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

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.

Analysis of the effect of the scorpion toxin AaH-II on action potential generation in the axon initial segment.

The toxin AaH-II, from the scorpion Androctonus australis Hector venom, is a 64 amino acid peptide that targets voltage-gated Na+ channels (VGNCs) and slows their inactivation. While at macroscopic cellular level AaH-II prolongs the action potential (AP), a functional analysis of the effect of the toxin in the axon initial segment (AIS), where VGNCs are highly expressed, was never performed so far. Here, we report an original analysis of the effect of AaH-II on the AP generation in the AIS of neocortical layer-5 pyramidal neurons from mouse brain slices. After determining that AaH-II does not discriminate between Nav1.2 and Nav1.6, i.e. between the two VGNC isoforms expressed in this neuron, we established that 7 nM was the smallest toxin concentration producing a minimal detectable deformation of the somatic AP after local delivery of the toxin. Using membrane potential imaging, we found that, at this minimal concentration, AaH-II substantially widened the AP in the AIS. Using ultrafast Na+ imaging, we found that local application of 7 nM AaH-II caused a large increase in the slower component of the Na+ influx in the AIS. Finally, using ultrafast Ca2+ imaging, we observed that 7 nM AaH-II produces a spurious slow Ca2+ influx via Ca2+-permeable VGNCs. Molecules targeting VGNCs, including peptides, are proposed as potential therapeutic tools. Thus, the present analysis in the AIS can be considered a general proof-of-principle on how high-resolution imaging techniques can disclose drug effects that cannot be observed when tested at the macroscopic level.

Spatial and temporal aspects of neuronal calcium and sodium signals measured with low-affinity fluorescent indicators.

Low-affinity fluorescent indicators for Ca2+ or Na+ allow measuring the dynamics of intracellular concentration of these ions with little perturbation from physiological conditions because they are weak buffers. When using synthetic indicators, which are small molecules with fast kinetics, it is also possible to extract spatial and temporal information on the sources of ion transients, their localization, and their disposition. This review examines these important aspects from the biophysical point of view, and how they have been recently exploited in neurophysiological studies. We first analyze the environment where Ca2+ and Na+ indicators are inserted, highlighting the interpretation of the two different signals. Then, we address the information that can be obtained by analyzing the rising phase and the falling phase of the Ca2+ and Na+ transients evoked by different stimuli, focusing on the kinetics of ionic currents and on the spatial interpretation of these measurements, especially on events in axons and dendritic spines. Finally, we suggest how Ca2+ or Na+ imaging using low-affinity synthetic fluorescent indicators can be exploited in future fundamental or applied research.

Nav1.2 and BK channel interaction shapes the action potential in the axon initial segment.

In neocortical layer-5 pyramidal neurons, the action potential (AP) is generated in the axon initial segment (AIS) when the membrane potential (Vm ) reaches the threshold for activation of the voltage-gated Na+ channels (VGNCs) Nav 1.2 and Nav 1.6. Yet, whereas these VGNCs are known to differ in spatial distribution along the AIS and in biophysical properties, our understanding of the functional differences between the two channels remains elusive. Here, using ultrafast Na+ , Vm and Ca2+ imaging in combination with partial block of Nav 1.2 by the peptide G1 G4 -huwentoxin-IV, we demonstrate an exclusive role of Nav 1.2 in shaping the generating AP. Precisely, we show that selective block of ∼30% of Nav 1.2 widens the AP in the distal part of the AIS and we demonstrate that this effect is due to a loss of activation of BK Ca2+ -activated K+ channels (CAKCs). Indeed, Ca2+ influx via Nav 1.2 activates BK CAKCs, determining the amplitude and the early phase of repolarization of the AP in the AIS. By using control experiments using 4,9-anhydrotetrodotoxin, a moderately selective inhibitor of Nav 1.6, we concluded that the Ca2+ influx shaping the early phase of the AP is exclusive of Nav 1.2. Hence, we mimicked this result with a neuron model in which the role of the different ion channels tested reproduced the experimental evidence. The exclusive role of Nav 1.2 reported here is important for understanding the physiology and pathology of neuronal excitability. KEY POINTS: We optically analysed the action potential generated in the axon initial segment of mouse layer-5 neocortical pyramidal neurons and its associated Na+ and Ca2+ currents using ultrafast imaging techniques. We found that partial selective block of the voltage-gated Na+ channel Nav 1.2, produced by a recently developed peptide, widens the shape of the action potential in the distal part of the axon initial segment. We demonstrate that this effect is due to a reduction of the Ca2+ influx through Nav 1.2 that activates BK Ca2+ -activated K+ channels. To validate our conclusions, we generated a neuron model that reproduces the ensemble of our experimental results. The present results indicate a specific role of Nav 1.2 in the axon initial segment for shaping of the action potential during its generation.

