Blockade of presynaptic K ATP channels reduces the zinc-mediated posttetanic depression at hippocampal mossy fiber synapses.

Zinc is one of the most abundant transition metals in the brain, being present in a variety of synaptic processes. The mossy fiber terminals in area CA3 of the hippocampus contain large amounts of vesicular zinc and have an extremely high density of ATP-sensitive potassium (KATP) channels. The activation of these channels by zinc leads to rapid hyperpolarization of these presynaptic terminals and inhibition of transmitter release. It has been previously shown that intense stimulation of the synapses between mossy fibers and CA3 pyramidal cells evokes a posttetanic depression of synaptic activity, accompanied by a decrease in presynaptic calcium and vesicular zinc signals. These results suggest a neuromodulatory role for zinc at these synapses, which could be mediated by inhibition of presynaptic voltage-dependent calcium channels (VDCCs) and/or activation of presynaptic KATP channels. In order to evaluate the contribution of the second mechanism we have applied multiple tetanic stimulations in the absence and presence of the KATP channel blocker tolbutamide. Under control conditions, it was observed that the delivery of six tetani caused a posttetanic depression of synaptic activity. In the presence of tolbutamide, the depression was smaller and had a shorter time course. A similar depression was also observed in the presynaptic zinc and calcium signals. These findings are in agreement with the hypothesis that the activation of KATP channels by tetanically released zinc leads to cell hyperpolarization and subsequent reduction of presynaptic calcium entry, followed by the inhibition of both zinc and glutamate release. Thus, these results suggest that the inhibition of mossy fiber synaptic transmission by intensely released zinc is partially mediated by the activation of KATP channels.

Automatic centerline extraction of irregular tubular structures using probability volumes from multiphoton imaging.

In this paper, we present a general framework for extracting 3D centerlines from volumetric datasets. Unlike the majority of previous approaches, we do not require a prior segmentation of the volume nor we do assume any particular tubular shape. Centerline extraction is performed using a morphology-guided level set model. Our approach consists of: i) learning the structural patterns of a tubular-like object, and ii) estimating the centerline of a tubular object as the path with minimal cost with respect to outward flux in gray level images. Such shortest path is found by solving the Eikonal equation. We compare the performance of our method with existing approaches in synthetic, CT, and multiphoton 3D images, obtaining substantial improvements, especially in the case of irregular tubular objects.

Imaging of 4-AP-induced, GABA(A)-dependent spontaneous synchronized activity mediated by the hippocampal interneuron network.

Under conditions of increased excitability, such as application of the K(+) channel blocker 4-aminopyridine (4-AP, 100 microM), interneurons in the hippocampal slice show an additional form of synchronized activity that is distinct from the ictal and interictal epileptiform activity induced by these manipulations. In principal neurons, i.e., pyramidal and granule cells, this synchronized interneuron activity (SIA) generates large, multi-component synaptic potentials, which have been termed long-lasting depolarizations (LLDs). These LLDs are dependent on GABA(A) receptor-mediated synaptic transmission but not on excitatory amino acid (EAA) receptors. Intracellular recordings from hilar interneurons have shown that depolarizing GABA(A) receptor-mediated synaptic potentials are also largely responsible for the synchronization of interneurons. The spatiotemporal characteristics of this interneuron activity have not been investigated previously. Using a voltage-sensitive dye and optical techniques that are capable of recording spontaneous synchronized activity, we have characterized the spatiotemporal pattern of SIA (in the presence of 4-AP + EAA receptor antagonists) and compared it with interictal epileptiform activity (in 4-AP only). Like interictal activity, SIA could be observed throughout the hippocampal slice. Unlike interictal activity, which originated in area CA2/CA3 and spread from there, SIA was most prominent in area CA1 and originated either there or in the subiculum. In CA1, interictal activity was largest in and near stratum pyramidale, while SIA was mainly located in s. lacunosum moleculare. Furthermore SIA was equally likely to propagate in either direction, and multiple patterns of propagation could be observed within a single brain slice. These studies suggest that hippocampal area CA1 has the highest propensity for SIA, that multiple locations can serve as the site of origin, and that interneurons located in s. lacunosum moleculare or interneurons that specifically project to this region may be particularly important for synchronized interneuron activity.

