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Optogenetics is based on the selective expression of exogenous opsins by neurons allowing experimental control of their electrical activity using visible light. The interpretation of the results of optogenetic experiments is based on the assumption that light stimulation selectively acts on those neurons expressing the exogenous opsins without perturbing the activity of naive ones. Here, we report that light stimulation, of wavelengths and power in the range of those normally used in optogenetic experiments, consistently reduces the firing activity of naive Mitral Cells (MCs) and Tufted Neurons in the olfactory bulb as well as in Medium Spiny Neurons (MSNs) in the striatum. No such effect was observed for cerebellar Purkinje and hippocampal CA1 neurons. The effects on MC firing appear to be mainly due to a light-induced increase in tissue temperature, between 0.1 and 0.4°C, associated with the generation of a hyperpolarizing current and a modification of action potential (AP) shape. Therefore, light in the visible range can affect neuronal physiology in a cell-specific manner. Beside the implications for optogenetic studies, our results pave the way to investigating the use of visible light for therapeutic purposes in pathologies associated with neuronal hyperexcitability., (© 2018 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

The scorpion toxin AmmTx3 is a specific blocker of K v 4 channels. It was shown to have interesting potential for neurological disorders. In this study, we report the first chemical synthesis of AmmTx3 by using the native chemical ligation strategy and validate its biological activity. We determined its 3D structure by nuclear magnetic resonance spectroscopy, and pointed out that AmmTx3 possesses the well-known CSαβ structural motif, which is found in a large number of scorpion toxins. Overall, this study establishes an easy synthetic access to biologically active AmmTx3 toxin., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Imaging the brain of living laboratory animals at a microscopic scale can be achieved by two-photon microscopy thanks to the high penetrability and low phototoxicity of the excitation wavelengths used. However, knowledge of the two-photon spectral properties of the myriad fluorescent probes is generally scarce and, for many, non-existent. In addition, the use of different measurement units in published reports further hinders the design of a comprehensive imaging experiment. In this review, we compile and homogenize the two-photon spectral properties of 280 fluorescent probes. We provide practical data, including the wavelengths for optimal two-photon excitation, the peak values of two-photon action cross section or molecular brightness, and the emission ranges. Beyond the spectroscopic description of these fluorophores, we discuss their binding to biological targets. This specificity allows in vivo imaging of cells, their processes, and even organelles and other subcellular structures in the brain. In addition to probes that monitor endogenous cell metabolism, studies of healthy and diseased brain benefit from the specific binding of certain probes to pathology-specific features, ranging from amyloid-β plaques to the autofluorescence of certain antibiotics. A special focus is placed on functional in vivo imaging using two-photon probes that sense specific ions or membrane potential, and that may be combined with optogenetic actuators. Being closely linked to their use, we examine the different routes of intravital delivery of these fluorescent probes according to the target. Finally, we discuss different approaches, strategies, and prerequisites for two-photon multicolor experiments in the brains of living laboratory animals.

In brain slices, resolving fast Ca 2+ fluorescence signals from submicron structures is typically achieved using 2-photon or confocal scanning microscopy, an approach that limits the number of scanned points. The novel multiplexing confocal system presented here overcomes this limitation. This system is based on a fast spinning disk, a multimode diode laser and a novel high-resolution CMOS camera. The spinning disk, running at 20 000 rpm, has custom-designed spiral pattern that maximises light collection, while rejecting out-of-focus fluorescence to resolve signals from small neuronal compartments. Using a 60× objective, the camera permits acquisitions of tens of thousands of pixels at resolutions of ~250 nm per pixel in the kHz range with 14 bits of digital depth. The system can resolve physiological Ca 2+ transients from submicron structures at 20 to 40 μm below the slice surface, using the low-affinity Ca 2+ indicator Oregon Green BAPTA-5N. In particular, signals at 0.25 to 1.25 kHz were resolved in single trials, or through averages of a few recordings, from dendritic spines and small parent dendrites in cerebellar Purkinje neurons. Thanks to an unprecedented combination of temporal and spatial resolution with relatively simple implementation, it is expected that this system will be widely adopted for multisite monitoring of Ca 2+ signals., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor electrical subthreshold events as they travel from synapses on distal dendrites and summate at particular locations to initiate action potentials. It is now possible to carry out these measurements at the scale of individual dendritic spines using voltage imaging. In these measurements, the voltage-sensitive probes can be thought of as transmembrane voltmeters with a linear scale, which directly monitor electrical signals. Grinvald et al. were important early contributors to the methodology of voltage imaging, and they pioneered some of its significant results. We combined voltage imaging and glutamate uncaging using computer-generated holography. The results demonstrated that patterned illumination, by reducing the surface area of illuminated membrane, reduces photodynamic damage. Additionally, region-specific illumination practically eliminated the contamination of optical signals from individual spines by the scattered light from the parent dendrite. Finally, patterned illumination allowed one-photon uncaging of glutamate on multiple spines to be carried out in parallel with voltage imaging from the parent dendrite and neighboring spines.

