Measuring synaptic efficacy and defining the rules for induction of synaptic plasticity at identified connections in the mammalian brain is essential for understanding how synapses contribute to learning and memory. This requires new approaches to selectively evoke presynaptic activity and measure postsynaptic responses with high spatiotemporal resolution and high sensitivity over long periods in vivo. Here we develop an all-optical approach to probe synaptic plasticity at identified cerebellar synapses in awake, behaving mice. We developed and applied JEDI-2Psub, a genetically encoded voltage indicator with increased sensitivity around resting membrane potentials, to record subthreshold and suprathreshold activity in Purkinje cell (PC) dendrites while selectively activating their granule cell (GrC) inputs using optogenetics and their climbing fiber (CF) inputs using sensory stimulation. We measured synaptic potentials and complex spike signals across the dendrites of multiple neighboring PCs, enabling us to examine correlations in voltage signals within and between neurons. We show how pairing GrC activity with sensory-evoked CF inputs can trigger long-term plasticity of inhibitory responses in PCs. These results provide a blueprint for defining the rules for plasticity induction at identified synapses in awake animals during behavior.

Volumetric functional imaging of transient cellular signaling and motion dynamics is often limited by hardware bandwidth and the scarcity of photons under short exposures. To overcome these challenges, we introduce squeezed light field microscopy (SLIM), a computational imaging approach that rapidly captures high-resolution three-dimensional light signals using only a single, low-format camera sensor. SLIM records over 1,000 volumes per second across a 550-µm diameter field of view and 300-µm depth, achieving 3.6-µm lateral and 6-µm axial resolution. Here we demonstrate its utility in blood cell velocimetry within the embryonic zebrafish brain and in freely moving tails undergoing high-frequency swings. Millisecond-scale temporal resolution further enables precise voltage imaging of neural membrane potentials in the leech ganglion and hippocampus of behaving mice. Together, these results establish SLIM as a versatile and robust tool for high-speed volumetric microscopy across diverse biological systems.

In vivo voltage imaging is a powerful tool for monitoring action potentials and dynamic electrical events in heterogeneous sensory neurons enabling the deciphering of rapid somatosensory information processing. Virus-driven expression of genetically encoded voltage indicator (GEVI) suffers from inconsistent expression levels and offers a limited time window for optimal voltage imaging. Here, we generated and characterized a knock-in mouse line with Pirt-driven expression of Marina, a positively tuned GEVI, in primary sensory neurons. Pirt-Marina mice enable optical reporting of touch, itch, and nociceptive sensations in vivo and distinct action potential patterns in the trigeminal and dorsal root ganglion neurons. Notably, Pirt-Marina mice display robust fluorescence signals in response to mechanical, thermal, or chemical stimuli, allowing visualization of transformations in sensory coding following inflammation and injury. This Pirt-Marina mouse line provides optical access to dynamic neuronal activity and plasticity in the peripheral nervous system (PNS) with high temporal accuracy, fidelity, and reliability.

Observing the activity of large populations of neurons in vivo is critical for understanding brain function and dysfunction. The use of fluorescent genetically-encoded calcium indicators (GECIs) in conjunction with miniaturized microscopes is an exciting emerging toolset for recording neural activity in unrestrained animals. Despite their potential, current miniaturized microscope designs are limited by using image sensors with low frame rates, sensitivity, and resolution. Beyond GECIs, there are many neuroscience applications which would benefit from the use of other emerging neural indicators, such as fluorescent genetically-encoded voltage indicators (GEVIs) that have faster temporal resolution to match neuron spiking, yet, require imaging at high speeds to properly sample the activity-dependent signals. We integrated an advanced CMOS image sensor into a popular open-source miniaturized microscope platform. MiniFAST is a fast and sensitive miniaturized microscope capable of 1080p video (1920x1080 pixels), 1.5 m resolution, frame rates up to 500 Hz (achieved with windowing: 1920 x 55 pixels height) and high gain ability (up to 70 dB) to image in extremely low light conditions. We report results of ~300 Hz in vivo imaging of freely behaving transgenic Thy1-GCaMP6f mice, high speed 500 Hz in vitro imaging of a GEVI and in vivo GEVI imaging in head-fixed mice. Our results extend miniaturized microscope capabilities in high-speed imaging, high sensitivity and increased resolution, opening the door for the open-source community to use fast and dim neural indicators.

