Synaptic Amplitude Research Articles

Beyond Two-Cell Networks: Experimental Measurement of Neuronal Responses to Multiple Synaptic Inputs

Oscillations of large populations of neurons are thought to be important in the normal functioning of the brain. We have used phase response curve (PRC) methods to characterize the dynamics of single neurons and predict population dynamics. Our past experimental work was limited to special circumstances (e.g., 2-cell networks of periodically firing neurons). Here, we explore the feasibility of extending our methods to predict the synchronization properties of stellate cells (SCs) in the rat entorhinal cortex under broader conditions. In particular, we test the hypothesis that PRCs in SCs scale linearly with changes in synaptic amplitude, and measure how well responses to Poisson process-driven inputs can be predicted in terms of PRCs. Although we see nonlinear responses to excitatory and inhibitory inputs, we find that models based on weak coupling account for scaling and Poisson process-driven inputs reasonably accurately.

Target-Specific Regulation of Synaptic Amplitudes in the Neocortex

In layers 2/3 in the rat visual cortex, glutamatergic synapses, between pyramidal neurons and GABAergic interneurons, show target-specific depression or facilitation. To study the mechanisms regulating these short-term synaptic modifications, we recorded from synaptically connected pyramidal neurons (presynaptic) and multipolar or bitufted interneurons (postsynaptic). Evoked AMPA receptor (AMPAR)- or NMDA receptor (NMDAR)-mediated EPSCs were pharmacologically isolated at these pyramidal-to-interneuron synapses while altering release probability (P(r)) by changing the extracellular Ca2+ concentration ([Ca2+]o). At the pyramidal-to-multipolar synapse, which shows paired-pulse depression, elevation of [Ca2+]o from physiological concentrations (2 mm) to 3 mm increased the amplitude of the initial AMPAR-mediated EPSC and enhanced paired-pulse depression. In contrast, the initial NMDAR-mediated EPSC did not change in amplitude with raised P(r) nor was paired-pulse depression altered. This lack of an increase of NMDAR-mediated currents is not a result of Ca2+-dependent effects on the NMDAR. Rather, at the pyramidal-to-multipolar synapse, raised P(r) increases the transient glutamate concentration at individual release sites, possibly reflecting multivesicular release. In contrast, at the pyramidal-to-bitufted synapse, which shows facilitation, AMPAR- and NMDAR-meditated EPSCs showed parallel increases in response to raised P(r). Thus, our results reveal differential recruitment of AMPA and NMDARs at depressing and facilitating synapses in layers 2/3 of the cortex and suggest that the mechanisms regulating dynamic aspects of synaptic transmission are target specific.

Variance–mean analysis: a simple and reliable approach for investigating synaptic transmission and modulation

The mechanisms underlying synaptic plasticity can be investigated by analyzing synaptic amplitude fluctuations before and after a synaptic modulation. However, many older fluctuation analysis techniques rely on models of synaptic transmission that incorporate unrealistic simplifying assumptions or have too many free parameters. As a result, these techniques have sometimes produced counterintuitive or contradictory results. In contrast, the variance–mean ( V– M) technique requires fewer assumptions and is more robust than previous approaches. It achieves these improvements by focusing on two key parameters of synaptic transmission, the average probability that a vesicle is released from a synaptic terminal following a presynaptic stimulus ( P av), and the average amplitude of the postsynaptic response to a vesicle of transmitter ( Q av). To apply V– M analysis, a fluctuating postsynaptic current (PSC) is recorded at several different extracellular Ca 2+ or Cd 2+ concentrations. The variance of the PSC amplitude is plotted against the mean amplitude at each concentration, forming a parabola. The degree of parabolic curvature estimates P av, and the limiting slope under low release conditions estimates Q av. The shape of the V– M parabola changes in characteristic ways following each of the three standard forms of synaptic modulation: a change in Q av (postsynaptic), a change in P av (presynaptic), or a change in the number of terminals ( N). The approach does not require specialized software, and can even be implemented as a purely graphical technique. V– M analysis has been used to investigate the site of expression of long-term potentiation and the mechanisms underlying paired-pulse depression. This report presents a detailed mathematical development of the technique, and explores the limiting conditions under which it can confidently be applied. V– M analysis requires fewer than 100 PSC amplitude measurements to accurately estimate P av and Q av, and it can reliably identify whether a synaptic modulation occurs at a pre- or postsynaptic site. In contrast to other techniques, V– M analysis is largely insensitive to recording noise, nonuniform modulation and intrinsic variability of the unitary synaptic amplitude.