Poly(ethylenimine)-mediated transfection: a new paradigm for gene delivery.

Poly(ethylenimine) (PEI) is a synthetic polycation that has been used successfully for gene delivery both in vitro and in vivo due, in theory, to a form of protection that is afforded to the carried plasmids. In this study the stability of PEI/DNA complexes was demonstrated using deoxyribonuclease (DNase) 1 and DNase 2, various levels of pH, and increasing exposure times. DNA that was complexed with PEI was not degraded when exposed to at least 25 Units of either enzyme for 24 h while uncomplexed forms of the same plasmid were digested when exposed to 0.010 Units of DNase 1 for 0.05 h or 0. 003 Units of DNase 2 for 1 h. For further comparison, the stability of complexes made with poly(L-lysine) (PLL) and DNA was examined and found to be lower than that of PEI/DNA complexes; PLL-complexed DNA was digested on exposure to 1.25 Units of DNase 1 for 3 min. Cells were transfected with PEI/DNA complexes and, by using a pH indicator and optical recording techniques, it was found that the normal lysosomal pH value of 5.0 was not altered, bringing into question PEI's hypothesized lysosomal entry. Confocal microscopy showed that PEI/DNA complexes and lysosomes do not merge during transfection (although PLL/DNA complexes do). The lack of lysosomal involvement in PEI-mediated transfection is surprising because it goes against the conventional wisdom that has attempted to explain how PEI functions during transfection. PEI forms a stable complex with DNA, which moves from endocytosis to nuclear entry without significant cellular obstacles.

Activity-dependent intracellular acidification correlates with the duration of seizure activity.

Synchronized neuronal activity (seizures) can appear in the presence or absence of synaptic transmission. Mechanisms of seizure initiation in each of these conditions have been studied, but relatively few studies have addressed seizure termination. In particular, how are seizures terminated in the absence of synaptic activity where there is no loss of excitatory drive or augmentation of inhibitory inputs? We have studied dynamic activity-dependent changes of intracellular pH in the absence of synaptic transmission using the fluorescent pH indicator carboxylseminaphthorhodafluo-1. During epileptiform activity we observed intracellular acidification, whereas between seizures the intracellular pH recovered. Experimental conditions that shortened the epileptiform discharge correlated with more rapid intracellular acidification. On the other hand, experimental manipulation of intracellular pH altered the duration of the seizure discharge, with acidification resulting in early termination of the epileptiform activity. These data show a direct relationship between the level of intracellular acidification and the duration of the seizures, suggesting that an intracellular pH-dependent process can terminate nonsynaptic neuronal synchronization.

Activity-dependent modulation of K+ currents at presynaptic terminals of mammalian central synapses.

1. The activity-dependent regulation of presynaptic K+ currents at the CA3-CA1 synapse in the rat hippocampus was investigated during a train of evoked afferent action potentials. The waveforms of presynaptic compound action potentials (cAPs) and presynaptic Ca2+ transients ([Ca2+]pre,t) were measured with fluorescent voltage-sensitive and Ca2+-sensitive indicators in rat brain slices. 2. Under control conditions, presynaptic cAPs and the accompanying [Ca2+]pre,t displayed similar amplitudes for each stimulus, suggesting that there was no cumulative change of K+ and Ca2+ currents during the test train. However, when a subgroup of presynaptic K+ channels was blocked by a low concentration of 4-aminopyridine (4-AP, 40 microM), a significant facilitation of the [Ca2+]pre,t was observed. 3. This phenomenon was not due to a direct action of 4-AP on presynaptic Ca2+ channels, but to cumulative suppression of the K+ conductance as indicated by the corresponding change in waveforms of the cAP and presynaptic fibre volley. The observed facilitation was not an artifact by virtue of increased fibre recruitment, nor was it related to the accumulation of extracellular K+; rather, it was dependent on Ca2+ influx and stimulation frequency. The time course of recovery from facilitation was closely related to the decay of the intracellular Ca2+ concentration. 4. The facilitation was not blocked by a saturating concentration of 4-AP (8 mM) but was reduced during the application of the K+ channel blocker tetraethylammonium (TEA, 10 mM), implicating the involvement of TEA-sensitive K+ channels. Such activity-dependent suppression of presynaptic K+ conductance could lead to excessive transmitter release and might explain the hippocampal epileptiform activity that can be induced by application of 4-AP.