Background: Fast Ca(2+) imaging using low-affinity fluorescent indicators allows tracking Ca(2+) neuronal influx at high temporal resolution. In some systems, where the Ca(2+)-bound indicator is linear with Ca(2+) entering the cell, the Ca(2+) current has same kinetics of the fluorescence time derivative. In other systems, like cerebellar Purkinje neuron dendrites, the time derivative strategy fails since fluorescence kinetics is affected by Ca(2+) binding proteins sequestering Ca(2+) from the indicator., New Method: Our novel method estimates the kinetics of the Ca(2+) current in cells where the time course of fluorescence is not linear with Ca(2+) influx. The method is based on a two-buffer and two-indicator model, with three free parameters, where Ca(2+) sequestration from the indicator is mimicked by Ca(2+)-binding to the slower buffer. We developed a semi-automatic protocol to optimise the free parameters and the kinetics of the input current to match the experimental fluorescence change with the simulated curve of the Ca(2+)-bound indicator., Results: We show that the optimised input current is a good estimate of the real Ca(2+) current by validating the method both using computer simulations and data from real neurons. We report the first estimates of Ca(2+) currents associated with climbing fibre excitatory postsynaptic potentials in Purkinje neurons., Comparison With Existing Methods: The present method extends the possibility of studying Ca(2+) currents in systems where the existing time derivative approach fails., Conclusions: The information available from our technique allows investigating the physiological behaviour of Ca(2+) channels under all possible conditions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Key Points: In neurons, the Ca(2+) signal associated with the dendritic back-propagating action potential codes a chemical message to the different dendritic sites, playing a crucial role in electrical signalling, synaptic transmission and synaptic plasticity. The study of the underlying Ca(2+) current, mediated by different types of voltage-gated Ca(2+) channels, cannot be achieved by using the patch clamp technique. In this article, we used a recently developed cutting-edge optical technique to investigate the physiological behaviour of local Ca(2+) currents along the apical dendrite of CA1 hippocampal pyramidal neurons. We directly measure, for the first time, the synergistic activation and deactivation of the diverse dendritic voltage-gated Ca(2+) channels operating during bursts of back-propagating action potentials to precisely control the Ca(2+) signal. We demonstrate that the Ca(2+) loss via high-voltage-activated channels is compensated by the Ca(2+) entry via the other channels translating in high fidelity of Ca(2+) signalling., Abstract: In CA1 hippocampal pyramidal neurons, the dendritic Ca(2+) signal associated with somatic firing represents a fundamental activation code for several proteins. This signal, mediated by voltage-gated Ca(2+) channels (VGCCs), varies along the dendrites. In this study, using a recent optical technique based on the low-affinity indicator Oregon Green 488 BAPTA-5N, we analysed how activation and deactivation of VGCCs produced by back-propagating action potentials (bAPs) along the apical dendrite shape the Ca(2+) signal at different locations in CA1 hippocampal pyramidal neurons of the mouse. We measured, at multiple dendritic sites, the Ca(2+) transients and the changes in membrane potential associated with bAPs at 50 μs temporal resolution and we estimated the kinetics of the Ca(2+) current. We found that during somatic bursts, the bAPs decrease in amplitude along the apical dendrite but the amplitude of the associated Ca(2+) signal in the initial 200 μm dendritic segment does not change. Using a detailed pharmacological analysis, we demonstrate that this effect is due to the perfect compensation of the loss of Ca(2+) via high-voltage-activated (HVA) VGCCs by a larger Ca(2+) component via low-voltage-activated (LVA) VGCCs, revealing a mechanism coupling the two VGCC families of K(+) channels. More distally, where the bAP does not activate HVA-VGCCs, the Ca(2+) signal is variable during the burst. Thus, we demonstrate that HVA- and LVA-VGCCs operate synergistically to stabilise Ca(2+) signals associated with bAPs in the most proximal 200 μm dendritic segment., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