Respiratory sinus arrhythmia: endogenous activation of nicotinic receptors mediates respiratory modulation of brainstem cardioinhibitory parasympathetic neurons.

The heart rate increases during inspiration and decreases during expiration. This respiratory sinus arrhythmia (RSA) occurs by modulation of premotor cardioinhibitory parasympathetic neuron (CPN) activity. However, RSA has not been fully characterized in rats, and despite the critical role of CPNs in the generation of RSA, little is known about the mechanisms that mediate this cardiorespiratory interaction. This study demonstrates that RSA in conscious rats is similar to that in other species. The mechanism of RSA was then examined in vitro. Rhythmic inspiratory-related activity was recorded from the hypoglossal rootlet of 700- to 800-microm medullary sections. CPNs were identified by retrograde fluorescent labeling, and neurotransmission to CPNs was examined using patch-clamp electrophysiological techniques. During inspiratory bursts, the frequency of both spontaneous gamma-aminobutyric acidergic (GABAergic) and spontaneous glycinergic synaptic events in CPNs was significantly increased. Focal application of the nicotinic antagonist dihydro-beta-erythroidine in an alpha4beta2-selective concentration (3 micromol/L) abolished the respiratory-evoked increase in GABAergic frequency. In contrast, the increase in glycinergic frequency during inspiration was not altered by nicotinic antagonists. Prenatal nicotine exposure exaggerated the increase in GABAergic frequency during inspiration and enhanced GABAergic synaptic amplitude both between and during inspiratory events. Glycinergic synaptic frequency and amplitude were unchanged by prenatal nicotine exposure. This study establishes a neurochemical link between neurons essential for respiration and CPNs, reveals a functional role for endogenous acetylcholine release and the activation of nicotinic receptors in the generation of RSA, and demonstrates that this cardiorespiratory interaction is exaggerated in rats prenatally exposed to nicotine.

Differential Control of Vesicle Priming and Short-Term Plasticity by Munc13 Isoforms

Presynaptic short-term plasticity is an important adaptive mechanism regulating synaptic transmitter release at varying action potential frequencies. However, the underlying molecular mechanisms are unknown. We examined genetically defined and functionally unique axonal subpopulations of synapses in excitatory hippocampal neurons that utilize either Munc13-1 or Munc13-2 as synaptic vesicle priming factor. In contrast to Munc13-1-dependent synapses, Munc13-2-driven synapses show pronounced and transient augmentation of synaptic amplitudes following high-frequency stimulation. This augmentation is caused by a Ca 2+-dependent increase in release probability and releasable vesicle pool size, and requires phospholipase C activity. Thus, differential expression of Munc13 isoforms at individual synapses represents a general mechanism that controls short-term plasticity and contributes to the heterogeneity of synaptic information coding.

Synaptojanin 1 Contributes to Maintaining the Stability of GABAergic Transmission in Primary Cultures of Cortical Neurons

Inhibitory synapses in the CNS can exhibit a considerable stability of neurotransmission over prolonged periods of high-frequency stimulation. Previously, we showed that synaptojanin 1 (SJ1), a presynaptic polyphosphoinositide phosphatase, is required for normal synaptic vesicle recycling (Cremona et al., 1999). We asked whether the stability of inhibitory synaptic responses was dependent on SJ1. Whole-cell patch-clamp recordings of unitary IPSCs were obtained in primary cortical cultures between cell pairs containing a presynaptic, fast-spiking inhibitory neuron (33.5-35 degrees C). Prolonged presynaptic stimulation (1000 stimuli, 2-20 Hz) evoked postsynaptic responses that decreased in size with a bi-exponential time course. A fast component developed within a few stimuli and was quantified with paired-pulse protocols. Paired-pulse depression (PPD) appeared to be independent of previous GABA release at intervals of >/=100 msec. The characteristics of PPD, and synaptic depression induced within the first approximately 80 stimuli in the trains, were unaltered in SJ1-deficient inhibitory synapses. A slow component of depression developed within hundreds of stimuli, and steady-state depression showed a sigmoidal dependence on stimulation frequency, with half-maximal depression at 6.0 +/- 0.5 Hz. Slow depression was increased when release probability was augmented, and there was a small negative correlation between consecutive synaptic amplitudes during steady-state depression, consistent with a presynaptic depletion process. Slow depression was increased in SJ1-deficient synapses, with half-maximal depression at 3.3 +/- 0.9 Hz, and the recovery was retarded approximately 3.6-fold. Our studies establish a link between a distinct kinetic component of physiologically monitored synaptic depression and a molecular modification known to affect synaptic vesicle reformation.