Simultaneous optical recording of membrane potential and intracellular calcium from brain slices.

Optical recording techniques provide a constantly evolving and increasingly powerful set of tools for investigations of cellular physiology. These techniques rely on the use of optical indicators, molecules that change their optical properties depending on the cellular parameter of interest. In this paper we discuss some of the general considerations involved in recording optical signals from multiple indicators. Specifically, we describe a technique for simultaneously recording transients of membrane potential and intracellular calcium concentration, two parameters that have a very complex interrelationship in neuronal functioning. This technique relies on the use of two fluorescent indicators (the voltage-sensitive dye RH-414 and the calcium-sensitive dye Calcium Orange) that have overlapping excitation spectra but separable emission spectra. This fact, in combination with the use of fast, spatially resolving photodetectors (10 x 10-element photodiode matrices), allows for truly simultaneous recording of these transients from brain slices with high spatial ( approximately 200 x 200 microm with a 10x microscope objective) and temporal ( approximately 500 micros) resolution. Furthermore, the quality of the signals obtained is sufficient to allow for recording of spontaneous synchronized activity such as epileptiform activity induced by the potassium channel blocker 4-aminopyridine. The nature of the signals obtained by these indicators recorded from guinea pig hippocampal slices and some applications of this technique are discussed.

High-speed, random-access fluorescence microscopy: II. Fast quantitative measurements with voltage-sensitive dyes.

An improved method for making fast quantitative determinations of membrane potential with voltage-sensitive dyes is presented. This method incorporates a high-speed, random-access, laser-scanning scheme (Bullen et al., 1997. Biophys. J. 73:477-491) with simultaneous detection at two emission wavelengths. The basis of this ratiometric approach is the voltage-dependent shift in the emission spectrum of the voltage-sensitive dye di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS). Optical measurements are made at two emission wavelengths, using secondary dichroic beamsplitting and dual photodetectors (<570 nm and >570 nm). Calibration of the ratiometric measurements between signals at these wavelengths was achieved using simultaneous optical and patch-clamp measurements from adjacent points. Data demonstrating the linearity, precision, and accuracy of this technique are presented. Records obtained with this method exhibited a voltage resolution of approximately 5 mV, without any need for temporal or spatial averaging. Ratiometric recordings of action potentials from isolated hippocampal neurons are used to illustrate the usefulness of this approach. This method is unique in that it is the first to allow quantitative determination of dynamic membrane potential changes in a manner optimized for both high spatiotemporal resolution (2 micrometers and <0.5 ms) and voltage discrimination.

Modulation of transmitter release by action potential duration at the hippocampal CA3-CA1 synapse.