The combination of fluorescence measurements of membrane potential and intracellular [Formula: see text] 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 [Formula: see text] channels using the foreseen possibility to extract [Formula: see text] currents from imaging experiments. The kinetics of the [Formula: see text] current, mediated by voltage-gated [Formula: see text] channels, can be accurately derived from the [Formula: see text] fluorescence measurement using [Formula: see text] indicators with [Formula: see text] that equilibrate in [Formula: see text]. 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 [Formula: see text] fluorescence change, including a method to calibrate the signal to charge flux density. Finally, we show an example of simultaneous membrane potential and [Formula: see text] 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.

A central question in neuronal network analysis is how the interaction between individual neurons produces behavior and behavioral modifications. This task depends critically on how exactly signals are integrated by individual nerve cells functioning as complex operational units. Regional electrical properties of branching neuronal processes which determine the input-output function of any neuron are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin (synaptic contacts on distal dendrites) and summate at particular locations to influence action potential initiation. It became possible recently to carry out this type of measurement using high-resolution multisite recording of membrane potential changes with intracellular voltage-sensitive dyes. This chapter reviews the development and foundation of the method of voltage-sensitive dye recording from individual neurons. Presently, this approach allows monitoring membrane potential transients from all parts of the dendritic tree as well as from axon collaterals and individual dendritic spines.

Membrane potential imaging using voltage-sensitive dyes can be combined with other optical techniques for a variety of applications. Combining voltage imaging with Ca2+ imaging allows correlating membrane potential changes with intracellular Ca2+ signals or with Ca2+ currents. Combining voltage imaging with uncaging techniques allows analyzing electrical signals elicited by photorelease of a particular molecule. This approach is also a useful tool to calibrate the change in fluorescence intensity in terms of membrane potential changes from different sites permitting spatial mapping of electrical activity. Finally, combining voltage imaging with optogenetics, in particular with channelrhodopsin stimulation, opens the gate to novel investigations of brain circuitries by allowing measurements of synaptic signals mediated by specific sets of neurons. Here we describe in detail the methods of membrane potential imaging in combination with other optical techniques and discus some important applications.

Information processing in the central nervous system makes use of densely woven networks of neurons with complex dendritic and axonal arborizations. Studying signaling in such a network requires precise control over the activity of specific neurons and an understanding how the synaptic signals are integrated. We established a system using a recently published red-shifted voltage sensitive dye in slices from mice expressing channelrhodopsin (Ch) in GABAergic neurons. Using a focused 473 nm laser for Ch activation and 635 nm laser wide field illumination for voltage sensitive dye excitation we were able to simultaneously measure dendritic voltage transients and stimulate inhibitory synaptic connections. The combination of these techniques provides excellent spatiotemporal control over neuron activation and high resolution information on dendritic signal processing.

