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Optical assays of synaptic strength could facilitate studies of neuronal transmission and its dysregulation in disease. Here we introduce a genetic toolbox for all-optical interrogation of synaptic electrophysiology (synOptopatch) via mutually exclusive expression of a channelrhodopsin actuator and an archaerhodopsin-derived voltage indicator. Optically induced activity in the channelrhodopsin-expressing neurons generated excitatory and inhibitory postsynaptic potentials that we optically resolved in reporter-expressing neurons. We further developed a yellow spine-targeted Ca2+ indicator to localize optogenetically triggered synaptic inputs. We demonstrated synOptopatch recordings in cultured rodent neurons and in acute rodent brain slice. In synOptopatch measurements of primary rodent cultures, acute ketamine administration suppressed disynaptic inhibitory feedbacks, mimicking the effect of this drug on network function in both rodents and humans. We localized this action of ketamine to excitatory synapses onto interneurons. These results establish an in vitro all-optical model of disynaptic disinhibition, a synaptic defect hypothesized in schizophrenia-associated psychosis.

Perineal urethrostomy in cats is indicated for urethral pathologies located distal to the bulbourethral glands. The description of the bulbourethral glands as the cranial landmark when performing a PU is based on the increased urethral diameter at this location, rather than on an anatomical limitation. This suggests that urethral pathologies cranial to the bulbourethral glands could potentially be treated with PU. At present, the extent to which the pelvic urethra can be mobilized is unknown. Characterization and quantification of the effect of PU on the pelvic urethra is required prior to attempting to define the location of the pelvic urethra, cranial to the bulbourethral glands, which can be exteriorized when performing a PU. Our aim was to characterize and quantify the effect of performing a PU on the location and length of the pelvic urethra. Methods: Ten male feline cadavers were used, and four markers were placed on the pelvic urethra via a ventral approach to the peritoneal and pelvic cavities. Two orthogonal radiographic views were acquired prior and subsequent to performing a PU. The distance of each marker to a predefined landmark/origin and the distances of the markers relative to each other were measured on all radiographs. Results: PU resulted in significant caudal translation of the markers relative to the predefined landmark on all radiographic views; however, PU did not result in a significant change in the distances between the markers. Conclusions: Performing a PU results in caudal translation and minimal stretching of the mobilized pelvic urethra.

Sensory inputs from the nasal epithelium to the olfactory bulb (OB) are organized as a discrete map in the glomerular layer (GL). This map is then modulated by distinct types of local neurons and transmitted to higher brain areas via mitral and tufted cells. Little is known about the functional organization of the circuits downstream of glomeruli. We used in vivo two-photon calcium imaging for large scale functional mapping of distinct neuronal populations in the mouse OB, at single cell resolution. Specifically, we imaged odor responses of mitral cells (MCs), tufted cells (TCs) and glomerular interneurons (GL-INs). Mitral cells population activity was heterogeneous and only mildly correlated with the olfactory receptor neuron (ORN) inputs, supporting the view that discrete input maps undergo significant transformations at the output level of the OB. In contrast, population activity profiles of TCs were dense, and highly correlated with the odor inputs in both space and time. Glomerular interneurons were also highly correlated with the ORN inputs, but showed higher activation thresholds suggesting that these neurons are driven by strongly activated glomeruli. Temporally, upon persistent odor exposure, TCs quickly adapted. In contrast, both MCs and GL-INs showed diverse temporal response patterns, suggesting that GL-INs could contribute to the transformations MCs undergo at slow time scales. Our data suggest that sensory odor maps are transformed by TCs and MCs in different ways forming two distinct and parallel information streams.

EmrE is an Escherichia coli H⁺-coupled multidrug transporter that provides a unique experimental paradigm because of its small size and stability, and because its activity can be studied in detergent solution. In this work, we report a study of the transient kinetics of substrate binding and substrate-induced proton release in EmrE. For this purpose, we measured transient changes in the tryptophan fluorescence upon substrate binding and the rates of substrate-induced proton release. The fluorescence of the essential and fully conserved Trp residue at position 63 is sensitive to the occupancy of the binding site with either protons or substrate. The maximal rate of binding to detergent-solubilized EmrE of TPP⁺, a high-affinity substrate, is 2 x 10⁷ M⁻¹·s⁻¹, a rate typical of diffusion-limited reactions. Rate measurements with medium- and low-affinity substrates imply that the affinity is determined mainly by the koff of the substrate. The rates of substrate binding and substrate-induced release of protons are faster at basic pHs and slower at lower pHs. These findings imply that the substrate-binding rates are determined by the generation of the species capable of binding; this is controlled by the high affinity to protons of the glutamate at position 14, because an Asp replacement with a lower pK is faster at the same pHs.