Presynaptic Ca2+ influx through voltage-dependent Ca2+ channels triggers neurotransmitter release. Action potential duration plays a determinant role in the dynamics of presynaptic Ca2+ influx. In this study, the presynaptic Ca2+ influx was optically measured with a low-affinity Ca2+ indicator (Furaptra). The effect of action potential duration on Ca2+ influx and transmitter release was investigated. The K+ channel blocker 4-aminopyridine (4-AP) was applied to broaden the action potential and thereby increase presynaptic Ca2+ influx. This increase of Ca2+ influx appeared to be much less effective in enhancing transmitter release than raising the extracellular Ca2+ concentration. 4-AP did not change the Ca2+ dependence of transmitter release but instead shifted the synaptic transmission curve toward larger total Ca2+ influx. These results suggest that changing the duration of Ca2+ influx is not equivalent to changing its amplitude in locally building up an effective Ca2+ concentration near the Ca2+ sensor of the release machinery. Furthermore, in the presence of 4-AP, the N-type Ca2+ channel blocker omegaCgTx GVIA was much less effective in blocking transmitter release. This phenomenon was not simply due to a saturation of the release machinery by the increased overall Ca2+ influx because a similar reduction of Ca2+ influx by application of the nonspecific Ca2+ channel blocker Cd2+ resulted in much more inhibition of transmitter release. Rather, the different potencies of omega-CgTx GVIA and Cd2+ in inhibiting transmitter release suggest that the Ca2+ sensor is possibly located at a distance from a cluster of Ca2+ channels such that it is sensitive to the location of Ca2+ channels within the cluster.

Acousto-optic random-access laser scanning microscopy: fundamentals and applications to optical recording of neuronal activity.

A novel approach to laser scanning microscopy is presented that utilizes diffraction-based scanning principles to achieve fast random-access positioning of a focused laser beam. This non-imaging approach overcomes the speed limitation of present reflection-based scanning microscopes while maintaining high spatial resolution. The presented system combines conventional video microscopy with fast non-imaging scanning microscopy. Together with readily available optical indicators of neuronal activity, this system permits multi-site optical recording from living brain tissue. In this paper, we will review the underlying principles of laser scanning microscopy and the steps in development that led to the current acousto-optic scanning system. We will present typical signals recorded with the current system, and we will outline ongoing extensions of the system. We will also discuss the present limitation of this instrumentation and look into directions of future development.

Indicators and optical configuration for simultaneous high-resolution recording of membrane potential and intracellular calcium using laser scanning microscopy.

The instrumental design and experimental conditions for high-speed, simultaneous optical recording of membrane potential and intracellular Ca2+ with subcellular resolution are presented. This method employs an extended version of a high-speed, random-access, laser-scanning fluorescence microscope designed to record fast physiological signals from small neuronal structures with high spatiotemporal resolution (Bullen, Patel, Saggau, Biophys J 73:477-491, 1997). With this instrument, imaging and optical recording functions are conducted separately allowing frame rates up to 3 kHz. Individual scanning points are selected interactively from a reference image collected with differential interference contrast (DIC) optics. At each recording site, fluorescence from two indicators is measured simultaneously by independent photodetectors. To optimize signal strength, spectral separation and the achievable signal-to-noise ratio, several combinations of voltage-sensitive dye, Ca2+ indicator and optical elements (dichroic mirrors, filters, etc.) were considered. The best results were achieved from the combination of the intracellular voltage-sensitive dye Di-2-ANEPEQ and the Ca2+ indicator Calcium Green-1. These indicators have overlapping absorption spectra allowing simultaneous excitation with a single laser line (488 nm). Spectral separation of the fluorescence from these two indicators was accomplished using a secondary dichroic mirror (DCLP580) and emission filters (535/45 and OG590). Representative records obtained with this instrument and this combination of indicators demonstrate the feasibility of simultaneous high fidelity measurements of membrane potential and intracellular Ca2+ from the same point at high spatial (2 micrometer) and temporal (

Presynaptic inhibition of synaptic transmission in the rat hippocampus by activation of muscarinic receptors: involvement of presynaptic calcium influx.

