Development of Ca 2+ hotspots between Lymnaea neurons during synaptogenesis (original) (raw)
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The Journal of Neuroscience, 1998
Whole-cell recordings and Ca 2ϩ flux measurements were made at a giant calyx-type synapse in rat brainstem slices to determine the contribution of glutamate receptor (GluR) channels and voltage-dependent Ca 2ϩ channels (VDCCs) to postsynaptic Ca 2ϩ influx during synaptic transmission. A single presynaptic action potential (AP) evoked an EPSP, followed by a single AP. The EPSP-AP sequence caused a postsynaptic Ca 2ϩ influx of ϳ3.0 pC, primarily through VDCCs (ϳ70%) and NMDA-type (up to 30%) channels but also through AMPA-type (Ͻ5%) GluR channels. At Ϫ80 mV, the fractional Ca 2ϩ current (P f) mediated by AMPA receptor (AMPAR) and NMDA receptor (NMDAR) channels was 1.3 and 11-12%, respectively. Simulations of the time course of Ca 2ϩ influx through GluR channels suggested that the small contribution of AMPAR channels occurred only during the first few milliseconds of an EPSP, whereas influx through NMDAR channels dominated later. The NMDARmediated Ca 2ϩ influx was localized in regions covered by the presynaptic terminal, whereas the Ca 2ϩ influx mediated by VDCCs was more homogeneously distributed. Because of the temporal and spatial differences, calcium ions entering through the three different pathways are likely to activate different intracellular targets in the postsynaptic cell. Key words: action potential; fura-2; fractional Ca 2ϩ current; postsynaptic Ca 2ϩ influx; medial nucleus of the trapezoid body; calyx of Held; glutamate receptors; Ca 2ϩ channels; Ca 2ϩ imaging
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1998
Whole-cell recordings and Ca2+ flux measurements were made at a giant calyx-type synapse in rat brainstem slices to determine the contribution of glutamate receptor (GluR) channels and voltage-dependent Ca2+ channels (VDCCs) to postsynaptic Ca2+ influx during synaptic transmission. A single presynaptic action potential (AP) evoked an EPSP, followed by a single AP. The EPSP-AP sequence caused a postsynaptic Ca2+ influx of approximately 3.0 pC, primarily through VDCCs ( approximately 70%) and NMDA-type (up to 30%) channels but also through AMPA-type (<5%) GluR channels. At -80 mV, the fractional Ca2+ current (Pf) mediated by AMPA receptor (AMPAR) and NMDA receptor (NMDAR) channels was 1.3 and 11-12%, respectively. Simulations of the time course of Ca2+ influx through GluR channels suggested that the small contribution of AMPAR channels occurred only during the first few milliseconds of an EPSP, whereas influx through NMDAR channels dominated later. The NMDAR-mediated Ca2+ influx wa...
Synaptic Transmission in Pair Recordings From CA3 Pyramidal Cells in Organotypic Culture
Journal of Neurophysiology, 1999
We performed simultaneous whole cell recordings from pairs of monosynaptically coupled hippocampal CA3 pyramidal neurons in organotypic slices. Stimulation of an action potential in a presynaptic cell resulted in an AMPA-receptor-mediated excitatory postsynaptic current (EPSC) in the postsynaptic cell that averaged ϳ34 pA. The average size of EPSCs varied in amplitude over a 20-fold range across different pairs. Both paired-pulse facilitation and depression were observed in the synaptic current in response to two presynaptic action potentials delivered 50 ms apart, but the average usually was dominated by depression. In addition, the amplitude of the second EPSC depended on the amplitude of the first EPSC, indicating competition between successive events for a common resource that is not restored within the 50-ms interpulse interval. Variation in the synaptic strength among pairs could arise from a variety of sources. Our data from anatomic reconstruction, 1/CV 2 analysis, paired-pulse analysis, and manipulations of calcium/magnesium ratio suggest that differences in quantal size and release probability do not appear to vary sufficiently to fully account for the observed differences in amplitude. Thus it seems most likely that the variability in EPSC amplitude between pairs arises primarily from differences in the number of functional synapses. Injections of the calcium chelator bis-(o-aminophenoxy)-N,N,NЈ,NЈ-tetraacetic acid into the presynaptic neuron resulted in a rapid and nearly complete block of transmission, whereas injection of the slower-acting chelator EGTA resulted in a variable and partial block. In addition to demonstrating the feasibility of manipulating the intracellular presynaptic environment by injection into the presynaptic soma, these data, and the EGTA results in particular may suggest variability in the linkage between calcium entry sites an release sites in these synapses.
Imaging calcium entry sites and ribbon structures in two presynaptic cells
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2003
We investigated the location of calcium entry sites and synaptic ribbons in the type-Mb goldfish bipolar neuron and the bullfrog saccular hair cell. Cells were loaded with a fast calcium indicator (Fluo-3 or Fluo-5F) and an excess of a high-affinity but slow Ca buffer (EGTA). The cell surface was imaged by evanescent field microscopy. Small fluorescent "hot spots" representing calcium entry sites appeared abruptly when a voltage step opened Ca channels and disappeared or dimmed abruptly when Ca channels closed. In bipolar cells, the fluorescence of hot spots tracked the calcium influx. Hair cells showed similar Ca hot spots. Synaptic ribbons or dense bodies were labeled by immunofluorescence with an antibody that recognizes the ribbon protein ribeye. The antibody labeled punctate structures beneath the plasma membrane. In both bipolar neurons and hair cells, the number of Ca entry sites was similar or identical to that of ribbons or dense bodies, consistent with the idea t...