Mechanism and impact of allosteric AMPA receptor modulation by the Ampakine TM CX546

Glutamate release at central synapses is transduced into a characteristic fast postsynaptic response by AMPA receptor gating and agonist affinity. The effect of two classes of modulators of AMPA receptor desensitization, the benzothiadiazides (cyclothiazide and IDRA 21) and the benzoylpiperidines (CX516 and CX546), were studied on gating kinetics of recombinant, native AMPA receptors and on synaptic currents. CX546 reduced the degree of desensitization more potently than CX516 or IDRA 21, but not as efficiently as cyclothiazide. In presence of CX516/CX546, desensitization of GluR2 flip receptors was inhibited more than of GluR1 flip, whereas they had no effect upon response shape or conductance. CX546 increased agonist affinity threefold on nondesensitizing AMPA receptors by slowing agonist unbinding. Analysis of modulatory action suggests that, in contrast to cyclothiazide or IDRA 21, the Ampakine TM CX546 binds specifically to the agonist bound nondesensitized receptor, most likely acting by destabilizing the desensitized receptor conformation. All modulators tested showed higher efficiency on native receptors as compared to homomeric receptors. At the glutamatergic synapse, evoked synaptic amplitudes were weakly potentiated, while EPSC decay was slowed by nearly a factor of three in the presence of CX546 or cyclothiazide. In the presence of CX546, the current induced by short pulses of glutamate from recombinant GluR2 receptors decayed with a time course that was approximately twentyfold faster than EPSCs. The unique properties of CX546 may be beneficial for therapeutical use.

The transient precision of integrate and fire neurons: effect of background activity and noise.

We study the response of an integrate and fire neuron to a randomly timed step stimulus. We calculate the latency to the first spike after stimulus onset and its jitter. Background activity, seen in most neurons, reduces latency but causes substantial jitter in the response, indicating a tradeoff between timing precision and latency. The effect of intrinsic noise and synaptic noise on this tradeoff is studied. For synaptic noise we find that, unexpectedly, jitter does not increase for larger synaptic amplitudes, instead, jitter is practically independent of synaptic amplitude. Constant intrinsic noise interacts counterintuitively with latency and jitter, and depending on the stimulus strength, noise shifts the tradeoff in either direction.

The Control of Rate and Timing of Spikes in the Deep Cerebellar Nuclei by Inhibition

Cerebellar nucleus neurons were recorded in vitro, and dynamic clamping was used to simulate inhibitory synaptic input from Purkinje cells likely to occur in vivo. Inhibitory input patterns with varying synaptic amplitudes and synchronicity were applied to determine how spike rate and spike timing can be controlled by inhibition. The excitatory input conductance was held constant to isolate the effect of dynamic inhibitory inputs on spiking. We found that the timing of individual spikes was controlled precisely by short decreases in the inhibitory conductance that were the consequence of synchronization between many inputs. The spike rate of nucleus neurons was controlled in a linear way by the rate of inhibitory inputs. The spike rate, however, also depended strongly on the amount of synchronicity present in the inhibitory inputs. An irregular spike train similar to in vivo data resulted from applied synaptic conductances when the conductance was large enough to overcome intrinsic pacemaker currents. In this situation subthreshold fluctuations in membrane potential closely followed the time course of the combined reversal potential of excitation and inhibition. This indicates that the net synaptic driving force for realistic input levels in vivo may be small and that synaptic input may operate primarily by shunting. The accurate temporal control of output spiking by inhibitory input that can be achieved in this way in the deep cerebellar nuclei may be particularly important to allow fine temporal control of movement via inhibitory output from cerebellar cortex.