1. Modulation of presynaptic voltage-dependent calcium channels (VDCCs) by muscarinic receptors at the CA3-CA1 synapse of rat hippocampal slices was investigated by using the calcium indicator fura-2. Stimulation-evoked presynaptic calcium transients ([Ca(pre)]t) and field excitatory postsynaptic potentials (fe.p.s.ps) were simultaneously recorded. The relationship between presynaptic calcium influx and synaptic transmission was studied. 2. Activation of muscarinic receptors inhibited [Ca(pre)]t, thereby reducing synaptic transmission. Carbachol (CCh, 10 microM) inhibited [Ca(pre)]t by 35% and reduced fe.p.s.p. by 85%. The inhibition was completely antagonized by 1 microM atropine. An approximate 4th power relationship was found between presynaptic calcium influx and postsynaptic responses. 3. Application of the N-type VDCC-blocking peptide toxin omega-conotoxin GVIA (omega-CTx GVIA, 1 microM) inhibited [Ca(pre)]t and fe.p.s.ps by 21% and 49%, respectively, while the P/Q-type VDCC blocker omega-agatoxin IVA (omega)-Aga IVA, 1 microM) reduced [Ca(pre)]t and fe.p.s.ps by 35% and 85%, respectively. 4. Muscarinic receptor activation differentially inhibited distinct presynaptic VDCCs. Omega-CTx GVIA-sensitive calcium channels were inhibited by muscarinic receptors, while omega-Aga IVA-sensitive channels were not. The percentage inhibition of omega-CTx GVIA-sensitive [Ca(pre)]t was about 63%. 5. Muscarinic receptors inhibited presynaptic VDCCs in a way similar to adenosine (Ad) receptors. The percentage inhibition of omega-CTx GVIA-sensitive [Ca(pre)]t by Ad (100 microM) was about 59%. There was no significant inhibition of omega-Aga IVA-sensitive channels by Ad. The inhibitions of [Ca(pre)]t by CCh and Ad were mutually occlusive. 6. These results indicate that inhibition of synaptic transmission by muscarinic receptors is mainly the consequence of a reduction of the [Ca(pre)]t due to inhibition of presynaptic VDCCs.

Inhibition of synaptic transmission by neuropeptide Y in rat hippocampal area CA1: modulation of presynaptic Ca2+ entry.

Neuropeptide Y (NPY) agonists inhibit glutamate release by a presynaptic action at the CA3-CA1 synapse of rat hippocampus. We have examined the relationship between [Capre]t via presynaptic, voltage-dependent calcium channels (VDCCs), measured optically by using the fluorescent calcium indicator fura-2, and transmitter release, measured electrophysiologically. Activation of presynaptic NPY Y2 receptors reduced [Capre]t and thereby inhibited synaptic transmission. Multiple calcium channels are involved in synaptic transmission at this synapse. Activation of Y2 receptors inhibits N-type, P/Q-type, and unidentified presynaptic VDCCs. The inhibition of each of these calcium channel types contributes to the reduction of [Capre]t by Y2 receptors. Activation of adenosine receptors fully occluded the inhibition of presynaptic calcium influx by Y2 receptors but not the inhibition by GABAB receptors, suggesting a convergent action for Y2 and adenosine receptors, probably by coupling to the same G-protein.

High-speed, random-access fluorescence microscopy: I. High-resolution optical recording with voltage-sensitive dyes and ion indicators.

The design and implementation of a high-speed, random-access, laser-scanning fluorescence microscope configured to record fast physiological signals from small neuronal structures with high spatiotemporal resolution is presented. The laser-scanning capability of this nonimaging microscope is provided by two orthogonal acousto-optic deflectors under computer control. Each scanning point can be randomly accessed and has a positioning time of 3-5 microseconds. Sampling time is also computer-controlled and can be varied to maximize the signal-to-noise ratio. Acquisition rates up to 200k samples/s at 16-bit digitizing resolution are possible. The spatial resolution of this instrument is determined by the minimal spot size at the level of the preparation (i.e., 2-7 microns). Scanning points are selected interactively from a reference image collected with differential interference contrast optics and a video camera. Frame rates up to 5 kHz are easily attainable. Intrinsic variations in laser light intensity and scanning spot brightness are overcome by an on-line signal-processing scheme. Representative records obtained with this instrument by using voltage-sensitive dyes and calcium indicators demonstrate the ability to make fast, high-fidelity measurements of membrane potential and intracellular calcium at high spatial resolution (2 microns) without any temporal averaging.

