Large-scale deep tissue voltage imaging with targeted-illumination confocal microscopy.

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.

The autism spectrum disorder risk gene NEXMIF over-synchronizes hippocampal CA1 network and alters neuronal coding.

Mutations in autism spectrum disorder (ASD) risk genes disrupt neural network dynamics that ultimately lead to abnormal behavior. To understand how ASD-risk genes influence neural circuit computation during behavior, we analyzed the hippocampal network by performing large-scale cellular calcium imaging from hundreds of individual CA1 neurons simultaneously in transgenic mice with total knockout of the X-linked ASD-risk gene NEXMIF (neurite extension and migration factor). As NEXMIF knockout in mice led to profound learning and memory deficits, we examined the CA1 network during voluntary locomotion, a fundamental component of spatial memory. We found that NEXMIF knockout does not alter the overall excitability of individual neurons but exaggerates movement-related neuronal responses. To quantify network functional connectivity changes, we applied closeness centrality analysis from graph theory to our large-scale calcium imaging datasets, in addition to using the conventional pairwise correlation analysis. Closeness centrality analysis considers both the number of connections and the connection strength between neurons within a network. We found that in wild-type mice the CA1 network desynchronizes during locomotion, consistent with increased network information coding during active behavior. Upon NEXMIF knockout, CA1 network is over-synchronized regardless of behavioral state and fails to desynchronize during locomotion, highlighting how perturbations in ASD-implicated genes create abnormal network synchronization that could contribute to ASD-related behaviors.

Theta and gamma rhythmic coding through two spike output modes in the hippocampus during spatial navigation.

Hippocampal CA1 neurons generate single spikes and stereotyped bursts of spikes. However, it is unclear how individual neurons dynamically switch between these output modes and whether these two spiking outputs relay distinct information. We performed extracellular recordings in spatially navigating rats and cellular voltage imaging and optogenetics in awake mice. We found that spike bursts are preferentially linked to cellular and network theta rhythms (3-12 Hz) and encode an animal's position via theta phase precession, particularly as animals are entering a place field. In contrast, single spikes exhibit additional coupling to gamma rhythms (30-100 Hz), particularly as animals leave a place field. Biophysical modeling suggests that intracellular properties alone are sufficient to explain the observed input frequency-dependent spike coding. Thus, hippocampal neurons regulate the generation of bursts and single spikes according to frequency-specific network and intracellular dynamics, suggesting that these spiking modes perform distinct computations to support spatial behavior.

Parvalbumin neurons enhance temporal coding and reduce cortical noise in complex auditory scenes.

Cortical representations supporting many cognitive abilities emerge from underlying circuits comprised of several different cell types. However, cell type-specific contributions to rate and timing-based cortical coding are not well-understood. Here, we investigated the role of parvalbumin neurons in cortical complex scene analysis. Many complex scenes contain sensory stimuli which are highly dynamic in time and compete with stimuli at other spatial locations. Parvalbumin neurons play a fundamental role in balancing excitation and inhibition in cortex and sculpting cortical temporal dynamics; yet their specific role in encoding complex scenes via timing-based coding, and the robustness of temporal representations to spatial competition, has not been investigated. Here, we address these questions in auditory cortex of mice using a cocktail party-like paradigm, integrating electrophysiology, optogenetic manipulations, and a family of spike-distance metrics, to dissect parvalbumin neurons' contributions towards rate and timing-based coding. We find that suppressing parvalbumin neurons degrades cortical discrimination of dynamic sounds in a cocktail party-like setting via changes in rapid temporal modulations in rate and spike timing, and over a wide range of time-scales. Our findings suggest that parvalbumin neurons play a critical role in enhancing cortical temporal coding and reducing cortical noise, thereby improving representations of dynamic stimuli in complex scenes.

Deep brain stimulation creates informational lesion through membrane depolarization in mouse hippocampus.

Deep brain stimulation (DBS) is a promising neuromodulation therapy, but the neurophysiological mechanisms of DBS remain unclear. In awake mice, we performed high-speed membrane voltage fluorescence imaging of individual hippocampal CA1 neurons during DBS delivered at 40 Hz or 140 Hz, free of electrical interference. DBS powerfully depolarized somatic membrane potentials without suppressing spike rate, especially at 140 Hz. Further, DBS paced membrane voltage and spike timing at the stimulation frequency and reduced timed spiking output in response to hippocampal network theta-rhythmic (3-12 Hz) activity patterns. To determine whether DBS directly impacts cellular processing of inputs, we optogenetically evoked theta-rhythmic membrane depolarization at the soma. We found that DBS-evoked membrane depolarization was correlated with DBS-mediated suppression of neuronal responses to optogenetic inputs. These results demonstrate that DBS produces powerful membrane depolarization that interferes with the ability of individual neurons to respond to inputs, creating an informational lesion.

Spike ripples in striatum correlate with seizure risk in two mouse models.

Epilepsy biomarkers from electroencephalogram recordings are routinely used to assess seizure risk and localization. Two widely adopted biomarkers include: (i) interictal spikes, and (ii) high frequency ripple oscillations. The combination of these two biomarkers, ripples co-occurring with spikes (spike ripples), has been proposed as an improved biomarker for the epileptogenic zone and epileptogenicity in humans and rodent models. Whether spike ripples translate to predict seizure risk in rodent seizure models is unknown. Further, recent evidence suggests ictal networks can include deep gray nuclei in humans. Whether pathologic spike ripples and seizures are also observed in the basal ganglia in rodent models has not been explored. We addressed these questions using local field potential recordings from mice with and without striatal seizures after carbachol or 6-hydroxydopamine infusions into the striatum. We found increased spike ripples in the interictal and ictal periods in mice with seizures compared to pre-infusion and post-infusion seizure-free recordings. These data provide evidence of electrographic seizures involving the striatum in mice and support the candidacy of spike ripples as a translational biomarker for seizure risk in mouse models.