The current understanding of Ca(2+) channel function is derived from the use of the patch-clamp technique. In particular, the measurement of fast cellular Ca(2+) currents is routinely achieved using whole-cell voltage-clamp recordings. However, this experimental approach is not applicable to the study of local native Ca(2+) channels during physiological changes of membrane potential in complex cells, since the voltage-clamp configuration constrains the membrane potential to a given value. Here, we report for the first time to our knowledge that Ca(2+) currents from individual cells can be quantitatively measured beyond the limitations of the voltage-clamp approach using fast Ca(2+) imaging with low-affinity indicators. The optical measurement of the Ca(2+) current was correlated with the membrane potential, simultaneously measured with a voltage-sensitive dye to investigate the activation of Ca(2+) channels along the apical dendrite of the CA1 hippocampal pyramidal neuron during the back-propagation of an action potential. To validate the method, we analyzed the voltage dependence of high- and low-voltage-gated Ca(2+) channels. In particular, we measured the Ca(2+) current component mediated by T-type channels, and we investigated the mechanisms of recovery from inactivation of these channels. This method is expected to become a reference approach to investigate Ca(2+) channels in their native physiological environment., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Little is known about how neuron firing recorded in vivo retrogradely influences synaptic strength. We injected the firing of a rat hippocampal neurogliaform cell (NGFC), a widely expressed GABAergic neuron type, detected in vivo during theta rhythm, into NGFCs of rat or neuronal nitric oxide synthase (nNOS)-Cre-tdTomato mouse recorded in vitro. We found that the "in vivo firing pattern" produced a transient firing-induced suppression of synaptic inhibition (FSI) evoked by a presynaptic NGFC. Imaging experiments demonstrate that FSI was associated with action potential backpropagation (bAP) and a supralinear increase in dendritic Ca(2+). The application of the L-type Ca(2+) channel antagonist nimodipine blocked FSI. Further pharmacological experiments, such as the application of a nitric oxide-sensitive guanylyl cyclase (NO-sGC) receptor antagonist, a NOS inhibitor, and NO donors, suggested that NO released from postsynaptic cells mediated FSI and likely activated presynaptic receptors to inhibit GABA release. The in vivo firing pattern modulated the size of unitary EPSPs impinging on NGFCs through FSI and not via a direct effect on excitatory synaptic transmission. Our data demonstrate: (1) retrograde signaling initiated by in vivo firing pattern, (2) interneuron bAPs detected with fast temporal resolution, and (3) a novel role for NO expressed by specific interneuron types.

Ca(2+) imaging is a commonly used approach for measuring Ca(2+) signals at high spatial resolution. The method is often combined with electrode recordings to correlate electrical and chemical signals or to investigate Ca(2+) signals following an electrical stimulation. To obtain information on electrical activity at the same spatial resolution, Ca(2+) imaging must be combined with membrane potential imaging. Similarly, stimulation of subcellular compartments requires photostimulation. Thus, combining Ca(2+) imaging with an additional optical technique facilitates the study of a number of physiological questions. The aim of this article is to introduce some basic principles regarding the combination of Ca(2+) imaging with other optical techniques. We discuss the design of the optics, the design of experimental protocols, the optical characteristics of Ca(2+) indicators used in combination with an optical probe, and the affinity of the Ca(2+) indicator in relation to the type of measurement. This information will enable the reader to devise an optimal strategy for combined optical experiments.

The ability to monitor Ca(2+) signals and membrane potential simultaneously at multiple locations on the same neuron facilitates further progress in our understanding of neuronal function. In particular, this method allows correlation of electrical and chemical signals from multiple sites, including those inaccessible to microelectrodes. This protocol describes a procedure for loading cells with two indicators, a Ca(2+)-sensitive Fura dye and voltage-sensitive JPW1114, together with the equipment required for detecting and imaging the two signals. Potential problems are discussed as well as the capabilities and limitations of the technique.

This article describes simple configurations and methods for measuring optical Ca(2+) signals in response to photorelease of -glutamate. This photostimulation allows activation of postsynaptic glutamate receptors without activation of voltage-gated Ca(2+) channels, thereby permitting the separation and analysis of different Ca(2+) components. We give details of basic microscopy configurations and recommend tools for efficiently illuminating the preparation while preserving the healthy condition of the tissue. We also suggest methodological procedures and discuss linear optics for achieving simultaneous imaging and uncaging using two-photon illumination.