Presynaptic inhibition of elicited neurotransmitter release.

Activation of presynaptic receptors for a variety of neurotransmitters and neuromodulators inhibits transmitter release at many synapses. Such presynaptic inhibition might serve as a means of adjusting synaptic strength or preventing excessive transmitter release, or both. Previous evidence showed that presynaptic modulators inhibit Ca2+ channels and activate K+ channels at neuronal somata. These modulators also inhibit spontaneous transmitter release by mechanisms downstream of Ca2+ entry. The relative contribution of the above mechanisms to the inhibition of elicited release has been debated for a long time. Recent evidence at synapses where the relationship between transmitter release and presynaptic Ca2+ influx has been well characterized suggests that inhibition of presynaptic voltage-dependent Ca2+ channels plays the major role in presynaptic inhibition of elicited neurotransmitter release. In addition, modulation of the release machinery might contribute to inhibition of elicited release.

Presynaptic calcium dynamics and transmitter release evoked by single action potentials at mammalian central synapses.

The relationship between presynaptic calcium transients ([Ca2+]t) and transmitter release evoked by a single stimulus was both investigated experimentally and modeled at a mammalian central synapse, the CA3 to CA1 pyramidal cell synapse in guinea pig hippocampal slices. In the present study, we compared the low-affinity calcium indicator furaptra with the higher-affinity indicator fura-2. The 10-90% rise time of the furaptra transient was 2.4 ms compared to 7.8 ms with fura-2; the half-decay time (tau 1/2) was 30 ms for furaptra, compared to 238 ms for fura-2. The half-width of the calcium influx was 1.8 ms with furaptra, which provides an upper limit to the duration of the calcium current (ICa) evoked by an action potential. Modeling the decay time course of the furaptra transients led to the conclusion that the predominant endogenous calcium buffer in these terminals must have relatively slow kinetics (kon < 10(5)/M.s), although the presence of small amounts of fast buffers cannot be excluded. The relationship between the [Ca2+]t measured with furaptra and the postsynaptic response was the same as previously observed with fura-2: the postsynaptic response was proportional to about the fourth power (m approximately 4) of the amplitude of either [Ca2+]t or calcium influx. Thus, although fura-2 may be locally saturated by the high local [Ca2+] responsible for transmitter release, the volume-averaged fura-2 signal accurately reflects changes in this local concentration. The result that both indicators gave similar values for the power m constrains the amplitude of calcium influx in our model: Ica < 1 pA for 1 ms.

Simultaneous optical recording of evoked and spontaneous transients of membrane potential and intracellular calcium concentration with high spatio-temporal resolution.

We have developed a system for simultaneous optical recording of transients of membrane potential and intracellular calcium concentration from mammalian brain slice preparations with high spatio-temporal resolution. Simultaneous recording was achieved by using two dedicated photodetectors together with two fluorescent indicators. Specifically, the calcium-sensitive dye Calcium Orange and the voltage-sensitive dye RH-414 were selected because they have overlapping excitation spectra, but separable emission spectra. Transverse guinea pig hippocampal slices were double-loaded by bath application of the membrane-permeant form of Calcium Orange and RH-414. Transients of intracellular calcium concentration and membrane potential associated with evoked neural activity in hippocampal areas CA1 and CA3 were recorded. Furthermore, we have recorded calcium and voltage transients associated with spontaneous epileptiform activity induced by bath application of an epileptogenic drug, 4-aminopyridine. The use of photodiode matrices (10 x 10 elements each) as detectors gives the high spatial (200 x 200 microns/element with a 10 x objective) and temporal resolution (570 microseconds/frame). The recording system also includes a CCD camera for obtaining images of the preparation and overlaying the image with the optically detected signals. A software package has been developed for setting up the experimental protocol(s) and for collecting, processing, displaying, and analyzing the data in an user-friendly, windows-based environment.