Postsynaptic expression of long-term potentiation in the rat dentate gyrus demonstrated by variance-mean analysis.

1. Long-term potentiation (LTP) of synaptic transmission is the putative mechanism underlying learning and memory. Despite intensive study, it remains controversial whether LTP is expressed at a pre- or postsynaptic locus. A new approach was used to investigate this question at excitatory synapses from the medial perforant path (MPP) onto granule cells in the hippocampal dentate gyrus. The variance of the evoked synaptic amplitude was plotted against mean synaptic amplitude at several different Cd2+ concentrations. The slope of the variance-mean plot estimates the average amplitude of the response following the release of a single vesicle of transmitter (Qav). A presynaptic modulation should not affect Qav, but a postsynaptic modulation should alter it. 2. The variance-mean technique was tested by applying the analysis before and after three different synaptic modulations: (i) a reduction in Qav by the addition of the competitive antagonist CNQX; (ii) a reduction in the average probability of transmitter release (Pav) by the addition of baclofen; and (iii) an increase in the number of active synaptic terminals (N) by increasing the stimulus strength. CNQX reduced the average synaptic amplitude and Qav to the same extent, consistent with a postsynaptic action. In contrast, neither a change in N nor Pav altered Qav. This confirms that the variance-mean technique can distinguish between a pre- and a postsynaptic site of modulation. 3. Induction of LTP increased EPSC amplitude by 50 +/- 0.4 % (n = 5) and, in the same cells, increased Qav by 47 +/- 0.6 %. There was no significant difference between the increase in EPSC amplitude and the increase in Qav. Thus, LTP of the MPP input to dentate granule cells can be explained by an increase in the postsynaptic response to transmitter.

Effects of transition metal ions on spontaneous electrical activity and chemical synaptic transmission of neurons in the medicinal leech.

Leech neurons exposed to salines containing inorganic Ca(2+)-channel blockers generate rhythmic bursts of impulses. According to an earlier model, these blockers unmask persistent Na+ currents that generate plateau-like depolarizations, each triggering a burst of impulses. The resulting increase in intracellular Na+ activates an outward Na+/K+ pump current that contributes to burst termination. We tested this model by examining systematically the effects of six transition metal ions (Co2+, Ni2+, Mn2+, Cd2+, La3+, and Zn2+) on the electrical activity of neurons in isolated leech ganglia. Each ion induced bursting activity, but the amplitude, form, and persistence of bursting differed with the ion used and its concentration relative to Ca2+. All ions tested suppressed chemical synaptic transmission between identified motor neurons, consistent with block of voltage-dependent Ca2+ currents in these cells. In addition, a strong correlation between suppression of synaptic transmission and burst amplitudes was obtained. Finally, burst duration was increased and the rate of repolarization decreased in reduced K+ saline, as expected for pump-dependent repolarization. These results provide further support for the hypothesis that a novel form of oscillatory electrical activity driven by persistent Na+ currents and the Na+/K+ pump occurs in leech ganglia exposed to Ca(2+)-channel blockers.

The effects of synaptic noise on measurements of evoked excitatory postsynaptic response amplitudes

Spontaneously occurring synaptic events (synaptic noise) recorded intracellularly are usually assumed to be independent of evoked postsynaptic responses and to contaminate measures of postsynaptic response amplitude in a roughly Gaussian manner. Here we derive analytically the expected noise distribution for excitatory synaptic noise and investigate its effects on amplitude histograms. We propose that some fraction of this excitatory noise is initiated at the same release sites that contribute to the evoked synaptic event and develop an analytical model of the interaction between this fraction of the noise and the evoked postsynaptic response amplitude. Recording intracellularly with sharp microelectrodes in the in vitro hippocampal slice preparation, we find that excitatory synaptic noise accounts for up to 70% of the intracellular recording noise, when inhibition is blocked pharmacologically. Up to 20% of this noise shows a significant correlation with the evoked event amplitude, and the behavior of this component of the noise is consistent with a model which assumes that each release site experiences a refractory period of approximately 60 ms after release. In contrast with classical models of quantal variance, our models predict that excitatory synaptic noise can cause the apparent variance of successive peaks in an excitatory synaptic amplitude histogram to decrease from left to right, and in some cases to be less than the variance of the measured noise.

