130 research outputs found

    Dendritic spike induction of postsynaptic cerebellar LTP

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    The architecture of parallel fiber (PF) axons contacting cerebellar Purkinje neurons (PNs) retains spatial information over long distances. PF synapses can trigger local dendritic calcium spikes, but whether and how this calcium signal leads to plastic changes that decode the PF input organization is unknown. By combining voltage and calcium imaging, we show that PF-elicited calcium signals, mediated by voltage-gated calcium channels, increase non-linearly during high-frequency bursts of electrically constant calcium spikes because they locally and transiently saturate the endogenous buffer. We demonstrate that these non-linear calcium signals, independently of NMDA or metabotropic glutamate receptor activation, can induce PF long-term potentiation (LTP). Two-photon imaging in coronal slices revealed that calcium signals inducing LTP can be observed by stimulating either the PF or the ascending fiber pathway. We propose that local dendritic calcium spikes, evoked by synaptic potentials, provide a unique mechanism to spatially decode PF signals into cerebellar circuitry changes

    On the Induction of Postsynaptic Granule Cell-Purkinje Neuron LTP and LTD

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    In the last decade, several experimental studies have demonstrated that particular patterns of synaptic activity can induce postsynaptic parallel fiber (PF) long-term potentiation (LTP). This form of plasticity can reverse postsynaptic PF long-term depression (LTD), which has been traditionally considered as the principal form of plasticity underlying cerebellar learning. Postsynaptic PF-LTP requires a transient increase in intracellular Ca2+ concentration and, in contrast to PF-LTD, is induced without concomitant climbing fiber (CF) activation. Thus, it has been postulated that the polarity of long-term synaptic plasticity is determined by the amplitude of the Ca2+ transient during the induction protocol, with PF-LTP induced by smaller Ca2+ signals without concomitant CF activation. However, this hypothesis is contradicted by recent studies. A quantitative analysis of Ca2+ signals associated with induction of PF-LTP indicates that the bidirectional induction of long-term plasticity is regulated by more complex mechanisms. Here we review the state-of-the-art of research on postsynaptic PF-LTP and PF-LTD and discuss the principal open questions on this topi

    Combining Voltage and Calcium Imaging from Neuronal Dendrites

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    The ability to monitor membrane potential (V m) and calcium (Ca2+) transients at multiple locations on the same neuron can facilitate further progress in our understanding of neuronal function. Here we describe a method to combine V m and Ca2+ imaging using styryl voltage sensitive dyes and Fura type UV-excitable Ca2+ indicators. In all cases V m optical signals are linear with membrane potential changes, but the calibration of optical signals on an absolute scale is presently possible only in some neurons. The interpretation of Ca2+ optical signals depends on the indicator Ca2+ buffering capacity relative to the cell endogenous buffering capacity. In hippocampal CA1 pyramidal neurons, loaded with JPW-3028 and 300ÎĽM Bis-Fura-2, V m optical signals cannot be calibrated and the physiological Ca2+ dynamics are compromised by the presence of the indicator. Nevertheless, at each individual site, relative changes in V m and Ca2+ fluorescence signals under different conditions can provide meaningful new information on local dendritic integration. In cerebellar Purkinje neurons, loaded with JPW-1114 and 1mM Fura-FF, V m optical signals can be calibrated in terms of mV and Ca2+ optical signals quantitatively reveal the physiological changes in free Ca2+. Using these two examples, the method is explained in detai

    Using simultaneous voltage and calcium imaging to study fast Ca 2+ channels

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    International audienceThe combination of fluorescence measurements of membrane potential and intracellular Ca2+ concentration allows correlating the electrical and calcium activity of a cell with spatial precision. The technical advances allowing this type of measurement were achieved only recently and represent an important step in the progress of the voltage imaging approach pioneered over 40 years ago by Lawrence B. Cohen. Here, we show how this approach can be used to investigate the function of Ca2+ channels using the foreseen possibility to extract Ca2+ currents from imaging experiments. The kinetics of the Ca2+ current, mediated by voltage-gated Ca2+ channels, can be accurately derived from the Ca2+ fluorescence measurement using Ca2+ indicators with KD>10  μM that equilibrate in <1  ms. In this respect, the imaging apparatus dedicated to this application is described in detail. Next, we illustrate the mathematical procedure to extract the current from the Ca2+ fluorescence change, including a method to calibrate the signal to charge flux density. Finally, we show an example of simultaneous membrane potential and Ca2+ optical measurement associated with an action potential at a CA1 hippocampal pyramidal neuron from a mouse brain slice. The advantages and limitations of this approach are discussed