A technology that simultaneously records membrane potential from multiple neurons in behaving animals will have a transformative effect on neuroscience research 1 , 2 . Genetically encoded voltage indicators are a promising tool for these purposes; however, these have so far been limited to single-cell recordings with a marginal signal-to-noise ratio in vivo 3 – 5 . Here we developed improved near-infrared voltage indicators, high-speed microscopes and targeted gene expression schemes that enabled simultaneous in vivo recordings of supra- and subthreshold voltage dynamics in multiple neurons in the hippocampus of behaving mice. The reporters revealed subcellular details of back-propagating action potentials and correlations in subthreshold voltage between multiple cells. In combination with stimulation using optogenetics, the reporters revealed changes in neuronal excitability that were dependent on the behavioural state, reflecting the interplay of excitatory and inhibitory synaptic inputs. These tools open the possibility for detailed explorations of network dynamics in the context of behaviour. Fig. 1 Photoactivated QuasAr3 (paQuasAr3) reports neuronal activity in vivo. a , Schematic of the paQuasAr3 construct. b , Photoactivation by blue light enhanced voltage signals excited by red light in cultured neurons that expressed paQuasAr3 (representative example of n  = 4 cells). c , Model of the photocycle of paQuasAr3. d , Confocal images of sparsely expressed paQuasAr3 in brain slices. Scale bars, 50 μm. Representative images, experiments were repeated in n  = 3 mice. e , Simultaneous fluorescence and patch-clamp recordings from a neuron expressing paQuasAr3 in acute brain slice. Top, magnification of boxed regions. Schematic shows brain slice, patch pipette and microscope objective. f , Simultaneous fluorescence and patch-clamp recordings of inhibitory post synaptic potentials in an L2–3 neuron induced by electrical stimulation of L5–6 in acute slice. g , Normalized change in fluorescence (Δ F / F ) and SNR of optically recorded post-synaptic potentials (PSPs) as a function of the amplitude of the post-synaptic potentials. The voltage sensitivity was Δ F / F  = 40 ± 1.7% per 100 mV. The SNR was 0.93 ± 0.07 per 1 mV in a 1-kHz bandwidth ( n  = 42 post-synaptic potentials from 5 cells, data are mean ± s.d.). Schematic shows brain slice, patch pipette, field stimulation electrodes and microscope objective. h , Optical measurements of paQuasAr3 fluorescence in the CA1 region of the hippocampus (top) and glomerular layer of the olfactory bulb (bottom) of anaesthetized mice (representative traces from n  = 7 CA1 cells and n  = 13 olfactory bulb cells, n  = 3 mice). Schematics show microscope objective and the imaged brain region. i , STA fluorescence from 88 spikes in a CA1 oriens neuron. j , Frames from the STA video showing the delay in the back-propagating action potential in the dendrites relative to the soma. k , Sub-Nyquist fitting of the action potential delay and width shows electrical compartmentalization in the dendrites. Experiments in k – m were repeated in n  = 2 cells from n  = 2 mice. A combination of improved near-infrared voltage indicators, high-speed microscopes and targeted gene expression schemes enabled simultaneous in vivo optogenetic control and recording of voltage dynamics in multiple neurons in the hippocampus of behaving mice.

Astrocytes are glial cells that interact with neuronal synapses via their distal processes, where they remove glutamate and potassium (K+) from the extracellular space following neuronal activity. Astrocyte clearance of both glutamate and K+ is voltage dependent, but astrocyte membrane potential (Vm) is thought to be largely invariant. As a result, these voltage dependencies have not been considered relevant to astrocyte function. Using genetically encoded voltage indicators to enable the measurement of Vm at peripheral astrocyte processes (PAPs) in mice, we report large, rapid, focal and pathway-specific depolarizations in PAPs during neuronal activity. These activity-dependent astrocyte depolarizations are driven by action potential-mediated presynaptic K+ efflux and electrogenic glutamate transporters. We find that PAP depolarization inhibits astrocyte glutamate clearance during neuronal activity, enhancing neuronal activation by glutamate. This represents a novel class of subcellular astrocyte membrane dynamics and a new form of astrocyte–neuron interaction.Using voltage imaging, Armbruster et al. show that neuronal activity induces large, rapid and synapse-specific astrocyte depolarizations that enhance synaptic glutamate signaling, representing a novel form of neuron–astrocyte communication.