Excitatory synaptic site heterogeneity during paired pulse plasticity in CA1 pyramidal cells in rat hippocampus in vitro.

1. The properties of individual excitatory synaptic sites onto adult CA1 hippocampal neurons were investigated using paired pulse minimal stimulation and low noise whole-cell recordings. Non-NMDA receptor-mediated synaptic responses were isolated using a pharmacological blockade of NMDA and GABAA receptors. Amongst the twenty-five stationary ensembles there were twelve showing paired pulse potentiation, two showing paired pulse depression and eleven with no significant net change. The signal-to-noise ratio averaged 4.5:1. There was no correlation between the amplitude of the first and second responses after separation of failures: the percentage of failures averaged 33.6% for the conditioning pulse and 31.7% for the test pulse. 2. Site-directed Bayesian statistical analysis was developed to predict the likely number of activated synapses, synaptic response amplitudes, probability of release and intrinsic variation at each individual synaptic site. Extensive simulations showed the usefulness of this model and defined appropriate parameters. These simulations demonstrated only small errors in estimating parameters of data sets with a small number of sites (< 10) and similar characteristics to the physiological data sets. 3. Physiological ensembles showed between one and three synaptic sites, which exhibited a wide range of values for release probability (0.03-0.99), synaptic amplitudes (1.46-16.8 pA; approximately 62% coefficient of variation between sites) and intrinsic variation over time (approximately 36%). Paired pulse plasticity occurred primarily from alterations in the release probabilities but a few ensembles also showed small changes in site amplitude. Initial release probability correlated negatively with the degree of paired pulse potentiation. Whilst it was possible to use simple assumptions regarding site homogeneity (such as required for a binomial process) for 48% (12 out of 25) of the data sets, the Bayesian analysis was necessary to reveal the complex changes and heterogeneity that occurred in the other 52% of the data sets. The Bayesian site analysis robustly indicated the presence of considerable site heterogeneity, significant intrinsic site variation over time and changes in parameters at individual synaptic sites with plasticity.

Regulation of excitatory input to inhibitory interneurons of the dentate gyrus during hypoxia.

The role of metabotropic glutamate receptors (mGluRs) and adenosine receptors in hypoxia-induced suppression of excitatory synaptic input to interneurons residing at the granule cell-hilus border in the dentate gyrus was investigated with the use of whole cell electrophysiological recording techniques in thin (250 microns) slices of immature rat hippocampus. Minimal stimulation evoked glutamatergic excitatory postsynaptic currents (EPSCs) in dentate interneurons in 68 +/- 4% (mean +/- SE) of trials during stimulation in the dentate granule cell layer (GCL) and 48 +/- 3% of trials during stimulation in CA3. Hypoxic episodes, produced by switching the perfusing solution from 95% O2-5% CO2 to a solution containing 95% N2-5% CO2 for 3-5 min, rapidly and reversibly decreased the synaptic reliability, or probability of evoking an EPSC, from either input without reducing EPSC amplitude, consistent with a presynaptic suppression of transmitter release. The mGluR antagonist (+)-alpha-methyl-4-carboxyphenylglycine [(+) MCPG; 500 microM] did not alter synaptic reliability or mean EPSC amplitude in either pathway. However, (+) MCPG significantly attenuated hypoxic suppression of input from both pathways, suggesting that mGluRs activated by release of glutamate partially mediate hypoxic suppression of EPSCs to dentate interneurons. The mGluR agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD; 100 microM) rapidly decreased the reliability of excitatory transmission from both the GCL (19 +/- 5% of control) and CA3 (39 +/- 15% of control). ACPD also increased the frequency of spontaneous EPSCs and evoked a slow inward current in dentate interneurons. Exogenous adenosine (10-300 microM) decreased synaptic reliability for both pathways and reduced the frequency of spontaneous EPSCs, but did not cause a decrease in the mean amplitude of evoked EPSCs, consistent with a presynaptic suppression of excitatory input to dentate interneurons. Conversely, the selective adenosine A1 receptor antagonists 8-cyclopentyl-1,3-dipropylxanthine (200 nM to 1 microM) and N6-cyclopentyl-9-methyladenine (1 microM) enhanced excitatory input to dentate interneurons by increasing synaptic reliability for both the GCL and CA3 inputs. Adenosine A1 receptor antagonists did not, however, reduce hypoxic suppression of excitatory input to dentate interneurons. These results indicate that hypoxia induces a presynaptic inhibition of excitatory input to dentate interneurons mediated in part by activation of mGluRs, but not adenosine A1 receptors, whereas both mGluRs and adenosine A1 receptors can depress excitatory input to dentate interneurons during normoxic stimulation. Regulation of excitatory input to dentate interneurons provides a mechanism to shape excitatory input to the hippocampus under both normal and pathological conditions.