Large-scale voltage imaging in behaving mice using targeted illumination.

Recent improvements in genetically encoded voltage indicators enabled optical imaging of action potentials and subthreshold transmembrane voltage in vivo. To perform high-speed voltage imaging of many neurons simultaneously over a large anatomical area, widefield microscopy remains an essential tool. However, the lack of optical sectioning makes widefield microscopy prone to background cross-contamination. We implemented a digital-micromirror-device-based targeted illumination strategy to restrict illumination to the cells of interest and quantified the resulting improvement both theoretically and experimentally with SomArchon expressing neurons. We found that targeted illumination increased SomArchon signal contrast, decreased photobleaching, and reduced background cross-contamination. With the use of a high-speed, large-area sCMOS camera, we routinely imaged tens of spiking neurons simultaneously over minutes in behaving mice. Thus, the targeted illumination strategy described here offers a simple solution for widefield voltage imaging of many neurons over a large field of view in behaving animals.

Distinct neuronal populations contribute to trace conditioning and extinction learning in the hippocampal CA1.

Trace conditioning and extinction learning depend on the hippocampus, but it remains unclear how neural activity in the hippocampus is modulated during these two different behavioral processes. To explore this question, we performed calcium imaging from a large number of individual CA1 neurons during both trace eye-blink conditioning and subsequent extinction learning in mice. Our findings reveal that distinct populations of CA1 cells contribute to trace conditioned learning versus extinction learning, as learning emerges. Furthermore, we examined network connectivity by calculating co-activity between CA1 neuron pairs and found that CA1 network connectivity patterns also differ between conditioning and extinction, even though the overall connectivity density remains constant. Together, our results demonstrate that distinct populations of hippocampal CA1 neurons, forming different sub-networks with unique connectivity patterns, encode different aspects of learning.

CaMKIIα-Positive Interneurons Identified via a microRNA-Based Viral Gene Targeting Strategy.

Single-cell analysis is revealing increasing diversity in gene expression profiles among brain cells. Traditional promotor-based viral gene expression techniques, however, cannot capture the growing variety among single cells. We demonstrate a novel viral gene expression strategy to target cells with specific miRNA expression using miRNA-guided neuron tags (mAGNET). We designed mAGNET viral vectors containing a CaMKIIα promoter and microRNA-128 (miR-128) binding sites, and labeled CaMKIIα+ cells with naturally low expression of miR-128 (Lm128C cells) in male and female mice. Although CaMKIIα has traditionally been considered as an excitatory neuron marker, our single-cell sequencing results reveal that Lm128C cells are CaMKIIα+ inhibitory neurons of parvalbumin or somatostatin subtypes. Further evaluation of the physiological properties of Lm128C cell in brain slices showed that Lm128C cells exhibit elevated membrane excitability, with biophysical properties closely resembling those of fast-spiking interneurons, consistent with previous transcriptomic findings of miR-128 in regulating gene networks that govern membrane excitability. To further demonstrate the utility of this new viral expression strategy, we expressed GCaMP6f in Lm128C cells in the superficial layers of the motor cortex and performed in vivo calcium imaging in mice during locomotion. We found that Lm128C cells exhibit elevated calcium event rates and greater intrapopulation correlation than the overall CaMKIIα+ cells during movement. In summary, the miRNA-based viral gene targeting strategy described here allows us to label a sparse population of CaMKIIα+ interneurons for functional studies, providing new capabilities to investigate the relationship between gene expression and physiological properties in the brain.SIGNIFICANCE STATEMENT We report the discovery of a class of CaMKIIα+ cortical interneurons, labeled via a novel miRNA-based viral gene targeting strategy, combinatorial to traditional promoter-based strategies. The fact that we found a small, yet distinct, population of cortical inhibitory neurons that express CaMKIIα demonstrates that CaMKIIα is not as specific for excitatory neurons as commonly believed. As single-cell sequencing tools are providing increasing insights into the gene expression diversity of neurons, including miRNA profile data, we expect that the miRNA-based gene targeting strategy presented here can help delineate many neuron populations whose physiological properties can be readily related to the miRNA gene regulatory networks.

A Viral Toolbox of Genetically Encoded Fluorescent Synaptic Tags.

Fibronectin intrabodies generated with mRNA display (FingRs) are a recently developed tool for labeling excitatory or inhibitory synapses, with the benefit of not altering endogenous synaptic protein expression levels or synaptic transmission. Here, we generated a viral vector FingR toolbox that allows for multi-color, neuron-type-specific labeling of excitatory or inhibitory synapses in multiple brain regions. We screened various fluorophores, FingR fusion configurations, and transcriptional control regulations in adeno-associated virus (AAV) and retrovirus vector designs. We report the development of a red FingR variant and demonstrated dual labeling of excitatory and inhibitory synapses in the same cells. Furthermore, we developed cre-inducible FingR AAV variants and demonstrated their utility, finding that the density of inhibitory synapses in aspiny striatal cholinergic interneurons remained unchanged in response to dopamine depletion. Finally, we generated FingR retroviral vectors, which enabled us to track the development of excitatory and inhibitory synapses in hippocampal adult-born granule cells.