In vivo spatiotemporal control of voltage-gated ion channels by using photoactivatable peptidic toxins.

Photoactivatable drugs targeting ligand-gated ion channels open up new opportunities for light-guided therapeutic interventions. Photoactivable toxins targeting ion channels have the potential to control excitable cell activities with low invasiveness and high spatiotemporal precision. As proof-of-concept, we develop HwTxIV-Nvoc, a UV light-cleavable and photoactivatable peptide that targets voltage-gated sodium (NaV) channels and validate its activity in vitro in HEK293 cells, ex vivo in brain slices and in vivo on mice neuromuscular junctions. We find that HwTxIV-Nvoc enables precise spatiotemporal control of neuronal NaV channel function under all conditions tested. By creating multiple photoactivatable toxins, we demonstrate the broad applicability of this toxin-photoactivation technology.

Ultrafast Sodium Imaging of the Axon Initial Segment of Neurons in Mouse Brain Slices.

Monitoring Na+ influx in the axon initial segment (AIS) at high spatial and temporal resolution is fundamental to understanding the generation of an action potential (AP). Here, we present protocols to obtain this measurement, focusing on the AIS of layer 5 (L5) somatosensory cortex pyramidal neurons in mouse brain slices. We first outline how to prepare slices for this application, how to select and patch neurons, and how to optimize the image acquisition. Specifically, we describe the preparation of optimal slices, patching and loading of L5 pyramidal neurons with the Na+ indicator ING-2, and Na+ imaging at 100 µs temporal resolution with a pixel resolution of half a micron. Then, we present a data analysis strategy in order to extract information on the kinetics of activated voltage-gated Na+ channels by determining the change in Na+ by compensating for bleaching and calculating the time derivative of the resulting fit. In sum, this approach can be widely applied when investigating the function of Na+ channels during initiation of an AP and propagation under physiological or pathological conditions in neuronal subtypes. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of cortical slices Basic Protocol 2: Selection, patching, and Na+ fluorescence recording of a neuron Support Protocol: Calibrating Na+ fluorescence Basic Protocol 3: Data analysis.

Cal-520FF is the Present Optimal Ca2+ Indicator for Ultrafast Ca2+ Imaging and Optical Measurement of Ca2+ Currents.

Ultrafast Ca2+ imaging using low-affinity fluorescent indicators allows the precise measurement of the kinetics of fast Ca2+ currents mediated by voltage-gated Ca2+ channels. Thus far, only a few indicators provided fluorescence transients with sufficient signal-to-noise ratio necessary to achieve this measurement, with Oregon Green BAPTA-5N exhibiting the best performance. Here we evaluated the performance of the low-affinity Ca2+ indicator Cal-520FF to record fast Ca2+ signals and to measure the kinetics of Ca2+ currents. Compared to Oregon Green BAPTA-5N and to Fluo4FF, Cal-520FF offers a superior signal-to-noise-ratio providing the optimal characteristics for this important type of biophysical measurement. This ability is the result of a relatively high fluorescence at zero Ca2+, necessary to detect enough photons at short exposure windows, and a high dynamic range leading to large fluorescence transients associated with short Ca2+ influx periods. We conclude that Cal-520FF is at present the optimal commercial low-affinity Ca2+ indicator for ultrafast Ca2+ imaging applications.

Optical measurement of physiological sodium currents in the axon initial segment.