The ventromedial hypothalamus (VMH) projects to the periaqueductal gray (PAG) and anterior hypothalamic nucleus (AHN), mediating freezing and escape behaviors, respectively. We investigated VMH collateral (VMH-coll) neurons, which innervate both PAG and AHN, to elucidate their role in postsynaptic processing and defensive behavior plasticity. Using all-optical voltage imaging of 22,151 postsynaptic neurons ex vivo, we found that VMH-coll neurons engage inhibitory mechanisms at both synaptic ends and can induce synaptic circuit plasticity. In vivo optogenetic activation of the VMH-coll somas induced escape behaviors. We identified an Esr1-expressing VMH-coll subpopulation with postsynaptic connectome resembling that of wild-type collaterals on the PAG side. Activation of Esr1+VMH-coll neurons evoked freezing and unexpected flattening behavior, previously not linked to the VMH. Neuropeptides such as PACAP and dynorphin modulated both Esr1+VMH-coll connectomes. In vivo kappa-opioid receptor antagonism impaired Esr1+VMH-coll-mediated defensive behaviors. These findings unveiled the central role of VMH-coll pathways in innate defensive behavior plasticity.

Fragile X autosomal homolog 1 (FXR1), a member of the fragile X messenger riboprotein 1 family, has been linked to psychiatric disorders including autism and schizophrenia. Parvalbumin (PV) interneurons play critical roles in cortical processing, and have been implicated in FXR1-linked mental illnesses. Targeted deletion of FXR1 from PV interneurons in mice has been shown to alter cortical excitability and elicit schizophrenia-like behavior. This indicates that FXR1 regulates behaviorally relevant electrophysiological functions in PV interneurons. We therefore expressed a genetically-encoded hybrid voltage sensor in PV interneurons, and used voltage imaging in slices of mouse somatosensory cortex to assess the impact of targeted FXR1 deletion. These experiments showed that PV interneurons lacking FXR1 had excitatory synaptic potentials with larger amplitudes and shorter latencies compared to wild type. Synaptic potential rise-times, decay-times, and half-widths were also impacted to degrees that varied between cortical layer and synaptic input. Thus, FXR1 modulates the responsiveness of PV interneurons to excitatory synaptic inputs. This will enable FXR1 to control cortical processing in subtle ways, with the potential to influence behavior and contribute to psychiatric dysfunction. Parvalbumin interneurons have been implicated in schizophrenia and autism. The RNA binding protein FXR1, a member of the fragile X protein family has been linked to mental illnesses and disabilities. Voltage imaging from parvalbumin interneurons in cortical slices revealed that targeted ablation of FXR1 from these neurons alters the amplitude and dynamics of their excitatory synaptic responses. These changes have the potential to alter circuit processing and behavior, and may be relevant to FXR1-linked mental illnesses.

Microbial rhodopsin-derived genetically encoded voltage indicators (GEVIs) are powerful tools for mapping bioelectrical dynamics in cell culture and in live animals. Förster resonance energy transfer (FRET)-opsin GEVIs use voltage-dependent quenching of an attached fluorophore, achieving high brightness, speed, and voltage sensitivity. However, the voltage sensitivity of most FRET-opsin GEVIs has been reported to decrease or vanish under two-photon (2P) excitation. Here, we investigated the photophysics of the FRET-opsin GEVIs Voltron1 and Voltron2. We found that the previously reported negative-going voltage sensitivities of both GEVIs came from photocycle intermediates, not from the opsin ground states. The voltage sensitivities of both GEVIs were nonlinear functions of illumination intensity; for Voltron1, the sensitivity reversed the sign under low-intensity illumination. Using photocycle-optimized 2P illumination protocols, we demonstrate 2P voltage imaging with Voltron2 in the barrel cortex of a live mouse. These results open the door to high-speed 2P voltage imaging of FRET-opsin GEVIs in vivo.

