Spike timing and reliability in cortical pyramidal neurons: effects of EPSC kinetics, input synchronization and background noise on spike timing

In vivo studies have shown that neurons in the neocortex can generate action potentials at high temporal precision. The mechanisms controlling timing and reliability of action potential generation in neocortical neurons, however, are still poorly understood. Here we investigated the temporal precision and reliability of spike firing in cortical layer V pyramidal cells at near-threshold membrane potentials. Timing and reliability of spike responses were a function of EPSC kinetics, temporal jitter of population excitatory inputs, and of background synaptic noise. We used somatic current injection to mimic population synaptic input events and measured spike probability and spike time precision (STP), the latter defined as the time window (Deltat) holding 80% of response spikes. EPSC rise and decay times were varied over the known physiological spectrum. At spike threshold level, EPSC decay time had a stronger influence on STP than rise time. Generally, STP was highest (6 ms) triggered spikes at lower temporal precision (>or=6.58 ms). We found an overall linear relationship between STP and spike delay. The difference in STP between fast and slow compound EPSCs could be reduced by incrementing the amplitude of slow compound EPSCs. The introduction of a temporal jitter to compound EPSCs had a comparatively small effect on STP, with a tenfold increase in jitter resulting in only a five fold decrease in STP. In the presence of simulated synaptic background activity, precisely timed spikes could still be induced by fast EPSCs, but not by slow EPSCs.     

Presynaptic adenosine A1 receptors regulate retinohypothalamic neurotransmission in the hamster suprachiasmatic nucleus

Adenosine has been implicated as a modulator of retinohypothalamic neurotransmission in the suprachiasmatic nucleus (SCN), the seat of the light-entrainable circadian clock in mammals. Intracellular recordings were made from SCN neurons in slices of hamster hypothalamus using the in situ whole-cell patch clamp method. A monosynaptic, glutamatergic, excitatory postsynaptic current (EPSC) was evoked by stimulation of the optic nerve. The EPSC was blocked by bath application of the adenosine A(1) receptor agonist cyclohexyladenosine (CHA) in a dose-dependent manner with a half-maximal concentration of 1.7 microM. The block of EPSC amplitude by CHA was antagonized by concurrent application of the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). The adenosine A(2A) receptor agonist CGS21680 was ineffective in attenuating the EPSC at concentrations up to 50 microM. Trains of four consecutive stimuli at 25 ms intervals usually depressed the EPSC amplitude. However, after application of CHA, consecutive responses displayed facilitation of EPSC amplitude. The induction of facilitation by CHA suggested a presynaptic mechanism of action. After application of CHA, the frequency of spontaneous EPSCs declined substantially, while their amplitude distribution was unchanged or slightly reduced, again suggesting a mainly presynaptic site of action for CHA. Application of glutamate by brief pressure ejection evoked a long-lasting inward current that was unaffected by CHA at concentrations sufficient to reduce the evoked EPSC amplitude substantially (1 to 5 microM), suggesting that postsynaptic glutamate receptor-gated currents were unaffected by the drug. Taken together, these observations indicate that CHA inhibits optic nerve-evoked EPSCs in SCN neurons by a predominantly presynaptic mechanism.     

Fast, automated implementation of temporally precise blind deconvolution of multiphasic excitatory postsynaptic currents

Records of excitatory postsynaptic currents (EPSCs) are often complex, with overlapping signals that display a large range of amplitudes. Statistical analysis of the kinetics and amplitudes of such complex EPSCs is nonetheless essential to the understanding of transmitter release. We therefore developed a maximum-likelihood blind deconvolution algorithm to detect exocytotic events in complex EPSC records. The algorithm is capable of characterizing the kinetics of the prototypical EPSC as well as delineating individual release events at higher temporal resolution than other extant methods. The approach also accommodates data with low signal-to-noise ratios and those with substantial overlaps between events. We demonstrated the algorithm's efficacy on paired whole-cell electrode recordings and synthetic data of high complexity. Using the algorithm to align EPSCs, we characterized their kinetics in a parameter-free way. Combining this approach with maximum-entropy deconvolution, we were able to identify independent release events in complex records at a temporal resolution of less than 250 μs. We determined that the increase in total postsynaptic current associated with depolarization of the presynaptic cell stems primarily from an increase in the rate of EPSCs rather than an increase in their amplitude. Finally, we found that fluctuations owing to postsynaptic receptor kinetics and experimental noise, as well as the model dependence of the deconvolution process, explain our inability to observe quantized peaks in histograms of EPSC amplitudes from physiological recordings.     