The variance of successive peaks in synaptic amplitude histograms: effects of inter-site differences in quantal size.

Variability in the measured amplitude of evoked synaptic events can arise from several factors, including: measurement noise, trial-to-trial variation in the amplitude of the response at a single release site, or variation between different release sites (inter-site variation) in the mean amplitude of the single quantal response. Classic (linear) models of variability include only the first two of these factors, although differences in the number of postsynaptic receptors or the degree of electrotonic attenuation for different release sites could cause substantial inter-site variations in quantal size. In this paper, the effect of inter-site variation on the variance of successive histogram peaks has been determined analytically and verified by computational studies. This effect is minimal at the edges of the histogram and contributes maximally to central peaks. Linear approximations to the variance of successive histogram peaks may therefore result in very poor fits to measured data if substantial inter-site variation in quantal size is involved. Our computational results indicate that for synaptic contacts with high release probabilities and substantial inter-site variation, the variance of histogram peaks will decrease with increasing quantal content.

Quantal analysis of synaptic transmission in the central nervous system

In the past year, important advances have been made in the understanding of quantal neurotransmission at central synapses. These include new statistical tests for the significance of quantal peaks in synaptic amplitude histograms, greater understanding of possible sources of quantal variance, and new attempts to undertake a rigorous quantal analysis of neurotransmission.

Paired individual and mean postsynaptic currents recorded in four-cell networks of Aplysia

1. Presynaptic neurons B4 and B5 of Aplysia buccal ganglia produce similar inhibitory postsynaptic currents (PSCs) in several postsynaptic follower cells. Two previous papers have characterized the variability of synaptic current amplitude and decay time both for individual PSCs and also for mean values characterizing synapses and have compared PSC amplitude and time course at different synapses sharing a common presynaptic or postsynaptic neuron. 2. To distinguish similarity in synaptic current amplitude or decay introduced by a common pre- or postsynaptic neuron from similarity because of factors common to the particular ganglion or animal, paired synapses were analyzed in four-cell networks in which each of two identified presynaptic neurons produces similar PSCs in each of two postsynaptic cells. Pairing the same synaptic data by common presynaptic or postsynaptic neuron tests if the presynaptic or postsynaptic element partially specifies a parameter; cross-pairing controls for more global factors. Paired values of peak conductance gpeak and decay time constant tau were compared for both individual sequential PSCs and for averages characterizing synapses. Analyses of individual PSCs examine processes affecting synaptic plasticity on a time scale of seconds to minutes, while average values compare more slowly varying factors. 3. Peak amplitudes were compared between individual PSCs in each of 24 paired sets. Correlations of gpeak fluctuations were significantly larger for PSCs produced by the same presynaptic neuron than for postsynaptic or cross pairings (P less than 0.05), consistent with partially correlated fluctuations in transmitter release at different presynaptic terminals. 4. Firing rates of individual presynaptic neurons were modulated to induce variability of test PSCs. These manipulations altered synaptic peak amplitudes in paired postsynaptic neurons, although not to the same degree. Manipulation of a single presynaptic neuron modulated input from that neuron alone to common postsynaptic cells without any effect on input from the paired presynaptic neuron. When fluctuations in the amplitude of gpeak were examined in runs incorporating presynaptic modulation, correlations were strong for sets of PSCs sharing a common presynaptic neuron (R = 0.87), significantly greater (P less than 0.001) than for other pairings. 5. In contrast to the partial presynaptic specification of fluctuations of individual PSCs, values of synaptic amplitude and time course averaged over 21-132 PSCs at a given synapse reflect postsynaptic determinants. Mean values of gpeak characterizing synapses paired by common postsynaptic cell are highly similar (P = 0.0001), in contrast to the lack of similarity seen when the same data are presynaptically (P = 0.11) or cross (P = 0.36) paired.(ABSTRACT TRUNCATED AT 400 WORDS)