ABSTRACT. In recent years, the use of light emitting diodes (LEDs) has become commonplace in fluorescence microscopy. LEDs are economical and easy to couple to commercial microscopes, and they provide powerful and stable light that can be triggered by transistor-transistor logic pulses in the range of tens of microseconds or shorter. LEDs are usually installed on the epifluorescence port of the microscope to obtain whole-field illumination, which is ideal for fluorescence imaging. In contrast, photolysis or channelrhodopsin stimulation often requires localized illumination, typically achieved using lasers. Here we show that insertion of a long-pass (>411  nm) filter with an appropriately sized pinhole in the epifluorescence pathway, combined with dual UV/visible illumination, can produce efficient whole-field visible illumination and spot UV illumination of 15 to 20 μm. We tested our system by performing calcium imaging experiments combined with L-glutamate or N-methyl-D-aspartic acid (NMDA) photorelease in hippocampal neurons from brain slices or dissociated cultures, demonstrating the ability to obtain local activation of NMDA receptors exclusively in the illuminated spot. The very inexpensive and simple system that we report here will allow many laboratories with limited budgets to run similar experiments in a variety of physiological applications.

Combining voltage and Ca(2+) imaging allows the correlation of electrical and chemical activity at sub-cellular level. Here we describe a novel apparatus designed to obtain simultaneous voltage and Ca(2+) measurements with single-trial resolution from sites as small as a few microns. These measurements can be obtained with negligible optical cross-talk between the two signals and negligible photo-damage of the preparation. The capability of the technique was assessed recording either from individual neurons in brain slices or from networks of cultured neurons. The present achievements open the gate to many novel physiological investigations requiring simultaneous measurement of voltage and Ca(2+) signals.

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.

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 Ca(2+) 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 Ca(2+) transient during the induction protocol, with PF-LTP induced by smaller Ca(2+) signals without concomitant CF activation. However, this hypothesis is contradicted by recent studies. A quantitative analysis of Ca(2+) 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 topic.

Studies of the spatio-temporal distribution of inhibitory postsynaptic potentials (IPSPs) in a neuron have been limited by the spatial information that can be obtained by electrode recordings. We describe a method that overcomes these limitations by imaging IPSPs with voltage-sensitive dyes. CA1 hippocampal pyramidal neurons from brain slices were loaded with the voltage-sensitive dye JPW-1114 from a somatic patch electrode in whole-cell configuration. After removal of the patch electrode, we found that neurons recover their physiological intracellular chloride concentration. Using an improved voltage-imaging technique, dendritic GABAergic IPSPs as small as 1 mV could be resolved optically from multiple sites with spatial averaging. We analyzed the sensitivity of the technique, in relation to its spatial resolution. We monitored the origin and the spread of IPSPs originating in different areas of the apical dendrite and reconstructed their spatial distribution. We achieved a clear discrimination of IPSPs from the dendrites and from the axon. This study indicates that voltage imaging is a uniquely suited approach for the investigation of several fundamental aspects of inhibitory synaptic transmission that require spatial information., (Copyright (c) 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Among the major families of voltage-gated Ca(2+) channels, the low-voltage-activated channels formed by the Ca(v)3 subunits, referred to as T-type Ca(2+) channels, have recently gained increased interest in terms of the intracellular Ca(2+) signals generated upon their activation. Here, we provide an overview of recent reports documenting that T-type Ca(2+) channels act as an important Ca(2+) source in a wide range of neuronal cell types. The work is focused on T-type Ca(2+) channels in neurons, but refers to non-neuronal cells in cases where exemplary functions for Ca(2+) entering through T-type Ca(2+) channels have been described. Notably, Ca(2+) influx through T-type Ca(2+) channels is the predominant Ca(2+) source in several neuronal cell types and carries out specific signaling roles. We also emphasize that Ca(2+) signaling through T-type Ca(2+) channels occurs often in select subcellular compartments, is mediated through strategically co-localized targets, and is exploited for unique physiological functions.

