Extracting structural and functional features of widely distributed biological circuits with single cell resolution via tissue clearing and delivery vectors

The scientific community has learned a great deal from imaging small and naturally transparent organisms such as nematodes and zebrafish. The consequences of genetic mutations on their organ development and survival can be visualized easily and with high-throughput at the organism-wide scale. In contrast, three-dimensional information is less accessible in mammalian subjects because the heterogeneity of light-scattering tissue elements renders their organs opaque. Likewise, genetically labeling desired circuits across mammalian bodies is prohibitively slow and costly via the transgenic route. Emerging breakthroughs in viral vector engineering, genome editing tools, and tissue clearing can render larger opaque organisms genetically tractable and transparent for whole-organ cell phenotyping, tract tracing and imaging at depth

Extracting structural and functional features of widely distributed biological circuits with single cell resolution via tissue clearing and delivery vectors

The scientific community has learned a great deal from imaging small and naturally transparent organisms such as nematodes and zebrafish. The consequences of genetic mutations on their organ development and survival can be visualized easily and with high-throughput at the organism-wide scale. In contrast, three-dimensional information is less accessible in mammalian subjects because the heterogeneity of light-scattering tissue elements renders their organs opaque. Likewise, genetically labeling desired circuits across mammalian bodies is prohibitively slow and costly via the transgenic route. Emerging breakthroughs in viral vector engineering, genome editing tools, and tissue clearing can render larger opaque organisms genetically tractable and transparent for whole-organ cell phenotyping, tract tracing and imaging at depth

Optical Deconstruction of Parkinsonian Neural Circuitry

Deep brain stimulation (DBS) is a therapeutic option for intractable neurological and psychiatric disorders, including Parkinson's disease and major depression. Because of the heterogeneity of brain tissues where electrodes are placed, it has been challenging to elucidate the relevant target cell types or underlying mechanisms of DBS. We used optogenetics and solid-state optics to systematically drive or inhibit an array of distinct circuit elements in freely moving parkinsonian rodents and found that therapeutic effects within the subthalamic nucleus can be accounted for by direct selective stimulation of afferent axons projecting to this region. In addition to providing insight into DBS mechanisms, these results demonstrate an optical approach for dissection of disease circuitry and define the technological toolbox needed for systematic deconstruction of disease circuits by selectively controlling individual components

eNpHR: a Natronomonas halorhodopsin enhanced for optogenetic applications

Temporally precise inhibition of distinct cell types in the intact nervous system has been enabled by the microbial halorhodopsin NpHR, a fast light-activated electrogenic Cl^− pump. While neurons can be optically hyperpolarized and inhibited from firing action potentials at moderate NpHR expression levels, we have encountered challenges with pushing expression to extremely high levels, including apparent intracellular accumulations. We therefore sought to molecularly engineer NpHR to achieve strong expression without these cellular side effects. We found that high expression correlated with endoplasmic reticulum (ER) accumulation, and that under these conditions NpHR colocalized with ER proteins containing the KDEL ER retention sequence. We screened a number of different putative modulators of membrane trafficking and identified a combination of two motifs, an N-terminal signal peptide and a C-terminal ER export sequence, that markedly promoted membrane localization and ER export defined by confocal microscopy and whole-cell patch clamp. The modified NpHR displayed increased peak photocurrent in the absence of aggregations or toxicity, and potent optical inhibition was observed not only in vitro but also in vivo with thalamic single-unit recording. The new enhanced NpHR (eNpHR) allows safe, high-level expression in mammalian neurons, without toxicity and with augmented inhibitory function, in vitro and in vivo

Hydrogel-Tissue Chemistry: Principles and Applications

Over the past five years, a rapidly developing experimental approach has enabled high-resolution and high-content information retrieval from intact multicellular animal (metazoan) systems. New chemical and physical forms are created in the hydrogel-tissue chemistry process, and the retention and retrieval of crucial phenotypic information regarding constituent cells and molecules (and their joint interrelationships) are thereby enabled. For example, rich data sets defining both single-cell-resolution gene expression and single-cell-resolution activity during behavior can now be collected while still preserving information on three-dimensional positioning and/or brain-wide wiring of those very same neurons—even within vertebrate brains. This new approach and its variants, as applied to neuroscience, are beginning to illuminate the fundamental cellular and chemical representations of sensation, cognition, and action. More generally, reimagining metazoans as metareactants—or positionally defined three-dimensional graphs of constituent chemicals made available for ongoing functionalization, transformation, and readout—is stimulating innovation across biology and medicine

Q&A: How can advances in tissue clearing and optogenetics contribute to our understanding of normal and diseased biology?

Mammalian organs comprise a variety of cells that interact with each other and have distinct biological roles. Access to evaluate and perturb intact biological systems at the cellular and molecular levels is essential to fully understand their functioning in normal and diseased conditions, yet technical limitations have constrained most research to small pieces of tissue. Tissue clearing and optogenetics can help overcome this hurdle: tissue clearing affords optical interrogation of whole organs at the molecular level, and optogenetics enables the scalable control and measurement of cellular activity with light. In this Q&A, we delineate recent advances and practical challenges associated with these two techniques when applied body-wide

Proceedings of the Sixth Deep Brain Stimulation Think Tank Modulation of Brain Networks and Application of Advanced Neuroimaging, Neurophysiology, and Optogenetics

The annual deep brain stimulation (DBS) Think Tank aims to create an opportunity for a multidisciplinary discussion in the field of neuromodulation to examine developments, opportunities and challenges in the field. The proceedings of the Sixth Annual Think Tank recapitulate progress in applications of neurotechnology, neurophysiology, and emerging techniques for the treatment of a range of psychiatric and neurological conditions including Parkinson’s disease, essential tremor, Tourette syndrome, epilepsy, cognitive disorders, and addiction. Each section of this overview provides insight about the understanding of neuromodulation for specific disease and discusses current challenges and future directions. This year’s report addresses key issues in implementing advanced neurophysiological techniques, evolving use of novel modulation techniques to deliver DBS, ans improved neuroimaging techniques. The proceedings also offer insights into the new era of brain network neuromodulation and connectomic DBS to define and target dysfunctional brain networks. The proceedings also focused on innovations in applications and understanding of adaptive DBS (closed-loop systems), the use and applications of optogenetics in the field of neurostimulation and the need to develop databases for DBS indications. Finally, updates on neuroethical, legal, social, and policy issues relevant to DBS research are discussed