Synchronous neuronal ensembles play a pivotal role in the consolidation of long-term memory in the hippocampus. However, their organization during the acquisition of spatial memory remains less clear. In this study, we used neuronal population voltage imaging to investigate the synchronization patterns of CA1 pyramidal neuronal ensembles during the exploration of a new environment, a critical phase for spatial memory acquisition. We found synchronous ensembles comprising approximately 40% of CA1 pyramidal neurons, firing simultaneously in brief windows (~25ms) during immobility and locomotion in novel exploration. Notably, these synchronous ensembles were not associated with ripple oscillations but were instead phase-locked to local field potential theta waves. Specifically, the subthreshold membrane potentials of neurons exhibited coherent theta oscillations with a depolarizing peak at the moment of synchrony. Among newly formed place cells, pairs with more robust synchronization during locomotion displayed more distinct place-specific activities. These findings underscore the role of synchronous ensembles in coordinating place cells of different place fields.

Bioelectricity is a fundamental biophysical phenomenon present in all cells, playing a crucial role in embryogenesis by regulating processes such as neuronal signaling, pattern formation, and cancer suppression. Precise monitoring of bioelectric signals and their dynamic changes throughout development is vital for advancing our understanding of higher organisms. However, the lack of suitable techniques for mapping bioelectric signals during early development has greatly limited our ability to interpret these mechanisms. To address this challenge, we developed an Ace2N-mNeon expression library in zebrafish, which exhibits membrane localization from 4 hours post-fertilization to at least 5 days post-fertilization and broad expression across multiple cell types throughout development. We validated the use of this library for studying bioelectric changes via voltage imaging to record signals in neurons and cardiomyocytes at different development stages. Through this approach, we found evidence of synchronized neuronal activity during early embryogenesis and observed faster voltage dynamics in cardiomyocytes as development progressed. Our results show that the Ace2N-mNeon library is a valuable tool for developmental bioelectric studies supporting advanced techniques such as voltage imaging and fluorescence lifetime imaging (FLIM). These methods enable non-invasive, dynamic monitoring of bioelectric signals across diverse cell types throughout development, significantly surpassing the capabilities of current electrophysiological techniques.

Active conductances tune the kinetics of axonal action potentials (APs) to support specialized functions of neuron types. However, the temporal characteristics of voltage signals strongly depend on the size of neuronal structures, as capacitive and resistive effects slow down voltage discharges in the membranes of small elements. Axonal action potentials are particularly sensitive to these inherent biophysical effects because of the large diameter variabilities within individual axons, potentially implying bouton size-dependent synaptic effects. However, using direct patch-clamp recordings and voltage imaging in small hippocampal axons in acute slices from rat brains, we demonstrate that AP shapes remain uniform within the same axons, even across an order of magnitude difference in caliber. Our results show that smaller axonal structures have more Kv1 potassium channels that locally re-accelerate AP repolarization and contribute to size-independent APs, while they do not preclude the plasticity of AP shapes. Thus, size-independent axonal APs ensure consistent digital signals for each synapse within axons of same types.

Voltage imaging holds great potential for biomedical research by enabling noninvasive recording of the electrical activity of excitable cells such as neurons or cardiomyocytes. Camera-based detection can record from hundreds of cells in parallel, but imaging entire volumes is limited by the need to focus through the sample at high speeds. Remote focusing techniques can remedy this drawback, but have so far been either too slow or light-inefficient. Here, we introduce flipped image remote focusing, a remote focusing method that doubles the light efficiency compared to conventional beamsplitter-based techniques and enables high-speed volumetric voltage imaging at 500 volumes/s. We show the potential of our approach by combining it with light sheet imaging in the zebrafish spinal cord to record from >100 spontaneously active neurons in parallel.

Voltage imaging measures neuronal activity directly and holds promise for understanding information processing within individual neurons and across populations. However, imaging voltage over large neuronal populations has been challenging owing to the simultaneous requirements of high imaging speed and signal-to-noise ratio, large volume coverage and low photobleaching rate. Here, to overcome this challenge, we developed a confocal light-field microscope that surpassed the traditional limits in speed and noise performance by incorporating a speed-enhanced camera, a fast and robust scanning mechanism, laser-speckle-noise elimination and optimized light efficiency. With this method, we achieved simultaneous recording from more than 300 spiking neurons within an 800-µm-diameter and 180-µm-thick volume in the mouse cortex, for more than 20 min. By integrating the spatial and voltage activity profiles, we have mapped three-dimensional neural coordination patterns in awake mouse brains. Our method is robust for routine application in volumetric voltage imaging.

