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Abstract Rodents continuously move their heads and whiskers in a coordinated manner while perceiving objects through whisker-touch. Studies in head-fixed rodents showed that the ventroposterior medial (VPM) and posterior medial (POm) thalamic nuclei code for whisker kinematics, with POm involvement reduced in awake animals. To examine VPM and POm involvement in coding head and whisker kinematics in awake, head-free conditions, we recorded thalamic neuronal activity and tracked head and whisker movements in male mice exploring an open arena. Using optogenetic tagging, we found that in freely moving mice, both nuclei equally coded whisker kinematics and robustly coded head kinematics. The fraction of neurons coding head kinematics increased after whisker trimming, ruling out whisker-mediated coding. Optogenetic activation of thalamic neurons evoked overt kinematic changes and increased the fraction of neurons leading changes in head kinematics. Our data suggest that VPM and POm integrate head and whisker information and can influence head kinematics during tactile perception.

Abstract In the classical model of the basal ganglia, direct pathway striatal projection neurons (dSPNs) send projections to the substantia nigra (SNr) and entopeduncular nucleus to regulate motor function. Recent studies have re-established that dSPNs also possess axon collaterals within the globus pallidus (GPe) (bridging collaterals), yet the significance of these collaterals for behavior is unknown. Here we use in vivo optical and chemogenetic tools combined with deep learning approaches in mice to dissect the roles of dSPN GPe collaterals in motor function. We find that dSPNs projecting to the SNr send synchronous motor-related information to the GPe via axon collaterals. Inhibition of native activity in dSPN GPe terminals impairs motor activity and function via regulation of Npas1 neurons. We propose a model by which dSPN GPe axon collaterals (striatopallidal Go pathway) act in concert with the canonical terminals in the SNr to support motor control by inhibiting Npas1 neurons.

Little is known about the synaptic organization of associative cortical structures such as the medial prefrontal cortex. Here, the authors use two-photon optogenetic stimulation to obtain a detailed cellular resolution map of functional synaptic connectivity of the mouse medial prefrontal cortex, finding unique spatial patterns of local-circuit connectivity in neurons that project to the basolateral amygdala.

Summary: Epithelial responses to the cytokine interleukin-13 (IL-13) cause airway obstruction in asthma. Here we utilized multiple genomic techniques to identify IL-13-responsive regulatory elements in bronchial epithelial cells and used these data to develop a CRISPR interference (CRISPRi)-based therapeutic approach to downregulate airway obstruction-inducing genes in a cell type- and IL-13-specific manner. Using single-cell RNA sequencing (scRNA-seq) and acetylated lysine 27 on histone 3 (H3K27ac) chromatin immunoprecipitation sequencing (ChIP-seq) in primary human bronchial epithelial cells, we identified IL-13-responsive genes and regulatory elements. These sequences were functionally validated and optimized via massively parallel reporter assays (MPRAs) for IL-13-inducible activity. The top secretory cell-selective sequence from the MPRA, a novel, distal enhancer of the sterile alpha motif pointed domain containing E-26 transformation-specific transcription factor (SPDEF) gene, was utilized to drive CRISPRi and knock down SPDEF or mucin 5AC (MUC5AC), both involved in pathologic mucus production in asthma. Our work provides a catalog of cell type-specific genes and regulatory elements involved in IL-13 bronchial epithelial response and showcases their use for therapeutic purposes.

Abstract Nullomers and nullpeptides are short DNA or amino acid sequences that are absent from a genome or proteome, respectively. One potential cause for their absence could be their having a detrimental impact on an organism. Results Here, we identify all possible nullomers and nullpeptides in the genomes and proteomes of thirty eukaryotes and demonstrate that a significant proportion of these sequences are under negative selection. We also identify nullomers that are unique to specific functional categories: coding sequences, exons, introns, 5′UTR, 3′UTR, promoters, and show that coding sequence and promoter nullomers are most likely to be selected against. By analyzing all protein sequences across the tree of life, we further identify 36,081 peptides up to six amino acids in length that do not exist in any known organism, termed primes. We next characterize all possible single base pair mutations that can lead to the appearance of a nullomer in the human genome, observing a significantly higher number of mutations than expected by chance for specific nullomer sequences in transposable elements, likely due to their suppression. We also annotate nullomers that appear due to naturally occurring variants and show that a subset of them can be used to distinguish between different human populations. Analysis of nullomers and nullpeptides across vertebrate evolution shows they can also be used as phylogenetic classifiers. Conclusions We provide a catalog of nullomers and nullpeptides in distinct functional categories, develop methods to systematically study them, and highlight the use of variability in these sequences in other analyses

The thalamus provides sensory input to the cortex, but many aspects of thalamocortical signaling remain unknown. Here, the authors reveal parallel non-overlapping thalamic pathways with distinct representations of tactile and decision-related information during a goal-directed sensorimotor task.