EmrE is a small H + ‐coupled multidrug transporter in Escherichia coli . Claims have been made for an antiparallel topology of this homodimeric protein. However, our own biochemical studies performed with detergent‐solubilized purified protein support a parallel topology of the protomers. We developed an alternative approach to constrain the relative topology of the protomers within the dimer so that their activity can be assayed also in vivo before biochemical handling. Tandem EmrE was built with two identical monomers genetically fused tail to head (C‐terminus of the first to N‐terminus of the second monomer) with hydrophilic linkers of varying length. All the constructs conferred resistance to ethidium by actively removing it from the cytoplasm. The purified proteins bound substrate and transported methyl viologen into proteoliposomes by a proton‐dependent mechanism. A tandem where one of the essential glutamates was replaced with glutamine transported only monovalent substrates and displayed a modified stoichiometry. The results support a parallel topology of the protomers in the functional dimer. The implications regarding insertion and evolution of membrane proteins are discussed.

Hippocampal place cells (PCs) are important for spatial coding and episodic memory. PCs’ representations are modulated upon transitioning between environments (global remapping) but also change with repeated exposure to familiar spaces (representational drift). To gain insights into the mechanistic basis for this unique balance between circuit plasticity and stability, we used voltage imaging to longitudinally record the subthreshold and spiking activity of pyramidal neurons (PNs) and somatostatin-positive (SST) interneurons in CA1 during virtual navigation. A fraction of cells from both populations showed spatial representations, but many SSTs were speed-tuned or fired uniformly across space. Intracellular recordings revealed increased theta power and asymmetric ramp-like depolarization in PN place fields, while SSTs exhibited symmetric depolarization with no theta increase. Longitudinal recordings across weeks demonstrated representational drifts in both populations, although SSTs exhibited remarkably stable firing and subthreshold properties. Transition to a novel environment induced remapping in both populations, accompanied by increase in SST activity and reduction in PNs. These results provide new insights into how hippocampal circuits balance representational stability with experience-dependent plasticity.

Understanding how neuronal integration is modulated by behavior is a fundamental goal in neuroscience. We combined voltage imaging with optogenetic depolarization to reveal how changes in excitatory (E) and inhibitory (I) inputs, modulate the spiking output, subthreshold dynamics, and gain of key genetically defined cell types in the CA1 region of the hippocampus. We imaged pyramidal cells (PCs), vasoactive intestinal peptide (VIP), somatostatin (SST), and parvalbumin (PV) interneurons (INs) and found that locomotion reduced firing in PCs and VIP INs while increasing activity in SST and PV cells. Prolonged optical depolarization experiments and simulations revealed that intracellular theta oscillations are predominantly driven by inhibitory inputs in PCs and VIP cells. Firing rate-laser intensity (F-I) curves revealed distinct gain modulation across cell types, with a divisive gain reduction in PC bursting during locomotion, while simple spikes are unaffected. A two-compartment model suggested that this results from a balanced E/I input increase to somatic and dendritic compartments. These findings reveal how behavioral state-dependent coordination of excitation and inhibition governs hippocampal neuronal dynamics and output-specific gain modulation.

Genetically encoded voltage indicators (GEVIs) are powerful tools for monitoring neuronal activity, but their application, particularly for long-term recordings in vivo, is often limited by photobleaching under the required high illumination intensities. This constraint restricts the total duration of continuous or trial-based experiments, crucial for studying processes like synaptic plasticity or circuit dynamics during behavior. Here, we introduce ElectraON and ElectraOFF, a pair of green fluorescent eFRET-based GEVIs engineered by incorporating a photostability-enhanced derivative of the bright monomeric fluorescent protein mBaoJin with Ace opsin variants. Critically, Electras demonstrate over 6-fold improved photostability compared to state-of-the-art eFRET GEVIs, pAce, and Ace-mNeon2, under one-photon illumination, while characterized by bright green fluorescence, millisecond kinetics, and good membrane localization. This enhanced stability translates to a 3-to >10-fold extension in functional recording duration, maintaining reliable spike detection in both cultured neurons in vitro and sparsely labeled neurons in the awake mouse cortex in vivo. We demonstrated sustained in vivo recordings exceeding 30 minutes, with instances surpassing one hour. Furthermore, Electras show functionality under scanless two-photon excitation in cultured cells. These highly photostable indicators significantly extend the temporal window for voltage imaging, broadening the scope of accessible biological questions.