A statistical test for demonstrating a presynaptic site of action for a modulator of synaptic amplitude

A statistical technique for demonstrating a presynaptic site of action for a modulator of synaptic amplitude was developed and tested. It requires that multiple measurements of peak synaptic amplitude be made under control and test conditions. The ratio of the coefficients of variation (CV) obtained under test and control conditions is calculated. A method was developed for determining the confidence interval for the CV ratio (CVR) statistic based on the null hypothesis that the synaptic modulation is purely postsynaptic. If the measured CVR falls outside the confidence interval, this implies that the modulator of synaptic amplitude is, at least in part, acting at a presynaptic site. The effectiveness of the technique and its limitations were investigated using Monte Carlo simulations. It was found to be sensitive and reliable under a variety of realistic recording conditions. The test was effective even in the presence of simulated presynaptic rundown of the synaptic response. Conventional deconvolution analysis was also applied to the Monte Carlo simulations and was found to be an inadequate indicator of the site of synaptic modulation when the discrete amplitude components were not well resolved. The CVR technique was applied to excitatory postsynaptic currents (epsc) recorded between pairs of cultured hippocampal neurones in control and test media containing 1 mM Ca 2+ and 2 mM Ca 2+, respectively. Test conditions increased the average synaptic amplitude, and the statistical analysis indicated that this modulation was produced by an action at a presynaptic site.

Quantal analysis of synaptic potentials in neurons of the central nervous system.

This review concentrates on the results of quantal analysis at central synapses, particularly where the morphology and the electrophysiology of the same synaptic connection has been investigated. Results from quantal analysis at peripheral synapses are drawn on to help resolve some issues. The major topics covered in this review are the properties of the quantal synaptic potential and the junctional mechanisms involved in its generation

Quantal nature of neuromuscular transmission in crayfish phasic abdominal flexors

1. The electrophysiology and structure of the crayfish (Procambarus clarkii) deep abdominal flexor neuromuscular system were studied at several stages of development for the purpose of establishing and analyzing the quantal nature of synaptic transmission in these muscles. 2. In adult crayfish we were unable to obtain evidence for quantized neurotransmitter release in this system; spontaneous miniature synaptic potentials were never observed and evoked synaptic potential amplitude fluctuations suggested a mean quantal size of no more than a few microvolts. By recording from very young crayfish, so as to select fibers of higher input resistance, we were able to reveal the existence of a population of miniature potentials with a mean amplitude of less than 100 μV. Their amplitude distributions were always positively skewed, with the mode buried in the background noise. The time intervals between successive events were approximately exponentially distributed. 3. The extremely small quantal size was accounted for in terms of the input resistance (RN) of the muscle fibers. The exceptionally lowRN resulted from a low specific membrane resistivity combined with an unusual organization of the muscle fibers into a system of electrically coupled subunits. Thus the total membrane area of each fiber is disproportionately large for its diameter. The developmental origin and functional significance of this form of neuromuscular organization are discussed. 4. The mean quantal content per muscle fiber was estimated to be several thousand in adult animals, an order of magnitude higher than has been reported for other crayfish muscles. The large quantal content appears to result from a greater than usual total number of synaptic contacts. 5. Synapses formed by the motor giant (MoG) and non-giant (NG) motor neurons displayed the same use-dependent labilities in young crayfish as previously reported in adults. With repetitive stimulation at low to moderate rates the NG synapses facilitated mildly due to an increase in mean quantal content, while the MoG synapses depressed even at rates of once per minute or less.