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.     

A Functional Study of Single Mammalian CNS “Synaptic Bouton”

Single CNS neurons could be dissociated with adherent functional synaptic boutons without using any enzyme, namely when preparing a “synaptic bouton.” This allows experimenters to investigate the effects of presynaptic modulators of synaptic transmission with unprecedented case and accuracy. Moreover, a single bouton can be visualized using fluorescent markers and can also be focally stimulated with electrical pulses. In this communication, high voltage-dependent Ca²⁺ channels of nerve endings, as one of experimental examples using the “synaptic bouton” preparation, are described. Ca²⁺ channels belonging to different subtypes, which trigger GABA release from nerve terminals (boutons) projecting to rat hippocampal CA1 pyramidal neurons, were studied. GABA-ergic evoked inhibitory postsynaptic currents (eIPSCs) were recorded; these currents were evoked by focal stimulation of single boutons in mechanically dissociated neurons and by stimulation of a nerve bundle in slice preparations. Nilvadipine, an L-type Ca²⁺ channel blocker, completely inhibited eIPSCs evoked by stimulation of single boutons but exerted no effect on eIPSCs evoked by low-frequency stimulation of the nerve bundle. Nilvadipine did, however, prevent potentiation of the eIPSC amplitude following high-frequency stimulation of the nerve bundles in slice preparations. ω-Conotoxin-GVIA, an N-type Ca²⁺ channel blocker, and ω-Agatoxin-IVA, a P/Q-type Ca²⁺ channel blocker, completely inhibited the eIPSCs in 33.3 and 83.3% of the recordings from single boutons, respectively. In response to low-frequency nerve bundle stimulation in the slice preparation, both ω-Conotoxin-GVIA and ω-Agatoxin-IVA partially reduced the amplitude of eIPSC, and the residual component could be abolished by Cd²⁺. From these results, the following hypotheses could be drawn. (i) The distribution of P/Q- and N-type Ca²⁺ channels at a single bouton is nonuniform; (ii) when a focal stimulation is applied to a single bouton, L-type Ca²⁺ channels play a significant role in generation of action potentials, which subsequently activate P/Q- and N-type Ca²⁺ channels at GABA release sites; and (iii) action potentials conducted through axons in the slice preparation are sufficient to depolarize the bouton membrane, even when L-type Ca²⁺ channels are suppressed. Neirofiziologiya/Neurophysiology, Vol. 37, No. 2, pp. 181–183, March–April, 2005.     

Quantal release of glutamate generates pure kainate and mixed AMPA/kainate EPSCs in hippocampal neurons

The relative contribution of kainate receptors to ongoing glutamatergic activity is at present unknown. We report the presence of spontaneous, miniature, and minimal stimulation-evoked excitatory postsynaptic currents (EPSCs) that are mediated solely by kainate receptors (EPSC(kainate)) or by both AMPA and kainate receptors (EPSC(AMPA/kainate)). EPSC(kainate) and EPSC(AMPA/kainate) are selectively enriched in CA1 interneurons and mossy fibers synapses of CA3 pyramidal neurons, respectively. In CA1 interneurons, the decay time constant of EPSC(kainate) (circa 10 ms) is comparable to values obtained in heterologous expression systems. In both hippocampal neurons, the quantal release of glutamate generates kainate receptor-mediated EPSCs that provide as much as half of the total glutamatergic current. Kainate receptors are, therefore, key players of the ongoing glutamatergic transmission in the hippocampus.     

Determining the activation time course of synaptic AMPA receptors from openings of colocalized NMDA receptors.

Excitatory postsynaptic currents (EPSCs) in most mammalian central neurons have a fast alpha-amino-3-hydroxy-5-methyl-4-isoazole-proprionic acid (AMPA) receptor-mediated component, lasting a few milliseconds, and a slow N-methyl-D-aspartic acid (NMDA)-receptor-mediated component, lasting hundreds of milliseconds. The time course of the AMPA phase is crucial in the integrative function of neurons, but measuring it accurately is often confounded by cable filtering between the recording electrode and the synapse. We describe a method for recovering the AMPA phase of individual EPSCs by determining the impulse response of the cable filter from single NMDA channel transitions in the slow tails of the same EPSC, then deconvolving the measured AMPA current. Using simulations, we show that filtering of an AMPA conductance transient in a voltage-clamped dendrite behaves in an almost perfectly linear fashion. Expressions are derived for the time course of single channel transitions and the AMPA phase filtered through a voltage-clamped cable or a single exponential filter, using a kinetic model for AMPA receptor activation. Fitting these expressions to experimental records directly estimates the underlying kinetics of the AMPA phase. Example measurements of spontaneous EPSCs in cultured nonpyramidal rat cortical neurons yielded rising time constants of 0.2-0.8 ms, and decay time constants of 1.3-2 ms at 23-25 degrees C.     

