholine on miniature endplate currents in mice

The possibility of postsynaptic potentiation (PSP) and desensitization developing due to nonquantal acetylcholine (ACh) secretion was investigated in mouse diaphragm with reference to time-amplitude relationships of miniature endplate currents (MEPC). The H effect (which characterizes nonquantal secretion (NS) of ACh) fell to zero over 3 h under the action of armine-induced inhibition of acetylcholinesterase (AChE) at a temperature of 20°C. A decline in the decay time constant () of MEPC unaccompanied by observable alteration in MEPC amplitude occurred at the same time. This accelerated decay of MEPC was not observed in the absence of NS (the early stages of denervation). Start of NS did not show any effect on maximum retardation of MEPC decay due to AChE inhibition, indicating that no PSP sets in under the effects of non-quantal secretion. The effect of decline in accelerated with a rise in temperature; it could be reproduced with neostigmine replacing armine, while remained unchanged in the time spells investigated with AChE in its active state. Non-quantal ACh is not thought to produce substantial retardation of MEPC decay, although it does bring about desensitization, signs of which may be partially masked owing to concurrent onset of PSP.S. V. Kurashov Medical Institute, Kazan'. Translated from Neirofiziologiya, Vol. 22, No. 4, pp. 507–513, July–August, 1990.     

Phencyclidine (PCP) blocks glutamate-activated postsynaptic currents

Phencyclidine (PCP) was tested on the metathoracic tibialis muscles of Locusta migratoria. In physiological solution, the peak amplitude of the excitatory postsynaptic currents (EPSCs) evoked by nerve stimulation was linearly related to membrane potential between -50 and -150 mV. The decay time constant of the EPSC (tau EPSC) was exponentially dependent on voltage and decreased with hyperpolarization. The membrane potential change required to produce an e-fold change in tau EPSC was 315 mV. PCP (5-40 microM) produced a concentration-dependent depression of both EPSC peak amplitude and tau EPSC. A slight nonlinearity in the current-voltage relationship could be discerned at high concentrations of PCP. The shortening of the decay time constant of EPSC (tau EPSC) occurred without significant change in the voltage sensitivity observed under control conditions. Under all experimental conditions, the decay of the EPSCs remained a single exponential of time. Fluctuation analysis indicated that 5 microM PCP shortens the lifetime of the glutamate-activated channels by 25.7 +/- 3%. PCP (10-80 microM) did not induced desensitization of the glutamate receptors. These results suggest that PCP interacts with the open conformation of ion channels activated by the glutamate receptor.     

Modelling endplate currents: dependence on quantum secretion probability and decay of miniature current

Quantification of the time course and amplitude of endplate currents (EPC) was made with respect to dispersion of quanta secretion and to changes in the exponential decay of miniature endplate currents (_mepc). The relationship between RPC amplitude and _mepc follows a double-exponential curve with ₁= 0.3 ms and ₂ = 6 ms. If the amplitude of fully synchronised EPC is taken as 100%, then the loss of EPC amplitude is already 42% with physiological parameters of dispersion (the half-rise and decay constant of distribution of secretion probability = 0.5 ms, _mepc =1 ms). This loss is even more substantial if secretion is more dispersed or miniature endplate currents decay faster. Correspondence to: F. Vyskocil     

Desensitisation of postsynaptic membrane in the neuro-muscular junction due to an increase in spontaneous quantal release of a mediator

Dependence of the amplitude of miniature end-plate currents on frequency of spontaneous quantal release modulated by the elevation of K+ concentration was studied in the frog voltage clamped neuromuscular junctions. A sharp increase of mEPC frequency (not less than approximately 50 per sec) was followed by an obvious fall in both their amplitude and acceleration of decay only in the presence of 3 microM prostigmine (acetylcholinesterase inhibitor) and 5 microM proadiphene, these agents promoting a desensitization of cholinergic postsynaptic membrane. Probable depletion of transmitter store is not involved in the phenomenon observed which is mainly due to the repetitive activation of the postsynaptic zones and the increase of the desensitized cholinoreceptor number.     

Voltage clamp study of fast excitatory synaptic currents in bullfrog sympathetic ganglion cells

Excitatory postsynaptic currents (EPSCs) have been studied in voltage- clamped bullfrog sympathetic ganglion B cells. The EPSC was small, rose to a peak within 1-3 ms, and then decayed exponentially over most of its time-course. For 36 cells at --50 mV (21-23 degrees C), peak EPSC size was --6.5 +/- 3.5 nA (mean +/- SD), and the mean decay time constant tau was 5.3 +/- 0.9 ms. tau showed a small negative voltage dependence, which appeared independent of temperature, over the range -- 90 to --30 mV; the coefficient of voltage dependence was --0.0039 +/- 0.0014 mV-1 (n = 29). The peak current-voltage relationship was linear between --120 and --30 mV but often deviated from linearity at more positive potentials. The reversal potential determined by interpolation was approximately --5 mV. EPSC decay tau had a Q10 = 3. The commonly used cholinesterase inhibitors, neostigmine and physostigmine, exhibited complex actions at the ganglia. Neostigmine (1 X 10(-5)M) produced a time-dependent slowing of EPSC decay without consistent change in EPSC size. In addition, the decay phase often deviated from a single exponential function, although it retained its negative voltage dependence. With 1 x 10(-6) M physostigmine, EPSC decay was slowed by the decay phase remained exponential. At higher concentrations of physostigmine, EPSC decay was markedly prolonged and was composed of at least two decay components. High concentrations of atropine (10(-5) to 10(-4) M) produced complex alterations in EPSC decay, creating two or more exponential components; one decay component was faster and the other was slower than that observed in untreated cells. These results suggest that the time-course of ganglionic EPSC decay is primarily determined by the kinetics of the receptor-channel complex rather than hydrolysis or diffusion of transmitter away from the postsynaptic receptors.