GABAB receptor-mediated presynaptic inhibition in guinea-pig hippocampus is caused by reduction of presynaptic Ca2+ influx.

1. The hypothesis that activation of GABAB receptors inhibits evoked synaptic transmission by reducing the presynaptic Ca2+ influx was tested using a recently developed technique for simultaneously recording the presynaptic Ca2+ transient ([Ca2+]t) and the field excitatory postsynaptic potential (fEPSP) evoked by a single electrical stimulus at CA3 to CA1 synapses of guinea-pig hippocampus. 2. The GABAB receptor agonist baclofen reversibly blocked, in a dose-dependant manner, both the fEPSP and the presynaptic [Ca2+]t with similar time courses. During application of baclofen, the fEPSP was proportional to about the fourth power of the presynaptic [Ca2+]t, and the presynaptic fibre volley and the resting Ca2+ level did not change. These results are similar to those we previously observed following application of several voltage-dependent Ca2+ channel blockers, suggesting that baclofen inhibits the fEPSP by blocking the presynaptic Ca2+ influx. 3. The inhibition by baclofen of both the fEPSP and the presynaptic [Ca2+]t was blocked by the GABAB receptor antagonist CGP 35348, consistent with the causal relationship between the GABAB receptor-mediated presynaptic inhibition of the [Ca2+]t and the fEPSP. 4. The inhibition by baclofen of the [Ca2+]t was partially occluded by application of the voltage-dependent Ca2+ channel blocker omega-conotoxin-GVIA (omega-CgTX-GVIA), but not omega-agatoxin-IVA (omega-AgaTX-IVA), suggesting that baclofen reduces the presynaptic [Ca2+]t by blocking Ca2+ channels including the omega-CgTX-GVIA-sensitive type. 5. We conclude that baclofen inhibits evoked transmitter release by reducing presynaptic Ca2+ influx.(ABSTRACT TRUNCATED AT 250 WORDS)

Block of multiple presynaptic calcium channel types by omega-conotoxin-MVIIC at hippocampal CA3 to CA1 synapses.

1. The effect of the voltage-dependent Ca channel (VDCC) antagonist omega-conotoxin-MVIIC (omega-CTx-MVIIC) on the presynaptic Ca influx and synaptic transmission was studied in area CA1 of guinea pig hippocampus. The presynaptic Ca transient ([Ca]t) and the field excitatory postsynaptic potential (fEPSP) evoked by a single electrical stimulus were simultaneously recorded at CA3 to CA1 synapses. 2. omega-CTx-MVIIC dose dependently blocked the fEPSP and the presynaptic [Ca]t without affecting the presynaptic fiber volley and the presynaptic resting Ca level. During application of omega-CTx-MVIIC, the decrease of both the fEPSP and the presynaptic [Ca]t had a similar time course, and the initial slope of the fEPSP is proportional to about the fourth power of the amplitude of the presynaptic [Ca]t. These results strongly suggest that omega-CTx-MVIIC inhibits the fEPSP by blocking presynaptic Ca channels at hippocampal CA3 to CA1 synapses. 3. Sequential application of high concentrations of omega-CTx-MVIIC (10 microM) and other VDCC blockers including omega-conotoxin-GVIA (omega-CTx-GVIA, 1 microM) and omega-agatoxin-IVA (omega-Aga-IVA, 1 microM) showed that omega-CTx-MVIIC significantly occludes the effects of omega-CTx-GVIA and omega-Aga-IVA. Combined application of omega-CTx-GVIA (1 microM) and omega-Aga-IVA (1 microM) largely but not completely occluded the effect of omega-CTx-MVIIC.(ABSTRACT TRUNCATED AT 250 WORDS)