This chapter presents three examples of imaging brain activity with voltage- or calcium-sensitive dyes. Because experimental measurements are limited by low sensitivity, the chapter then discusses the methodological aspects that are critical for optimal signal-to-noise ratio. Two of the examples use wide-field (1-photon) imaging and the third uses two-photon scanning microscopy. These methods have relatively high temporal resolution ranging from 10 to 10,000 Hz. The three examples are the following: (1) Internally injected voltage-sensitive dye can be used to monitor membrane potential in the dendrites of invertebrate and vertebrate neurons in in vitro preparations. These experiments are directed at understanding how individual neurons convert the complex input synaptic activity into the output spike train. (2) Recently developed methods for staining many individual cells in the mammalian brain with calcium-sensitive dyes together with two-photon microscopy made it possible to follow the spike activity of many neurons simultaneously while in vivo preparations are responding to stimulation. (3) Calcium-sensitive dyes that are internalized into olfactory receptor neurons in the nose will, after several days, be transported to the nerve terminals of these cells in the olfactory bulb glomeruli. There, the population signals can be used as a measure of the input from the nose to the bulb. Three kinds of noise in measuring light intensity are discussed: (1) Shot noise from the random emission of photons from the preparation. (2) Extraneous (technical) noise from external sources. (3) Noise that occurs in the absence of light, the dark noise. In addition, we briefly discuss the light sources, the optics, and the detectors and cameras. The commonly used organic voltage and ion sensitive dyes stain all of the cell types in the preparation indiscriminately. A major effort is underway to find methods for staining individual cell types in the brain selectively. Most of these efforts center around fluorescent protein activity sensors because transgenic methods can be used to express them in individual cell types.

The ability to monitor membrane potential (V(m)) and calcium (Ca(2+)) 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 Ca(2+) imaging using styryl voltage sensitive dyes and Fura type UV-excitable Ca(2+) 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 Ca(2+) optical signals depends on the indicator Ca(2+) buffering capacity relative to the cell endogenous buffering capacity. In hippocampal CA1 pyramidal neurons, loaded with JPW-3028 and 300 microM Bis-Fura-2, V(m) optical signals cannot be calibrated and the physiological Ca(2+) dynamics are compromised by the presence of the indicator. Nevertheless, at each individual site, relative changes in V (m) and Ca(2+) fluorescence signals under different conditions can provide meaningful new information on local dendritic integration. In cerebellar Purkinje neurons, loaded with JPW-1114 and 1 mM Fura-FF, V(m) optical signals can be calibrated in terms of mV and Ca(2+) optical signals quantitatively reveal the physiological changes in free Ca(2+). Using these two examples, the method is explained in detail.

T-type Ca2+ channels (T channels) underlie rhythmic burst discharges during neuronal oscillations that are typical during sleep. However, the Ca2+-dependent effectors that are selectively regulated by T currents remain unknown. We found that, in dendrites of nucleus reticularis thalami (nRt), intracellular Ca2+ concentration increases were dominated by Ca2+ influx through T channels and shaped rhythmic bursting via competition between Ca2+-dependent small-conductance (SK)-type K+ channels and Ca2+ uptake pumps. Oscillatory bursting was initiated via selective activation of dendritically located SK2 channels, whereas Ca2+ sequestration by sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) and cumulative T channel inactivation dampened oscillations. Sk2-/- (also known as Kcnn2) mice lacked cellular oscillations, showed a greater than threefold reduction in low-frequency rhythms in the electroencephalogram of non-rapid-eye-movement sleep and had disrupted sleep. Thus, the interplay of T channels, SK2 channels and SERCAs in nRt dendrites comprises a specialized Ca2+ signaling triad to regulate oscillatory dynamics related to sleep.

The architecture of parallel fiber axons contacting cerebellar Purkinje neurons retains spatial information over long distances. Parallel fiber synapses can trigger local dendritic calcium spikes, but whether and how this calcium signal leads to plastic changes that decode the parallel fiber input organization is unknown. By combining voltage and calcium imaging, we show that calcium signals, elicited by parallel fiber stimulation and 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 parallel fiber long-term potentiation. Two-photon imaging in coronal slices revealed that calcium signals inducing long-term potentiation can be observed by stimulating either the parallel fiber or the ascending fiber pathway. We propose that local dendritic calcium spikes, evoked by synaptic potentials, provide a unique mechanism to spatially decode parallel fiber signals into cerebellar circuitry changes.