In calcium imaging studies, Ca transients are commonly interpreted as neuronal action potentials (APs). However, our findings demonstrate that robust optical Ca transients primarily stem from complex "AP-Plateaus", while simple APs lacking underlying depolarization envelopes produce much weaker photonic signatures. Under challenging in vivo conditions, these "AP-Plateaus" are likely to surpass noise levels, thus dominating the Ca recordings. In spontaneously active neuronal culture, optical Ca transients (OGB1-AM, GCaMP6f) exhibited approximately tenfold greater amplitude and twofold longer half-width compared to optical voltage transients (ArcLightD). The amplitude of the ArcLightD signal exhibited a strong correlation with the duration of the underlying membrane depolarization, and a weaker correlation with the presence of a fast sodium AP. Specifically, ArcLightD exhibited robust responsiveness to the slow "foot" but not the fast "trunk" of the neuronal AP. Particularly potent stimulators of optical signals in both Ca and voltage imaging modalities were APs combined with plateau potentials (AP-Plateaus), resembling dendritic Ca spikes or "UP states" in pyramidal neurons. Interestingly, even the spikeless plateaus (amplitude > 10 mV, duration > 200 ms) could generate conspicuous Ca optical signals in neurons. Therefore, in certain circumstances, Ca transients should not be interpreted solely as indicators of neuronal AP firing.

Sensory stimulations at 40 Hz gamma (but not any other frequency), have shown promise in reversing Alzheimer's disease (AD)-related pathologies. What distinguishes 40 Hz? We hypothesized that stimuli at 40 Hz might summate more efficiently (temporal summation) or propagate more efficiently between cortical layers (vertically), or along cortical laminas (horizontally), compared to inputs at 20 or 83 Hz. To investigate these hypotheses, we used brain slices from AD mouse model animals (5xFAD). Extracellular (synaptic) stimuli were delivered in cortical layer 4 (L4). Leveraging a fluorescent voltage indicator (VSFP) expressed in cortical pyramidal neurons, we simultaneously monitored evoked cortical depolarizations at multiple sites, at 1 kHz sampling frequency. Experimental groups (AD-Female, CTRL-Female, AD-Male, and CTRL-Male) were tested at three stimulation frequencies (20, 40, and 83 Hz). Despite our initial hypothesis, two parameters-temporal summation of voltage waveforms and the strength of propagation through the cortical neuropil-did not reveal any distinct advantage of 40 Hz stimulation. Significant physiological differences between AD and Control mice were found at all stimulation frequencies tested, while the 40 Hz stimulation frequency was not remarkable.

Voltage imaging is an important complement to traditional methods for probing cellular physiology, such as electrode-based patch clamp techniques. Unlike the related Ca imaging, voltage imaging provides a direct visualization of bioelectricity changes. We have been exploring the use of sulfonated silicon rhodamine dyes (Berkeley Red Sensor of Transmembrane potential, BeRST) for voltage imaging. In this study, we explore the effect of converting BeRST to diEt BeRST, by replacing the dimethyl aniline of BeRST with a diethyl aniline group. The new dye, diEt BeRST, has a voltage sensitivity of 40% Δ/ per 100 mV, a 33% increase compared to the original BeRST dye, which has a sensitivity of 30% Δ/ per 100 mV. In neurons, the cellular brightness of diEt BeRST is about 20% as bright as that of BeRST, which may be due to the lower solubility of diEt BeRST (300 μM) compared to that of BeRST (800 μM). Despite this lower cellular brightness, diEt BeRST is able to record spontaneous and evoked action potentials from multiple neurons simultaneously and in single trials. Far-red excitation and emission profiles enable diEt BeRST to be used alongside existing fluorescent indicators of cellular physiology, like Ca-sensitive Oregon Green BAPTA. In hippocampal neurons, simultaneous voltage and Ca imaging reveals neuronal spiking patterns and frequencies that cannot be resolved with traditional Ca imaging methods. This study represents a first step toward describing the structural features that define voltage sensitivity and brightness in silicon rhodamine-based BeRST indicators.