    Ca2+ signaling by T-type Ca2+ channels in neurons

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    Among the major families of voltage-gated Ca2+ channels, the low-voltage-activated channels formed by the Cav3 subunits, referred to as T-type Ca2+ channels, have recently gained increased interest in terms of the intracellular Ca2+ signals generated upon their activation. Here, we provide an overview of recent reports documenting that T-type Ca2+ channels act as an important Ca2+ source in a wide range of neuronal cell types. The work is focused on T-type Ca2+ channels in neurons, but refers to non-neuronal cells in cases where exemplary functions for Ca2+ entering through T-type Ca2+ channels have been described. Notably, Ca2+ influx through T-type Ca2+ channels is the predominant Ca2+ source in several neuronal cell types and carries out specific signaling roles. We also emphasize that Ca2+ signaling through T-type Ca2+ channels occurs often in select subcellular compartments, is mediated through strategically co-localized targets, and is exploited for unique physiological function

    Improved time-resolved measurements of inorganic ions in particulate matter by PILS-IC integrated with a sample pre-concentration system

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    A particle-into-liquid sampler coupled with ion chromatograph (PILS-IC) for the on-line measurement of inorganic ions has been modified by the insertion of two ion-exchange pre-concentration cartridges that enrich the sample during the period of the IC analysis. The limits of detection of the modified instrument were 10-15 times lower and the time coverage 24 times higher (from 2 to 48 min per hour) than those of the original PILS-IC setup. The instrumental performance in terms of recovery and break-through volume from the cartridges was satisfactory. The modified PILS-IC was operated in comparison with a diffusion denuder line and with a high-resolution time-of-flight aerosol mass spectrometer (HR-TOF-AMS) during a short intensive measurement period organized in the framework of the European Monitoring and Evaluation Programme (EMEP), a co-operative program for monitoring and evaluation of the long-range transmission of the air pollutants in Europe. The instrument showed a quantitative response in agreement with the results of the diffusion lines, and an ability to trace fine concentration variations not so different from the performance of the much more complex HR-TOF-AMS. From the time patterns of the ion concentrations measured by the modified PILS-IC, it was possible to obtain useful information about the variations in the air quality and in the strength of the particulate matter sourc

    Kinetics and functional consequences of BK channels activation by N-type Ca2+ channels in the dendrite of mouse neocortical layer-5 pyramidal neurons

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    The back-propagation of an action potential (AP) from the axon/soma to the dendrites plays a central role in dendritic integration. This process involves an intricate orchestration of various ion channels, but a comprehensive understanding of the contribution of each channel type remains elusive. In this study, we leverage ultrafast membrane potential recordings (Vm) and Ca2+ imaging techniques to shed light on the involvement of N-type voltage-gated Ca2+ channels (VGCCs) in layer-5 neocortical pyramidal neurons’ apical dendrites. We found a selective interaction between N-type VGCCs and large-conductance Ca2+-activated K+ channels (BK CAKCs). Remarkably, we observe that BK CAKCs are activated within a mere 500 μs after the AP peak, preceding the peak of the Ca2+ current triggered by the AP. Consequently, when N-type VGCCs are inhibited, the early broadening of the AP shape amplifies the activity of other VGCCs, leading to an augmented total Ca2+ influx. A NEURON model, constructed to replicate and support these experimental results, reveals the critical coupling between N-type and BK channels. This study not only redefines the conventional role of N-type VGCCs as primarily involved in presynaptic neurotransmitter release but also establishes their distinct and essential function as activators of BK CAKCs in neuronal dendrites. Furthermore, our results provide original functional validation of a physical interaction between Ca2+ and K+ channels, elucidated through ultrafast kinetic reconstruction. This insight enhances our understanding of the intricate mechanisms governing neuronal signaling and may have far-reaching implications in the field

    Combining Membrane Potential Imaging with l-Glutamate or GABA Photorelease

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    Combining membrane potential imaging using voltage sensitive dyes with photolysis of l-glutamate or GABA allows the monitoring of electrical activity elicited by the neurotransmitter at different sub-cellular sites. Here we describe a simple system and some basic experimental protocols to achieve these measurements. We show how to apply the neurotransmitter and how to vary the dimension of the area of photolysis. We assess the localisation of photolysis and of the recorded membrane potential changes by depolarising the dendrites of cerebellar Purkinje neurons with l-glutamate photorelease using different experimental protocols. We further show in the apical dendrites of CA1 hippocampal pyramidal neurons how l-glutamate photorelease can be used to calibrate fluorescence changes from voltage sensitive dyes in terms of membrane potential changes (in mV) and how GABA photorelease can be used to investigate the phenomenon of shunting inhibition. We also show how GABA photorelease can be used to measure chloride-mediated changes of membrane potential under physiological conditions originating from different regions of a neuron, providing important information on the local intracellular chloride concentrations. The method and the proof of principle reported here open the gateway to a variety of important applications where the advantages of this approach are necessary
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