The non-linear and spatially inhomogeneous interactions of dendritic membrane potential signals that represent the first step in the induction of activity-dependent long-term synaptic plasticity are not fully understood, particularly in dendritic regions which are beyond the reach of electrode measurements. We combined voltage-sensitive-dye recordings and Ca(2+) imaging of hippocampal CA1 pyramidal neurons to study large regions of the dendritic arbor, including branches of small diameter (distal apical and oblique dendrites). Dendritic membrane potential transients were monitored at high spatial resolution and correlated with supra-linear [Ca(2+)](i) changes during one cycle of a repetitive patterned stimulation protocol that typically results in the induction of long-term potentiation (LTP). While the increase in the peak membrane depolarization during coincident pre- and post-synaptic activity was required for the induction of supra-linear [Ca(2+)](i) signals shown to be necessary for LTP, the change in the baseline-to-peak amplitude of the backpropagating dendritic action potential (bAP) was not critical in this process. At different dendritic locations, the baseline-to-peak amplitude of the bAP could be either increased, decreased or unaltered at sites where EPSP-AP pairing evoked supra-linear summation of [Ca(2+)](i) transients. We suggest that modulations in the bAP baseline-to-peak amplitude by local EPSPs act as a mechanism that brings the membrane potential into the optimal range for Ca(2+) influx through NMDA receptors (0 to -15 mV); this may require either boosting or the reduction of the bAP, depending on the initial size of both signals.

Type 1 metabotropic glutamate receptors (mGluR1) in Purkinje neurones (PNs) are important for motor learning and coordination. Here, two divergent mGluR1 Ca2+-signalling pathways and the associated membrane conductances were distinguished kinetically and pharmacologically after activation by 1-ms photorelease of L-glutamate or by bursts of parallel fibre (PF) stimulation. A new, mGluR1-mediated transient K+ conductance was seen prior to the slow EPSC (sEPSC). It was seen only in PNs previously allowed to fire spontaneously or held at depolarized potentials for several seconds and was slowly inhibited by agatoxin IVA, which blocks P/Q-type Ca2+ channels. It peaked in 148 ms, had well-defined kinetics and, unlike the sEPSC, was abolished by the phospholipase C (PLC) inhibitor U73122. It was blocked by the BK Ca2+-activated K+ channel blocker iberiotoxin and unaffected by apamin, indicating selective activation of BK channels by PLC-dependent store-released Ca2+. The K+ conductance and underlying transient Ca2+ release showed a highly reproducible delay of 99.5 ms following PF burst stimulation, with a precision of 1-2 ms in repeated responses of the same PN, and a subsequent fast rise and fall of Ca2+ concentration. Analysis of Ca2+ signals showed that activation of the K+ conductance by Ca2+ release occurred in small dendrites and subresolution structures, most probably spines. The results show that PF burst stimulation activates two pathways of mGluR1 signalling in PNs. First, transient, PLC-dependent Ca2+ release from stores with precisely reproducible timing and second, slower Ca2+ influx in the cation-permeable sEPSC channel. The priming by prior Ca2+ influx in P/Q-type Ca2+ channels may determine the path of mGluR1 signalling. The precise timing of PLC-mediated store release may be important for interactions of PF mGluR1 signalling with other inputs to the PN.