All-optical methods for imaging and manipulating brain networks with high spatial resolution are fundamental to study how neuronal ensembles drive behavior. Stimulation of neuronal ensembles using two-photon holographic techniques requires high-sensitivity actuators to avoid photodamage and heating. Moreover, two-photon-excitable opsins should be insensitive to light at wavelengths used for imaging. To achieve this goal, we developed a novel soma-targeted variant of the large-conductance blue-light-sensitive opsin CoChR (stCoChR). In the mouse cortex in vivo, we combined holographic two-photon stimulation of stCoChR with an amplified laser tuned at the opsin absorption peak and two-photon imaging of the red-shifted indicator jRCaMP1a. Compared to previously characterized blue-light-sensitive soma-targeted opsins in vivo, stCoChR allowed neuronal stimulation with more than 10-fold lower average power and no spectral crosstalk. The combination of stCoChR, tuned amplified laser stimulation, and red-shifted functional indicators promises to be a powerful tool for large-scale interrogation of neural networks in the intact brain.

Abstract Genetic variants account for approximately half the cases of congenital and early‐onset deafness. Methods and technologies for viral delivery of genes into the inner ear have evolved over the past decade to render gene therapy a viable and attractive approach for treatment. Variants in SYNE4, encoding the protein nesprin‐4, a member of the linker of nucleoskeleton and cytoskeleton (LINC), lead to DFNB76 human deafness. Syne4−/− mice have severe‐to‐profound progressive hearing loss and exhibit mislocalization of hair cell nuclei and hair cell degeneration. We used AAV9‐PHP.B, a recently developed synthetic adeno‐associated virus, to deliver the coding sequence of Syne4 into the inner ears of neonatal Syne4−/− mice. Here we report rescue of hair cell morphology and survival, nearly complete recovery of auditory function, and restoration of auditory‐associated behaviors, without observed adverse effects. Uncertainties remain regarding the durability of the treatment and the time window for intervention in humans, but our results suggest that gene therapy has the potential to prevent hearing loss in humans with SYNE4 mutations.

Current optogenetic inhibition methods like light-controlled ion pumps require high-intensity light and disrupt physiological ion gradients. Here, the authors somatically target the anion-conducting opsin GtACR to eliminate spiking in distal axons and improve photocurrents, thus enhancing its utility.

Mouse primary somatosensory barrel cortex (wS1) processes whisker sensory information, receiving input from two distinct thalamic nuclei. The first-order ventral posterior medial (VPM) somatosensory thalamic nucleus most densely innervates layer 4 (L4) barrels, whereas the higher-order posterior thalamic nucleus (medial part, POm) most densely innervates L1 and L5A. We optogenetically stimulated VPM or POm axons, and recorded evoked excitatory postsynaptic potentials (EPSPs) in different cell-types across cortical layers in wS1. We found that excitatory neurons and parvalbumin-expressing inhibitory neurons received the largest EPSPs, dominated by VPM input to L4 and POm input to L5A. In contrast, somatostatin-expressing inhibitory neurons received very little input from either pathway in any layer. Vasoactive intestinal peptide-expressing inhibitory neurons received an intermediate level of excitatory input with less apparent layer-specificity. Our data help understand how wS1 neocortical microcircuits might process and integrate sensory and higher-order inputs.

Summary: Sensory information is encoded within the brain in distributed spatiotemporal patterns of neuronal activity. Understanding how these patterns influence behavior requires a method to measure and to bidirectionally perturb with high spatial resolution the activity of the multiple neuronal cell types engaged in sensory processing. Here, we combined two-photon holography to stimulate neurons expressing blue light-sensitive opsins (ChR2 and GtACR2) with two-photon imaging of the red-shifted indicator jRCaMP1a in the mouse neocortex in vivo. We demonstrate efficient control of neural excitability across cell types and layers with holographic stimulation and improved spatial resolution by opsin somatic targeting. Moreover, we performed simultaneous two-photon imaging of jRCaMP1a and bidirectional two-photon manipulation of cellular activity with negligible effect of the imaging beam on opsin excitation. This all-optical approach represents a powerful tool to causally dissect how activity patterns in specified ensembles of neurons determine brain function and animal behavior. : Forli et al. developed an all-optical method to image and bidirectionally manipulate brain networks with high spatial resolution and minimal crosstalk in the intact mammalian brain. They validate the method across cell types and layers in the mouse neocortex. Keywords: optogenetics, two-photon excitation, digital holography, patterned illumination, two-photon imaging

