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The small Ultra-Red Fluorescent Protein (smURFP) represents a new class of fluorescent protein with exceptional photostability and brightness derived from allophycocyanin in a previous directed evolution. Here, we report the smURFP crystal structure to better understand properties and enable further engineering of improved variants. We compare this structure to the structures of allophycocyanin and smURFP mutants to identify the structural origins of the molecular brightness. We then use a structure-guided approach to develop monomeric smURFP variants that fluoresce with phycocyanobilin but not biliverdin. Furthermore, we measure smURFP photophysical properties necessary for advanced imaging modalities, such as those relevant for two-photon, fluorescence lifetime, and single-molecule imaging. We observe that smURFP has the largest two-photon cross-section measured for a fluorescent protein, and that it produces more photons than organic dyes. Altogether, this study expands our understanding of the smURFP, which will inform future engineering toward optimal FPs compatible with whole organism studies.

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a popular chemogenetic technology for manipulation of neuronal activity in uninstrumented awake animals with potential for human applications as well. The prototypical DREADD agonist clozapine N-oxide (CNO) lacks brain entry and converts to clozapine, making it difficult to apply in basic and translational applications. Here we report the development of two novel DREADD agonists, JHU37152 and JHU37160, and the first dedicated 18F positron emission tomography (PET) DREADD radiotracer, [18F]JHU37107. We show that JHU37152 and JHU37160 exhibit high in vivo DREADD potency. [18F]JHU37107 combined with PET allows for DREADD detection in locally-targeted neurons, and at their long-range projections, enabling noninvasive and longitudinal neuronal projection mapping.

Optogenetic techniques provide genetically targeted, spatially and temporally precise approaches to correlate cellular activities and physiological outcomes. In the nervous system, G protein-coupled receptors (GPCRs) have essential neuromodulatory functions through binding extracellular ligands to induce intracellular signaling cascades. In this work, we develop and validate an optogenetic tool that disrupts Gαq signaling through membrane recruitment of a minimal regulator of G protein signaling (RGS) domain. This approach, Photo-induced Gα Modulator--Inhibition of Gαq (PiGM-Iq), exhibited potent and selective inhibition of Gαq signaling. Using PiGM-Iq we alter the behavior of Caenorhabditis elegans and Drosophila with outcomes consistent with GPCR-Gαq disruption. PiGM-Iq changes axon guidance in cultured dorsal root ganglia neurons in response to serotonin. PiGM-Iq activation leads to developmental deficits in zebrafish embryos and larvae resulting in altered neuronal wiring and behavior. Furthermore, by altering the minimal RGS domain, we show that this approach is amenable to Gαi signaling. Our unique and robust optogenetic Gα inhibiting approaches complement existing neurobiological tools and can be used to investigate the functional effects neuromodulators that signal through GPCR and trimeric G proteins. [ABSTRACT FROM AUTHOR]

Over the past 20 years, protein engineering has been extensively used to improve and modify the fundamental properties of fluorescent proteins (FPs) with the goal of adapting them for a fantastic range of applications. FPs have been modified by a combination of rational design, structure-based mutagenesis, and countless cycles of directed evolution (gene diversification followed by selection of clones with desired properties) that have collectively pushed the properties to photophysical and biochemical extremes. In this review, we provide both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems.