The slow EPSC (sEPSC) of cerebellar parallel fiber --> Purkinje neuron synapses is mediated by metabotropic glutamate receptor type 1 (mGluR1) activation of nonselective cation channels. Here, the channel properties were studied with uniform calibrated photorelease of L-glutamate with ionotropic receptors blocked, allowing isolation of postsynaptic processes, or with parallel fiber stimulation or mGluR1 agonist application. Evoked current and fluorescence from Ca(2+) indicators were recorded. Noise analysis of the mGluR1 current gave a single-channel conductance of 0.6 pS and showed low open probability at maximal mGluR1 activation. Similar small single-channel conductances were obtained with the mGluR1 agonist (S)-dihydroxyphenylglycine, with parallel fiber or climbing fiber stimulation. The mGluR1 current fluctuations were unaffected by potassium channel blockers. Photoreleased L-glutamate triggered a Ca(2+) concentration increase in the distal dendrites with a time course similar to that of the mGluR1 current. The proximal dendritic and somatic Ca(2+) changes were delayed with respect to the current. Ca(2+) channel blockers and the phospholipase Cdelta inhibitor 1-[6-[((17delta)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione, which inhibits mGluR1-activated intracellular Ca(2+) release, did not prevent the dendritic Ca(2+) concentration increase. Polyamine naphthylacetyl spermine and cationic adamantanes that block the pore of the channel were used to vary the mGluR1 current over a wide range in each cell but still at maximal mGluR1 activation. The Ca(2+) influx was inhibited in parallel with the current. The results show that the mGluR1-activated current and the sEPSC are attributable to small-conductance, low-open probability Ca(2+)-permeable cation channels that will mediate spine-specific Ca(2+) influx during the parallel fiber sEPSP.

Since constitutively-high intracellular Ca(2+) ([Ca(2+)](i)) may confer hypoglossal motoneurons special vulnerability to excitoxic damage, we investigated the spatiotemporal dynamics of [Ca(2+)](i) and its relation to spike firing of rat hypoglossal motoneurons recorded under whole-cell patch clamp coupled with high resolution [Ca(2+)](i) imaging. A rise in [Ca(2+)](i), appearing in conjunction with single action potentials and becoming larger during spike trains, was first detected immediately beneath the cell membrane area, peaked 10-20 ms after each spike, and propagated to the cell core with slow decay time. Depletion of ryanodine-sensitive [Ca(2+)](i) stores by caffeine increased background [Ca(2+)](i), augmented the spike medium afterhyperpolarization, slowed down the action potential firing rate and depolarized cells (after an early hyperpolarization). The decay time constant of [Ca(2+)](i) transients was more than doubled by caffeine, although peak [Ca(2+)](i) remained unchanged. It is suggested that the main role of caffeine-sensitive stores was to buffer [Ca(2+)](i) elevated by sustained firing and to control spike accommodation.

The slow EPSP (sEPSP) or slow EPSC (sEPSC) at parallel fiber to Purkinje neuron synapses is attributable to a nonselective cation channel coupled to activation of metabotropic type 1 glutamate receptors (mGluR1s). Photorelease of L-glutamate in 1 msec from 4-methoxy-7-nitroindolinyl-or 7-nitroindolinyl-caged glutamate in cerebellar slices was used to isolate and study postsynaptic mechanisms coupling mGluR1 to the cation channel. L-Glutamate immediately activated a glutamate transporter current, followed by the slow mGluR1-activated conductance. Inhibitors of kinases, phosphatases, and G-proteins were tested on the peak glutamate-evoked currents. No effects of the inhibitors were seen on the initial glutamate transporter currents. In contrast, the later mGluR1 currents were either unaffected or enhanced by the protein tyrosine kinase (PTK) inhibitors PP1, K252a, and staurosporine were diminished or blocked by phosphatase inhibitors but were unaffected by inhibitors of serine-threonine kinases PKA, PKC, or PKG. The selective src-PTK inhibitor PP1 (10 microm intracellularly) potentiated submaximal mGluR1 currents evoked by low L-glutamate concentrations but had no effect on maximal responses (80 or 160 microm L-glutamate). L-Glutamate-evoked mGluR1 currents and parallel fiber sEPSCs were reversibly and completely inhibited by protein tyrosine phosphatase (PTP) inhibitor bpV(phen) (50-200 microm) and by nonselective phosphatase inhibitor orthovanadate (0.5 or 1 mm). mGluR1 currents were completely inhibited by GDPbetaS applied intracellularly (5 mm). The results confirm a role for a GTPase postsynaptically, show that tyrosine phosphorylation inhibits mGluR1 coupling to the channel, and show that PTPs increase activation by tyrosine dephosphorylation most likely upstream of the sEPSP cation channel.