Abstract Background The quantitative relations between RNA and protein are fundamental to biology and are still not fully understood. Across taxa, it was demonstrated that the protein-to-mRNA ratio in steady state varies in a direction that lessens the change in protein levels as a result of changes in the transcript abundance. Evidence for this behavior in tissues is sparse. We tested this phenomenon in new data that we produced for the mouse auditory system, and in previously published tissue datasets. A joint analysis of the transcriptome and proteome was performed across four datasets: inner-ear mouse tissues, mouse organ tissues, lymphoblastoid primate samples and human cancer cell lines. Results We show that the protein levels are more conserved than the mRNA levels in all datasets, and that changes in transcription are associated with translational changes that exert opposite effects on the final protein level, in all tissues except cancer. Finally, we observe that some functions are enriched in the inner ear on the mRNA level but not in protein. Conclusions We suggest that partial buffering between transcription and translation ensures that proteins can be made rapidly in response to a stimulus. Accounting for the buffering can improve the prediction of protein levels from mRNA levels.

In vivo experimental evidence indicates that acute neuronal activation increases Aβ release from presynaptic terminals, whereas long-term effects of chronic synaptic activation on Aβ pathology remain unclear. To address this issue, we adopted optogenetics and transduced stabilized step-function opsin, a channelrhodopsin engineered to elicit a long-lasting neuronal hyperexcitability, into the hippocampal perforant pathway of APP transgenic mice. In vivo microdialysis revealed a ∼24% increase in the hippocampal interstitial fluid Aβ42 levels immediately after acute light activation. Five months of chronic optogenetic stimulation increased Aβ burden specifically in the projection area of the perforant pathway (i.e., outer molecular layer of the dentate gyrus) of the stimulated side by ∼2.5-fold compared with that in the contralateral side. Epileptic seizures were observed during the course of chronic stimulation, which might have partly contributed to the Aβ pathology. These findings implicate functional abnormalities of specific neuronal circuitry in Aβ pathology and Alzheimer disease.

Sodium pumping rhodopsins (NaRs) are a unique member of the microbial-type I rhodopsin family which actively transport Na+ and H+ depending on ionic condition. In this study, we surveyed 12 different NaRs from various sources of eubacteria for their electrophysiological as well as spectroscopic properties. In mammalian cells several of these NaRs exhibited a Na+ based pump photocurrent and four interesting candidates were chosen for further characterization. Voltage dependent photocurrent amplitudes revealed a membrane potential-sensitive turnover rate, indicating the presence of an electrically-charged intermediate(s) in the photocycle reaction. The NaR from Salinarimonas rosea DSM21201 exhibited a red-shifted absorption spectrum, and slower kinetics compared to the first described sodium pump, KR2. Although the ratio of Na+ to H+ ion transport varied among the NaRs we tested, the NaRs from Flagellimonas sp_DIK and Nonlabens sp_YIK_SED-11 showed significantly higher Na+ selectivity when compared to KR2. All four further investigated NaRs showed a functional expression in dissociated hippocampal neuron culture and hyperpolarizing activity upon light-stimulation. Additionally, all four NaRs allowed optical inhibition of electrically-evoked neuronal spiking. Although efficiency of silencing was 3-5 times lower than silencing with the enhanced version of the proton pump AR3 from Halorubrum sodomense, our data outlines a new approach for hyperpolarization of excitable cells without affecting the intracellular and extracellular proton environment.

In neurons and neuroendocrine cells, docked vesicles need to undergo priming to become fusion competent. Priming is a multi-step process that was shown to be associated with vesicle immobilization. However, it is not known whether vesicle immobilization is sufficient to acquire complete fusion competence. To extend our understanding of the physical manifestation of vesicle priming, we took advantage of tomosyn, a SNARE-related protein that specifically inhibits vesicle priming, and measured its effect on vesicle dynamics in live chromaffin cells using total internal reflection fluorescence microscopy. We show here that while in control cells vesicles undergo immobilization before fusion, vesicle immobilization is attenuated in tomosyn overexpressing cells. This in turn increases the turnover rate of vesicles near the membrane and attenuates the fusion of newcomer vesicles. Moreover, the release probability of immobile vesicles in tomosyn cells is significantly reduced, suggesting that immobilization is an early and necessary step in priming but is insufficient, as further molecular processes are needed to acquire complete fusion competence. Using tomosyn as a molecular tool we provide a mechanistic link between functional docking and priming and suggest that functional docking is the first step in vesicle priming, followed by molecular modifications that do not translate into changes in vesicle mobility.