Viral vectors and lipofection‐based gene therapies have dispersion‐dependent transduction/transfection profiles that thwart precise targeting. The study describes the development of focused close‐field gene electrotransfer (GET) technology, refining spatial control of gene expression. Integration of fluidics for precise delivery of "naked" plasmid deoxyribonucleic acid (DNA) in sucrose carrier within the focused electric field enables negative biasing of near‐field conductivity ("conductivity‐clamping"–CC), increasing the efficiency of plasma membrane molecular translocation. This enables titratable gene delivery with unprecedently low charge transfer. The clinic‐ready bionics‐derived CC‐GET device achieved neurotrophin‐encoding miniplasmid DNA delivery to the cochlea to promote auditory nerve regeneration; validated in deafened guinea pig and cat models, leading to improved central auditory tuning with bionics‐based hearing. The performance of CC‐GET is evaluated in the brain, an organ problematic for pulsed electric field‐based plasmid DNA delivery, due to high required currents causing Joule‐heating and damaging electroporation. Here CC‐GET enables safe precision targeting of gene expression. In the guinea pig, reporter expression is enabled in physiologically critical brainstem regions, and in the striatum (globus pallidus region) delivery of a red‐shifted channelrhodopsin and a genetically‐encoded Ca2+ sensor, achieved photoactivated neuromodulation relevant to the treatment of Parkinson's Disease and other focal brain disorders. [ABSTRACT FROM AUTHOR]

It has been proposed that memories are encoded by modification of synaptic strengths through cellular mechanisms such as long-term potentiation (LTP) and long-term depression (LTD). However, the causal link between these synaptic processes and memory has been difficult to demonstrate. Here we show that fear conditioning, a type of associative memory, can be inactivated and reactivated by LTD and LTP, respectively. We began by conditioning an animal to associate a foot shock with optogenetic stimulation of auditory inputs targeting the amygdala, a brain region known to be essential for fear conditioning. Subsequent optogenetic delivery of LTD conditioning to the auditory input inactivates memory of the shock. Then subsequent optogenetic delivery of LTP conditioning to the auditory input reactivates memory of the shock. Thus, we have engineered inactivation and reactivation of a memory using LTD and LTP, supporting a causal link between these synaptic processes and memory.

Optogenetics allows the manipulation of neural activity in freely moving animals with millisecond precision, but its application in Drosophila melanogaster has been limited. Here we show that a recently described red activatable channelrhodopsin (ReaChR) permits control of complex behavior in freely moving adult flies, at wavelengths that are not thought to interfere with normal visual function. This tool affords the opportunity to control neural activity over a broad dynamic range of stimulation intensities. Using time-resolved activation, we show that the neural control of male courtship song can be separated into (i) probabilistic, persistent and (ii) deterministic, command-like components. The former, but not the latter, neurons are subject to functional modulation by social experience, which supports the idea that they constitute a locus of state-dependent influence. This separation is not evident using thermogenetic tools, a result underscoring the importance of temporally precise control of neuronal activation in the functional dissection of neural circuits in Drosophila.

We present an optimized triple modality reporter construct combining a far-red fluorescent protein (E2-Crimson), enhanced firefly luciferase enzyme (Luc2), and truncated wild type herpes simplex virus I thymidine kinase (wttk) that allows for sensitive, long-term tracking of tumor growth in vivo by fluorescence, bioluminescence, and positron emission tomography. Two human cancer cell lines (MDA-MB-231 breast cancer and HT-1080 fibrosarcoma cancer) were successfully transduced to express this triple modality reporter. Fluorescence and bioluminescence imaging of the triple modality reporter were used to accurately quantify the therapeutic responses of MDA-MB-231 tumors to the chemotherapeutic agent monomethyl auristatin E in vivo in athymic nude mice. Positive correlation was observed between the fluorescence and bioluminescence signals, and these signals were also positively correlated with the ex vivo tumor weights. This is the first reported use of both fluorescence and bioluminescence signals from a multi-modality reporter construct to measure drug efficacy in vivo.

Channelrhodopsins (ChRs) are used to optogenetically depolarize neurons. We engineered a variant of ChR, denoted red-activatable ChR (ReaChR), that is optimally excited with orange to red light (λ ∼590-630 nm) and offers improved membrane trafficking, higher photocurrents and faster kinetics compared to existing red-shifted ChRs. Red light is less scattered by tissue and is absorbed less by blood than the blue to green wavelengths that are required by other ChR variants. We used ReaChR expressed in the vibrissa motor cortex to drive spiking and vibrissa motion in awake mice when excited with red light through intact skull. Precise vibrissa movements were evoked by expressing ReaChR in the facial motor nucleus in the brainstem and illumination with red light through the external auditory canal. Thus, ReaChR enables transcranial optical activation of neurons in deep brain structures without the need to surgically thin the skull, form a transcranial window or implant optical fibers.

