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Voltage imaging with cellular specificity has been made possible by advances in genetically encoded voltage indicators. However, the kilohertz rates required for voltage imaging lead to weak signals. Moreover, out-of-focus fluorescence and tissue scattering produce background that both undermines the signal-to-noise ratio and induces crosstalk between cells, making reliable in vivo imaging in densely labeled tissue highly challenging. We describe a microscope that combines the distinct advantages of targeted illumination and confocal gating while also maximizing signal detection efficiency. The resulting benefits in signal-to-noise ratio and crosstalk reduction are quantified experimentally and theoretically. Our microscope provides a versatile solution for enabling high-fidelity in vivo voltage imaging at large scales and penetration depths, which we demonstrate across a wide range of imaging conditions and different genetically encoded voltage indicator classes., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Genetically encoded voltage indicators (GEVIs) hold immense potential for monitoring neuronal population activity. To date, best-in-class GEVIs rely on one-photon excitation. However, GEVI imaging of dense neuronal populations remains difficult because out-of-focus background fluorescence produces low contrast and excess noise when paired with conventional one-photon widefield imaging methods. To address this challenge, we developed an imaging system capable of efficient, high-contrast GEVI imaging at near-kHz rates and demonstrate it for in vivo and ex vivo imaging applications in the mouse neocortex. Our approach uses simultaneous multiplane imaging to monitor activity within contiguous tissue volumes with no penalty in speed or requirement for high excitation power. This approach, multi-Z imaging with confocal detection (MuZIC), permits high signal-to-noise ratio voltage imaging in densely labeled neuronal populations and is compatible with imaging through micro-optics. Moreover, it minimizes artifacts associated with concurrent imaging and optogenetic photostimulation for all-optical electrophysiology., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability., Competing Interests: Declaration of interests A.S.A., L.D.L., and E.R.S. have filed for a patent on the chemigenetic voltage indicators. I.K. and C.R.F. are co-inventors on a patent describing pipette cleaning that is licensed by Sensapex. M.C. and L.C. have performed consulting services for Sensapex., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Objective: To evaluate the urine cortisol-to-creatinine ratio (UCCR) for the diagnosis of hypoadrenocorticism (HA) in dogs and to determine whether the method of urine cortisol measurement affects results., Animals: 41 dogs with naturally occurring HA and 107 dogs with nonadrenal illness., Procedures: Urine samples were prospectively collected from dogs undergoing testing for HA. Urine cortisol concentrations were measured at a veterinary diagnostic laboratory using either a radioimmunoassay (RIA) or a chemiluminescent immunoassay (CLIA). Receiver operating characteristic (ROC) curves were constructed to assess UCCR performance by both methods for HA diagnosis. Sensitivities, specificities, accuracies, and predictive values were calculated for various cutpoints., Results: The areas under the ROC curves for UCCR diagnosis of HA were 0.99 (95% CI, 0.98 to 1.00) and 1.00 (95% CI, 1.00 to 1.00) when urine cortisol was determined by RIA and CLIA, respectively. An RIA UCCR of ≤ 2 was 97.2% sensitive, 93.6% specific, and 94.7% accurate for HA diagnosis, whereas a CLIA UCCR of ≤ 10 was 100% sensitive, specific, and accurate. An RIA UCCR > 4 and a CLIA UCCR of > 10 had negative predictive values of 100%., Clinical Relevance: The UCCR was an accurate diagnostic test for HA in this study population, although equivocal results are possible. Case characteristics, method of cortisol measurement, and laboratory-specific cutpoints must be considered when interpreting results.

Many neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), lead to the selective degeneration of discrete cell types in the CNS despite the ubiquitous expression of many genes linked to disease. Therapeutic advancement depends on understanding the unique cellular adaptations that underlie pathology of vulnerable cells in the context of disease-causing mutations. Here, we employ bacTRAP molecular profiling to elucidate cell type-specific molecular responses of cortical upper motor neurons in a preclinical ALS model. Using two bacTRAP mouse lines that label distinct vulnerable or resilient projection neuron populations in motor cortex, we show that the regulation of oxidative phosphorylation (Oxphos) pathways is a common response in both cell types. However, differences in the baseline expression of genes involved in Stem and the handling of reactive oxygen species likely lead to the selective degeneration of the vulnerable cells. These results provide a framework to identify cell-type-specific processes in neurodegenerative disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Translational profiling methodologies enable the systematic characterization of cell types in complex tissues, such as the mammalian brain, where neuronal isolation is exceptionally difficult. Here, we report a versatile strategy for profiling CNS cell types in a spatiotemporally restricted fashion by engineering a Cre-dependent adeno-associated virus expressing an EGFP-tagged ribosomal protein (AAV-FLEX-EGFPL10a) to access translating mRNAs by translating ribosome affinity purification (TRAP). We demonstrate the utility of this AAV to target a variety of genetically and anatomically defined neural populations expressing Cre recombinase and illustrate the ability of this viral TRAP (vTRAP) approach to recapitulate the molecular profiles obtained by bacTRAP in corticothalamic neurons across multiple serotypes. Furthermore, spatially restricting adeno-associated virus (AAV) injections enabled the elucidation of regional differences in gene expression within this cell type. Altogether, these results establish the broad applicability of the vTRAP strategy for the molecular dissection of any CNS or peripheral cell type that can be engineered to express Cre., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

The medial prefrontal cortex (mPFC) of both rats and rabbits has been shown to support trace eyeblink conditioning, presumably by providing an input to the cerebellum via the pons that bridges the temporal gap between conditioning stimuli. The pons of rats and rabbits, however, shows divergence in gross anatomical organization, leaving open the question of whether the topography of prefrontal inputs to the pons is similar in rats and rabbits. To investigate this question, we injected anterograde tracer into the mPFC of rats and rabbits to visualize and map in 3D the distribution of labeled terminals in the pons. Effective mPFC injections showed labeled axons in the ipsilateral descending pyramidal tract in both species. In rats, discrete clusters of densely labeled terminals were observed primarily in the rostromedial pons. Clusters of labeled terminals were also observed contralateral to mPFC injection sites in rats, appearing as a less dense "mirror-image" of ipsilateral labeling. In rabbits, mPFC labeled corticopontine terminals were absent in the rostral pons, and instead were restricted to the intermediate pons. The densest terminal fields were typically observed in association with the ipsilateral pyramidal tract as it descended ventromedially through the rabbit pons. No contralateral terminal labeling was observed for any injections made in the rabbit mPFC. The results suggest the possibility that mPFC inputs to the pons may be integrated with different sources of cortical inputs between rats and rabbits. The resulting implications for mPFC or pons manipulations for studies of trace eyeblink in each species are discussed., (© 2014 Wiley Periodicals, Inc.)