Cerebral Cortex, 2005
To study the type of presynaptic calcium channels controlling transmitter release at synaptic connections displaying depression or facilitation, dual whole cell recordings combined with biocytin labelling were performed in acute slices from motor cortex of 17-to 22-day-old rats. Layer V postsynaptic interneurons displayed either fast spiking (FS) (n 5 12) or burst firing (BF) (n 5 12) behaviour. The axons of FS cells ramified preferentially around pyramidal cell somata, while BF cell axons ramified predominately around pyramidal cell dendrites. Synapses between pyramidal cells and FS cells displayed brief train depression (n 5 12). Bath application of v-Agatoxin IVA (0.5 mM), blocking P/Q-type calcium channels, decreased mean peak amplitudes of the EPSPs to 40% of control EPSPs (n 5 8). Failure rate of the EPSPs after the first presynaptic action potential increased from 9 6 11 to 28 6 15%. This was associated with an increase in paired pulse ratio of 152 6 44%. v-Conotoxin GVIA (1-10 mM), selectively blocking N-type calcium channels, had no effect on peak amplitudes or frequency dependent properties of these connections (n 5 5). Synapses from pyramidal cells to BF cells displayed brief train facilitation (n 5 8). Application of v-Conotoxin in these connections decreased peak amplitudes of the EPSPs to 15% of control EPSPs (n 5 6) and decreased the paired pulse ratio by 41 6 30%. v-Agatoxin did not have any significant effect on the EPSPs elicited in BF cells. This study indicates that P/Q-type calcium channels are associated with transmitter release at connections displaying synaptic depression, whereas N-type channels are predominantly associated with connections displaying facilitation.
Drosophila and originates two main products, DLGA and DLGS97 which differ by the presence of an L27 domain. Combining electrophysiology, immunostaining and genetic manipulation at the pre and postsynaptic compartments we study the DLG contribution to the basal synaptic-function at the Drosophila larval neuromuscular junction. Our results reveal a specific function of DLGS97 in the regulation of the size of GLUR fields and their subunit composition. Strikingly the absence of any of DLG proteins at the presynaptic terminal disrupts the clustering and localization of the calcium channel DmCa1A subunit (Cacophony), decreases the action potential-evoked release probability and alters short-term plasticity. Our results show for the first time a crucial role of DLG proteins in the presynaptic function in vivo.
Imaging and Analysis of Presynaptic Calcium Influx in Cultured Neurons Using synGCaMP6f
Frontiers in Synaptic Neuroscience
Presynaptic Ca 2+ influx through voltage-gated calcium channels (VGCCs) is a key step in synaptic transmission that links action potential (AP)-derived depolarization to vesicle release. However, investigation of presynaptic Ca 2+ influx by patch clamp recordings is difficult due to the small size of the majority of synaptic boutons along thin axons that hamper clamp control. Genetically encoded calcium indicators (GECIs) in combination with live cell imaging provide an alternative method to study Ca 2+ transients in individual presynaptic terminals. The indicator GCaMP6f was developed for fast speed and high sensitivity in detecting Ca 2+ transients even in subcellular compartments. We fused GCaMP6f to synaptophysin (synGCaMP6f) to enrich the calcium indicator in presynaptic boutons of transfected primary hippocampal neurons to study presynaptic Ca 2+ changes in response to individual APs or short bursts. Changes in fluorescence intensity were evaluated by normalization to control level or, alternatively, by normalization to maximal fluorescence using the calcium ionophore ionomycin. Measurements revealed robust Ca 2+ transients with amplitudes that depend on parameters like the number of APs, stimulation frequency or external calcium concentration. Our findings indicate an appropriate sensitivity of synGCaMP6f for studying total presynaptic Ca 2+ transients induced by single APs or short bursts that showed little rundown of the response after repeated bursts. Moreover, these recordings are fast enough to even study short-term plasticity like paired pulse facilitation (PPF) and frequency dependence of Ca 2+ transients. In addition, synGCaMP6f could be used to dissect the contribution of different subtypes of VGCCs to presynaptic Ca 2+ influx. Our results demonstrate that synGCaMP6f allows the reliable analysis of changes in presynaptic calcium concentration at many individual synaptic boutons in parallel and provides the possibility to study the regulation of this important step in synaptic transmission.
The concept of calcium concentration microdomains in synaptic transmission
Neuropharmacology, 1995
Ever since the initial measurements of presynaptic calcium currents it has been evident that calcium triggers transmitter release quite rapidly. Several models indicate, as did the initial voltage clamp measurements, that the calcium concentration triggering such release could be very high at the entry site and that this concentration should be very short lasting. In order to determine this time course, calcium entry was studied at the squid giant synapse by imaging light emission from n-aequorin-J, intracellularly injected into the presynaptic terminal. The imaging utilized a video system capable of acquiring 4000 frames per sec. The results indicate that the calcium entry, triggered by action potentials, reaches a peak within 200 psec and has an overall duration of close to 800 psec, closely matching the duration of the presynaptic calcium current determined by voltage clamp results under similar conditions. Keyword&quid giant synapse, n-aequorin-J, light emission, imaging.