Intracellular regulation of neuronal nicotinic cholinergic receptors

Effects of substances affecting intracellular secondary messengers on the membrane currents evoked by ionophoretic application of acetylcholine (ACh currents) and on the excitatory postsynaptic currents (EPSC) evoked by single stimuli applied to preganglionic nerve fibres, were studied in neurones of the rat isolated superior cervical ganglion. Forskolin, the protein kinase A activator, and isobutyl-methyxanthine, the phosphodiesterase inhibitor, decreased the ACh currents. Neither forskolin nor isobutyl-methylxanthine affected the EPSC amplitude or the EPSC decay time constant. Phorbol ester, the protein kinase C activator, decreased the ACh current but did not affect either EPSC amplitude or the EPSC decay time constant. Thapsigargin, the intracellular calcium releaser, decreased the ACh current and the EPSC amplitude but did not affect the EPSC decay time constant. The data obtained suggest that nicotinic acetylcholine receptors (nAChRs) of ganglion neurones are not modulated through the pathways involving protein kinase A or protein kinase C. The nAChRs sensitivity to both exogenous and nerve-released acetylcholine is reduced by intracellular calcium without affecting kinetics of their ionic channels.     

Control of excitatory synaptic transmission by capsaicin is unaltered in TRPV1 vanilloid receptor knockout mice

Several studies have shown that capsaicin could effectively regulate excitatory synaptic transmission in the central nervous system, but the assumption that this effect is mediated by TRPV1 vanilloid receptors (TRPV1Rs) has not been tested directly. To provide direct evidence, we compared the effect of capsaicin on excitatory synapses in wild type mice and TRPV1R knockouts. Using whole-cell patch-clamp techniques, excitatory postsynaptic currents (EPSCs) were recorded in granule cells of the dentate gyrus. First, we investigated the effect of capsaicin on EPSCs evoked by focal stimulation of fibers in the stratum moleculare. Bath application of 10 microM capsaicin reduced the amplitude of evoked EPSCs both in wild type and TRPV1R knockout animals to a similar extent. Treatment of the slices with the TRPV1R antagonist capsazepine (10 microM) alone, or together with the agonist capsaicin, also caused a decrease in the EPSC amplitude both in wild type and TRPV1R knockout animals. Both drugs appeared to affect the efficacy of excitatory synapses at presynaptic sites, since a significant increase was observed in paired-pulse ratio of EPSC amplitude after drug treatment. Next we examined the effect of capsaicin on spontaneously occurring EPSCs. This prototypic vanilloid ligand increased the frequency of events without changing their amplitude in wild type mice. Similar enhancement in the frequency without altering the amplitude of spontaneous EPSCs was observed in TRPV1R knockout mice. These data strongly argue against the hypothesis that capsaicin modulates excitatory synaptic transmission by activating TRPV1Rs, at least in the hippocampal network.     

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.     

Quantal current fields around individual boutons in sympathetic ganglia.

The release of a quantum of neurotransmitter from an active zone of a bouton is accompanied by the flow of extracellular current that creates a potential field about the site of transmitter action beneath the bouton. It is shown theoretically that the density of the field at the peak of the quantal current gives rise to an extracellular potential that declines to values of less than 5 microV at 1.3 microm distance in the circumferential direction around the neuron and equally rapidly in the radial direction away from the neuron. A loose-patch electrode placed over a bouton distorts the quantal field about the bouton and calculations show that under current-clamp conditions, potentials of over 40 microV can be recorded with an electrode of tip diameter 2 microm, provided the separation between the tip and the neuron's surface is about 0.1 microm. Quantal release recorded from visualized boutons on rat monopolar pelvic ganglion cells with loose-patch electrodes is in agreement with the properties of the quantal potential field given in the theoretical analysis.     

