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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.

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. [ABSTRACT FROM AUTHOR]

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. 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. 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 FROM AUTHOR]