Fast Neuronal Calcium Signals in Brain Slices Loaded With Fluo-4 AM Ester.

Staining brain slices with acetoxymethyl ester (AM) Ca dyes is a straightforward procedure to load multiple cells, and Fluo-4 is a commonly used high-affinity indicator due to its very large dynamic range. It has been shown that this dye preferentially stains glial cells, providing slow and large Ca transients, but it is questionable whether and at which temporal resolution it can also report Ca transients from neuronal cells. Here, by electrically stimulating mouse hippocampal slices, we resolved fast neuronal signals corresponding to 1%-3% maximal fluorescence changes.

Neuronal Imaging at 8-Bit Depth to Combine High Spatial and High Temporal Resolution With Acquisition Rates Up To 40 kHz.

A challenge in neuroimaging is acquiring frame sequences at high temporal resolution from the largest possible number of pixels. Measuring 1%-10% fluorescence changes normally requires 12-bit or higher bit depth, constraining the frame size allowing imaging in the kHz range. We resolved Ca or membrane potential signals from cell populations or single neurons in brain slices by acquiring fluorescence at 8-bit depth and by binning pixels offline, achieving unprecedented frame sizes at kHz rates.

Nitric oxide modelling and its bioavailability influenced by red blood cells.

Nitric oxide (NO) is an important vasodilator responsible for maintaining vascular tone in the human body. Its production in endothelial cells (ECs) is regulated by the rise of cytoplasmic Ca concentration and shear stress perceived by blood flow. The increase in cytoplasmic Ca concentration is mainly activated by adenosine triphosphate (ATP) released from red blood cells (RBCs) and ECs.

Physiological Features of the Neural Stem Cells Obtained from an Animal Model of Spinal Muscular Atrophy and Their Response to Antioxidant Curcumin.

The most prevalent rare genetic disease affecting young individuals is spinal muscular atrophy (SMA), which is caused by a loss-of-function mutation in the telomeric gene survival motor neuron () . The high heterogeneity of the SMA pathophysiology is determined by the number of copies of , a separate centromeric gene that can transcribe for the same protein, although it is expressed at a slower rate. SMA affects motor neurons.

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.

Kinetics and functional consequences of BK channels activation by N-type Ca 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 (V) and Ca imaging techniques to shed light on the involvement of N-type voltage-gated Ca channels (VGCCs) in layer-5 neocortical pyramidal neurons' apical dendrites.

Diaphragm Fatigue in SMNΔ7 Mice and Its Molecular Determinants: An Underestimated Issue.

Spinal muscular atrophy (SMA) is a genetic disorder characterized by the loss of spinal motor neurons leading to muscle weakness and respiratory failure. Mitochondrial dysfunctions are found in the skeletal muscle of patients with SMA. For obvious ethical reasons, the diaphragm muscle is poorly studied, notwithstanding the very important role that respiratory involvement plays in SMA mortality.

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

Low-affinity fluorescent indicators for Ca 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.

Structural and functional impairments of skeletal muscle in patients with postacute sequelae of SARS-CoV-2 infection.

Following acute coronavirus disease 2019 (COVID-19), a substantial proportion of patients showed symptoms and sequelae for several months, namely the postacute sequelae of COVID-19 (PASC) syndrome. Major phenomena are exercise intolerance, muscle weakness, and fatigue. We aimed to investigate the physiopathology of exercise intolerance in patients with PASC syndrome by structural and functional analyses of skeletal muscle.

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 (V ) reaches the threshold for activation of the voltage-gated Na channels (VGNCs) Na 1.2 and Na 1.6.

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 (Na) channels and validate its activity in vitro in HEK293 cells, ex vivo in brain slices and in vivo on mice neuromuscular junctions.

Exercise Preconditioning Blunts Early Atrogenes Expression and Atrophy in Gastrocnemius Muscle of Hindlimb Unloaded Mice.

A large set of FoxOs-dependent genes play a primary role in controlling muscle mass during hindlimb unloading. Mitochondrial dysfunction can modulate such a process. We hypothesized that endurance exercise before disuse can protect against disuse-induced muscle atrophy by enhancing peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) expression and preventing mitochondrial dysfunction and energy-sensing AMP-activated protein kinase (AMPK) activation.

Age-related decline in murine heart and skeletal muscle performance is attenuated by reduced Ahnak1 expression.

Background: Aging is associated with a progressive reduction in cellular function leading to poor health and loss of physical performance. Mitochondrial dysfunction is one of the hallmarks of aging; hence, interventions targeting mitochondrial dysfunction have the potential to provide preventive and therapeutic benefits to elderly individuals. Meta-analyses of age-related gene expression profiles showed that the expression of Ahnak1, a protein regulating several signal-transduction pathways including metabolic homeostasis, is increased with age, which is associated with low VO and poor muscle fitness.

Rebalancing expression of HMGB1 redox isoforms to counteract muscular dystrophy.

Muscular dystrophies (MDs) are a group of genetic diseases characterized by progressive muscle wasting associated to oxidative stress and persistent inflammation. It is essential to deepen our knowledge on the mechanism connecting these two processes because current treatments for MDs have limited efficacy and/or are associated with side effects. Here, we identified the alarmin high-mobility group box 1 (HMGB1) as a functional link between oxidative stress and inflammation in MDs.

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.

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

Ultrafast Ca imaging using low-affinity fluorescent indicators allows the precise measurement of the kinetics of fast Ca currents mediated by voltage-gated Ca 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 Ca indicator Cal-520FF to record fast Ca signals and to measure the kinetics of Ca currents.

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.

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

Two recent studies have demonstrated that the dendritic Ca signal associated with a climbing fibre (CF) input to the cerebellar Purkinje neuron (PN) depends on the membrane potential (V). Specifically, when the cell is hyperpolarised, this signal is mediated by T-type voltage-gated Ca channels; in contrast, when the cell is firing, the CF-PN signal is mediated by P/Q-type voltage-gated Ca 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 V at the time of the CF input, suggesting that the standing V is a critical parameter for the induction of PF synaptic plasticity.

The Origin of Physiological Local mGluR1 Supralinear Ca 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 Ca transients, associated with the CF synaptic potential and colocalized with the PF Ca influx, occurring only when PF activity precedes the CF input. Here, we unravel the biophysical determinants of supralinear Ca signals associated with paired PF-CF synaptic activity.

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 Ca currents from brain slices. The technique is based on combined fluorescence recordings using low-affinity Ca indicators possibly in combination with voltage sensitive dyes.

Ultra-fast force-clamp spectroscopy data on the interaction between skeletal muscle myosin and actin.

Ultrafast force-clamp spectroscopy is a single molecule technique based on laser tweezers with sub-millisecond and sub-nanometer resolution. The technique has been successfully applied to investigate the rapid conformational changes that occur when a myosin II motor from skeletal muscle interacts with an actin filament. Here, we share data on the kinetics of such interaction and experimental records collected under different forces [1].

Two Distinct Sets of Ca 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 Ca channels (VGCCs), voltage-gated K channels (VGKCs), and Ca-activated SK and BK K channels. The resulting membrane potential () and Ca 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 Ca and K channels activated by CF-EPSPs, based on optical measurements of and Ca transients and on a single-compartment NEURON model reproducing experimental data.