Chronic pregabalin inhibits synaptic transmission between rat dorsal root ganglion and dorsal horn neurons in culture

In this study, we have examined the properties of synaptic transmission between dorsal root ganglion (DRG) and dorsal horn (DH) neurons, placed in co-culture. We also examined the effect of the anti-hyperalgesic gabapentinoid drug pregabalin (PGB) at this pharmacologically relevant synapse. The main method used was electrophysiological recording of excitatory post synaptic currents (EPSCs) in DH neurons. Synaptic transmission between DRG and DH neurons was stimulated by capsaicin, which activates transient receptor potential vanilloid-1 (TRPV1) receptors on small diameter DRG neurons. Capsaicin (1 μM) application increased the frequency of EPSCs recorded in DH neurons in DRG-DH co-cultures, by about 3-fold, but had no effect on other measured properties of the EPSCs. There was also no effect of capsaicin in the absence of co-cultured DRGs. Application of PGB (100 μM) for 40-48 h caused a reduction in the capsaicin-induced increase in EPSC frequency by 57%. In contrast, brief preincubation of PGB had no significant effect on the capsaicin-induced increase in EPSC frequency. In conclusion, this study shows that PGB applied for 40-48 h, but not acute application inhibits excitatory synaptic transmission at DRG-DH synapses, in response to nociceptive stimulation, most likely by a presynaptic effect on neurotransmitter release from DRG presynaptic terminals.     

Chronic pregabalin inhibits synaptic transmission between rat dorsal root ganglion and dorsal horn neurons in culture

In this study, we have examined the properties of synaptic transmission between dorsal root ganglion (DRG) and dorsal horn (DH) neurons, placed in co-culture. We also examined the effect of the anti-hyperalgesic gabapentinoid drug pregabalin (PGB) at this pharmacologically relevant synapse. The main method used was electrophysiological recording of excitatory post synaptic currents (EPSCs) in DH neurons. Synaptic transmission between DRG and DH neurons was stimulated by capsaicin, which activates transient receptor potential vanilloid-1 (TRPV1) receptors on small diameter DRG neurons. Capsaicin (1 μM) application increased the frequency of EPSCs recorded in DH neurons in DRG-DH co-cultures, by about 3-fold, but had no effect on other measured properties of the EPSCs. There was also no effect of capsaicin in the absence of co-cultured DRGs. Application of PGB (100 μM) for 40–48 h caused a reduction in the capsaicin-induced increase in EPSC frequency by 57%. In contrast, brief preincubation of PGB had no significant effect on the capsaicin-induced increase in EPSC frequency. In conclusion, this study shows that PGB applied for 40–48 h, but not acute application inhibits excitatory synaptic transmission at DRG-DH synapses, in response to nociceptive stimulation, most likely by a presynaptic effect on neurotransmitter release from DRG presynaptic terminals.     

Drosophila VAP-33A directs bouton formation at neuromuscular junctions in a dosage-dependent manner

Aplysia VAP-33 (VAMP-associated protein) has been previously proposed to be involved in the control of neurotransmitter release. Here, we show that a Drosophila homolog of VAP-33, DVAP-33A, is localized to neuromuscular junctions. Loss of DVAP-33A causes a severe decrease in the number of boutons and a corresponding increase in bouton size. Conversely, presynaptic overexpression of DVAP-33A induces an increase in the number of boutons and a decrease in their size. Gain-of-function experiments show that the presynaptic dose of DVAP-33A tightly modulates the number of synaptic boutons. Our data also indicate that the presynaptic microtubule architecture is severely compromised in DVAP-33A mutants. We propose that a DVAP-33A-mediated interaction between microtubules and presynaptic membrane plays a pivotal role during bouton budding.     

Participation of the adenylate cyclase system in the postsynaptic mechanism of heterosynaptic facilitation

In an analysis of the postsynaptic mechanism of heterosynaptic facilitation, changes in the amplitude of the excitatory postsynaptic current (EPSC) and the current evoked by application of acetylcholine (ACh current), acting on the adenylate cyclase system of the LC-1 and RC-1 neurons of the molluskPlanorbis corneus, were compared. Both responses are n-cholinergic and depend on the membrane conductivity for Na⁺ and K⁺. Application of serotonin led to a 100–300% increase in the amplitude of the EPSC and (in most cases) the ACh current. However, in 30% of the cases, the increase in the EPSC was accompanied by a decrease in the ACh current. This is probably due to the different contributions of Na⁺ and K⁺ to the mechanism of activation of the conductivity of th channel-receptor complex of the nonsynaptic cell membrane. The influence of serotonin on the EPSC and ACh current was simulated by the action of phosphodiesterase blockers and adenylate cyclase activators. Phosphodiesterase activators and protein kinase blockers reversibly inhibited the EPSC and ACh current. Thus, activation of the adenylate cyclase system, mediated by the action of serotonin, promotes the development of a postsynaptic mechanism of formation of heterosynaptic facilitation of the EPSC in the command neurons of the mollusk.A. A. Bogomolets Institute of Physiology, Ukrainian Academy of Sciences, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 6, pp. 676–683, November–December, 1991.     