KEY POINTS: Τhe axonal Na+ fluorescence underlying an action potential in the axon initial segment was optically measured at unprecedented temporal resolution. The measurement allowed resolution of the kinetics of the Na+ current at different axonal locations. The distinct components of the Na+ current were correlated with the kinetics of the action potential. NEURON simulations from a modified published model qualitatively predicted the experimentally measured Na+ current. The present method permits the direct investigation of the kinetic behaviour of native Na+ channels under physiological and pathological conditions. ABSTRACT: In most neurons of the mammalian central nervous system, the action potential (AP) is generated in the axon initial segment (AIS) by a fast Na+ current mediated by voltage-gated Na+ channels. While the axonal Na+ signal associated with the AP has been measured using fluorescent Na+ indicators, the insufficient resolution of these recordings has not allowed tracking the Na+ current kinetics underlying this fundamental event. In this article, we report the first optical measurement of Na+ currents in the AIS of pyramidal neurons of layer 5 of the somatosensory cortex from brain slices of the mouse. This measurement was obtained by achieving a temporal resolution of 100 µs in the Na+ imaging technique, with a pixel resolution of 0.5 µm, and by calculating the time-derivative of the Na+ change corrected for longitudinal diffusion. We identified a subthreshold current before the AP, a fast-inactivating current peaking during the rise of the AP and a non-inactivating current during the AP repolarization. We established a correlation between the kinetics of the non-inactivating current at different distances from the soma and the kinetics of the somatic AP. We quantitatively compared the experimentally measured Na+ current with the current obtained by computer simulation of published NEURON models, demonstrating how the present approach can lead to the correct estimate of the native behaviour of Na+ channels. Finally, we discuss how the present approach can be used to investigate the physiological or pathological function of different channel types during AP initiation and propagation.

Is Purkinje Neuron Hyperpolarisation Important for Cerebellar Synaptic Plasticity? A Retrospective and Prospective Analysis.

Two recent studies have demonstrated that the dendritic Ca2+ signal associated with a climbing fibre (CF) input to the cerebellar Purkinje neuron (PN) depends on the membrane potential (Vm). Specifically, when the cell is hyperpolarised, this signal is mediated by T-type voltage-gated Ca2+ channels; in contrast, when the cell is firing, the CF-PN signal is mediated by P/Q-type voltage-gated Ca2+ channels. When the CF input is paired with parallel fibre (PF) activity, the signal is locally amplified at the sites of PF-activated synapses according to the Vm at the time of the CF input, suggesting that the standing Vm is a critical parameter for the induction of PF synaptic plasticity. In this review, I analyse how the Vm can potentially play a role in cerebellar learning focussing, in particular, on the hyperpolarised state that appears to occur episodically, since PNs are mostly firing under physiological conditions. By revisiting the recent literature reporting in vivo recordings and synaptic plasticity studies, I speculate on how a putative role of the PN Vm can provide an interpretation for the results of these studies.

The Origin of Physiological Local mGluR1 Supralinear Ca2+ Signals in Cerebellar Purkinje Neurons.

In mouse cerebellar Purkinje neurons (PNs), the climbing fiber (CF) input provides a signal to parallel fiber (PF) synapses, triggering PF synaptic plasticity. This signal is given by supralinear Ca2+ transients, associated with the CF synaptic potential and colocalized with the PF Ca2+ influx, occurring only when PF activity precedes the CF input. Here, we unravel the biophysical determinants of supralinear Ca2+ signals associated with paired PF-CF synaptic activity. We used membrane potential (Vm) and Ca2+ imaging to investigate the local CF-associated Ca2+ influx following a train of PF synaptic potentials in two cases: (1) when the dendritic Vm is hyperpolarized below the resting Vm, and (2) when the dendritic Vm is at rest. We found that supralinear Ca2+ signals are mediated by type-1 metabotropic glutamate receptors (mGluR1s) when the CF input is delayed by 100-150 ms from the first PF input in both cases. When the dendrite is hyperpolarized only, however, mGluR1s boost neighboring T-type channels, providing a mechanism for local coincident detection of PF-CF activity. The resulting Ca2+ elevation is locally amplified by saturation of endogenous Ca2+ buffers produced by the PF-associated Ca2+ influx via the mGluR1-mediated nonselective cation conductance. In contrast, when the dendritic Vm is at rest, mGluR1s increase dendritic excitability by inactivating A-type K+ channels, but this phenomenon is not restricted to the activated PF synapses. Thus, Vm is likely a crucial parameter in determining PF synaptic plasticity, and the occurrence of hyperpolarization episodes is expected to play an important role in motor learning.SIGNIFICANCE STATEMENT In Purkinje neurons, parallel fiber synaptic plasticity, determined by coincident activation of the climbing fiber input, underlies cerebellar learning. We unravel the biophysical mechanisms allowing the CF input to produce a local Ca2+ signal exclusively at the sites of activated parallel fibers. We show that when the membrane potential is hyperpolarized with respect to the resting membrane potential, type-1 metabotropic glutamate receptors locally enhance Ca2+ influx mediated by T-type Ca2+ channels, and that this signal is amplified by saturation of endogenous buffer also mediated by the same receptors. The combination of these two mechanisms is therefore capable of producing a Ca2+ signal at the activated parallel fiber sites, suggesting a role of Purkinje neuron membrane potential in cerebellar learning.

