Marco Canepari

T-type Ca2+ channels, SK2 channels and SERCAs gate sleep-related oscillations in thalamic dendrites

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.

Dendritic signals from rat hippocampal CA1 pyramidal neurons during coincident pre‐ and post‐synaptic activity: a combined voltage‐ and calcium‐imaging study

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 Ca2+ 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 [Ca2+]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 [Ca2+]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 [Ca2+]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 Ca2+ 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.

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

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 functions.

Imaging Inhibitory Synaptic Potentials Using Voltage Sensitive Dyes

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.

Dendritic Spike Saturation of Endogenous Calcium Buffer and Induction of Postsynaptic Cerebellar LTP

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.

On the induction of postsynaptic granule cell-Purkinje neuron LTP and LTD.

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 topic.

Combining Membrane Potential Imaging with l-Glutamate or GABA Photorelease

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.

Imaging membrane potential changes from dendritic spines using computer-generated holography

Abstract. 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.

Combined voltage and calcium imaging and signal calibration

Voltage imaging using fluorescent voltage-sensitive dyes can be combined with other optical measurements, in particular with Ca2+ imaging, allowing for correlation of membrane potential changes with intracellular Ca2+ signals. Calibration of fluorescence voltage signals permits the comparison of membrane potential changes from different sites, allowing spatial mapping of membrane potential changes. These two technical aspects enhance the capability of voltage imaging to address several fundamental problems of neurobiology. Here we discuss how to combine voltage imaging with the optical measurement of intracellular Ca2+ transients and different approaches to calibrate voltage-sensitive dye signals on an absolute scale.