Computation of action potential propagation and presynaptic bouton activation in terminal arborizations of different geometries.

Action potential propagation in axons with bifurcations involving short collaterals with synaptic boutons has been simulated using SPICE, a general purpose electrical circuit simulation program. The large electrical load of the boutons may lead to propagation failure at otherwise uncritical geometric ratios. Because the action potential gradually fails while approaching the branch point, the electrotonic spread of the failing action potential cannot depolarize the terminal boutons above an assumed threshold of 20 mV (Vrest = 0 mV) for the presynaptic calcium inflow, and therefore fails to evoke transmitter release even for boutons attached at short collaterals. For even shorter collaterals the terminal boutons can again be activated by the spread of passive current reflected at the sealed end of the bouton which increases the membrane potential above firing threshold. The action potential is then propagated in anterograde fashion into the main axon and may activate the terminal bouton on the other collateral. Differential activation of the synaptic boutons can be observed without repetitive activation of the main axon and with the assumption of uniform membrane properties. Axon enlargements above a critical size at branch points can increase the safety factor for propagation significantly and may serve a double function: they can act both as presynaptic boutons and as boosters, facilitating invasion of the action potential into the terminal arborizations. The architecture of the terminal arborizations has a profound effect on the activation pattern of synapses, suggesting that terminal arborizations not only distribute neural information to postsynaptic cells but may also be able to process neural information presynaptically.     

Kinetic constants of the acetylcholine (ACh) receptor reaction deduced from the rise in open probability after steps in ACh concentration.

Outside-out patches of enzymatically dissociated adult and denervated mouse muscle fibers were superfused repetitively by pulses of acetylcholine (ACh) containing solution. Up to 300 channels opened simultaneously 300 microseconds after the beginning of a 1,000 microM ACh pulse corresponding to a peak current i of almost -1 nA. Single responses to ACh were averaged and the concentration dependence of i and of the rise time tr from 0.1 i to 0.9 i was measured. In adult receptors, i increased proportional to the second to third power of ACh concentration, whereas in embryonic-type receptors it was proportional to the first to the second power. tr increased from approximately 0.3 ms at 1,000 microM ACh to a plateau value of approximately 5 ms for adult and of approximately 10 ms for embryoniclike receptors at concentrations less than 10 microM ACh. The concentration dependence of i and tr was simulated using the standard model of ACh binding with different combinations of rate constants and two and three binding sites for ACh. The calculated curves were compared to the measurements and a set of well fitting rate constants was determined for adult and embryoniclike receptors. Three binding sites for ACh were necessary to fit the dose response for i for adult receptors. A method for deriving rate constants in a model of ACh-receptor interaction is described that avoids analysis of open-closed kinetics of single channels, which in rapid systems, as the ones studied here, are at the limit of the frequency response of the current measurement.     

The nerve-muscle synapse of the garter snake

Snake nerve-muscle preparations are well-suited for study of both motor innervation patterns at the systems level and NMJ function at the cellular level. Their small size (～100 myofibers) and thinness (one fiber) allows access to all NMJs in one muscle. Snake NMJs are of three types, two twitch subtypes and a single tonic type. Properties of the NMJs supplied by a particular motor neuron, and of the motor unit fibers they innervate, are precisely regulated by the motor neuron in a manner consistent with the Henneman Size Principle. Unlike its amphibian or mammalian cousins, the snake NMJ comprises ～50 (twitch) or ～20 (tonic) individual one-bouton synapses, similar to synapses found in the central nervous system. Each bouton releases a few quanta per stimulus. Larger fibers, which require more synaptic current to initiate contraction, receive nerve terminals that contain more boutons and express receptor patches with higher sensitivity to transmitter. Quantal analysis suggests that transmitter release sites in one bouton do not behave independently; rather, they may cooperate to reduce fluctuations and enhance reliability. After release, two mechanisms coexist for retrieval and reprocessing of spent vesicles–one involving clathrin-mediated endocytosis, the other macropinocytosis. Unanswered questions include how each mechanism is regulated in a use-dependent manner.