Imaging Native Calcium Currents in Brain Slices.

Imaging techniques may overcome the limitations of electrode techniques to measure locally not only membrane potential changes, but also ionic currents. Here, we review a recently developed approach to image native neuronal Ca2+ currents from brain slices. The technique is based on combined fluorescence recordings using low-affinity Ca2+ indicators possibly in combination with voltage sensitive dyes. We illustrate how the kinetics of a Ca2+ current can be estimated from the Ca2+ fluorescence change and locally correlated with the change of membrane potential, calibrated on an absolute scale, from the voltage fluorescence change. We show some representative measurements from the dendrites of CA1 hippocampal pyramidal neurons, from olfactory bulb mitral cells and from cerebellar Purkinje neurons. We discuss the striking difference in data analysis and interpretation between Ca2+ current measurements obtained using classical electrode techniques and the physiological currents obtained using this novel approach. Finally, we show how important is the kinetic information on the native Ca2+ current to explore the potential molecular targets of the Ca2+ flux from each individual Ca2+ channel.

Two Distinct Sets of Ca2+ and K+ Channels Are Activated at Different Membrane Potentials by the Climbing Fiber Synaptic Potential in Purkinje Neuron Dendrites.

In cerebellar Purkinje neuron dendrites, the transient depolarization associated with a climbing fiber (CF) EPSP activates voltage-gated Ca2+ channels (VGCCs), voltage-gated K+ channels (VGKCs), and Ca2+-activated SK and BK K+ channels. The resulting membrane potential (Vm) and Ca2+ transients play a fundamental role in dendritic integration and synaptic plasticity of parallel fiber inputs. Here we report a detailed investigation of the kinetics of dendritic Ca2+ and K+ channels activated by CF-EPSPs, based on optical measurements of Vm and Ca2+ transients and on a single-compartment NEURON model reproducing experimental data. We first measured Vm and Ca2+ transients associated with CF-EPSPs at different initial Vm, and we analyzed the changes in the Ca2+ transients produced by the block of each individual VGCCs, of A-type VGKCs and of SK and BK channels. Then, we constructed a model that includes six active ion channels to accurately match experimental signals and extract the physiological kinetics of each channel. We found that two different sets of channels are selectively activated. When the dendrite is hyperpolarized, CF-EPSPs mainly activate T-type VGCCs, SK channels, and A-type VGKCs that limit the transient Vm ∼ <0 mV. In contrast, when the dendrite is depolarized, T-type VGCCs and A-type VGKCs are inactivated and CF-EPSPs activate P/Q-type VGCCs, high-voltage activated VGKCs, and BK channels, leading to Ca2+ spikes. Thus, the potentially activity-dependent regulation of A-type VGKCs, controlling the activation of this second set of channels, is likely to play a crucial role in signal integration and plasticity in Purkinje neuron dendrites.SIGNIFICANCE STATEMENT The climbing fiber synaptic input transiently depolarizes the dendrite of cerebellar Purkinje neurons generating a signal that plays a fundamental role in dendritic integration. This signal is mediated by two types of Ca2+ channels and four types of K+ channels. Thus, understanding the kinetics of all of these channels is crucial for understanding PN function. To obtain this information, we used an innovative strategy that merges ultrafast optical membrane potential and Ca2+ measurements, pharmacological analysis, and computational modeling. We found that, according to the initial membrane potential, the climbing fiber depolarizing transient activates two distinct sets of channels. Moreover, A-type K+ channels limit the activation of P/Q-type Ca2+ channels and associated K+ channels, thus preventing the generation of Ca2+ spikes.

Opto nongenetics inhibition of neuronal firing.

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.

Synthesis by native chemical ligation and characterization of the scorpion toxin AmmTx3.

The scorpion toxin AmmTx3 is a specific blocker of Kv4 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 CSαß 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.

Two-photon probes for in vivo multicolor microscopy of the structure and signals of brain cells.

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-ß 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.

A novel multisite confocal system for rapid Ca2+ imaging from submicron structures in brain slices.

In brain slices, resolving fast Ca2+ 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 Ca2+ transients from submicron structures at 20 to 40 µm below the slice surface, using the low-affinity Ca2+ 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 Ca2+ signals.

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

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.