The hypothalamus responds to circulating leptin and insulin in the control of food intake and body weight. A number of neurotransmitters in the hypothalamus, including [gamma]-aminobutyric acid (GABA), also have key roles in feeding. Huntingtin-associated protein 1 (Hap1) is expressed more abundantly in the hypothalamus than in other brain regions, and lack of Hap1 in mice leads to early postnatal death. Hap1 is also involved in intracellular trafficking of the GABA[sub.A] receptor. Here, we report that fasting upregulates the expression of Hap1 in the rodent hypothalamus, whereas intracerebroventricular administration of insulin downregulates Hap1 by increasing its degradation through ubiquitination. Decreasing the expression of mouse hypothalamic Hap1 by siRNA reduces the level and activity of hypothalamic GABA[sub.A] receptors and causes a decrease in food intake and body weight. These findings provide evidence linking hypothalamic Hap1 to GABA in the stimulation of feeding and suggest that this mechanism is involved in the feeding-inhibitory actions of insulin in the brain., Author(s): Guoqing Sheng [1]; Guo-qing Chang [2]; John Y Lin [3]; Zhao-Xue Yu [1]; Zhi-Hui Fang [1]; Juan Rong [1]; Stuart A Lipton [3, 4]; Shi-Hua Li [1]; Gang Tong [...]

Cortical cells integrate synaptic input from multiple sources, but how these different inputs are distributed across individual neurons is largely unknown. Differences in input might account for diverse responses in neighboring neurons during behavior. We present a strategy for comparing the strengths of multiple types of input onto the same neuron. We developed methods for independent dualchannel photostimulation of synaptic inputs using ChR2 together with ReaChR, a red-shifted channelrhodopsin. We used dual-channel photostimulation to probe convergence of sensory information in the mouse primary motor cortex. Input from somatosensory cortex and thalamus converges in individual neurons. Similarly, inputs from distinct somatotopic regions of the somatosensory cortex are integrated at the level of single motor cortex neurons. We next developed a ReaChR transgenic mouse under the control of both Flp- and Cre-recombinases that is an effective tool for circuit mapping. Our approach to dual-channel photostimulation enables quantitative comparison of the strengths of multiple pathways across all length scales of the brain. [ABSTRACT FROM AUTHOR]

We present an optimized triple modality reporter construct combining a far-red fluorescent protein (E2-Crimson), enhanced firefly luciferase enzyme (Luc2), and truncated wild type herpes simplex virus I thymidine kinase (wttk) that allows for sensitive, long-term tracking of tumor growth in vivo by fluorescence, bioluminescence, and positron emission tomography. Two human cancer cell lines (MDA-MB-231 breast cancer and HT-1080 fibrosarcoma cancer) were successfully transduced to express this triple modality reporter. Fluorescence and bioluminescence imaging of the triple modality reporter were used to accurately quantify the therapeutic responses of MDA-MB-231 tumors to the chemotherapeutic agent monomethyl auristatin E in vivo in athymic nude mice. Positive correlation was observed between the fluorescence and bioluminescence signals, and these signals were also positively correlated with the ex vivo tumor weights. This is the first reported use of both fluorescence and bioluminescence signals from a multi-modality reporter construct to measure drug efficacy in vivo. [ABSTRACT FROM AUTHOR]

The hypothalamus responds to circulating leptin and insulin in the control of food intake and body weight. A number of neurotransmitters in the hypothalamus, including γ-aminobutyric acid (GABA), also have key roles in feeding. Huntingtin-associated protein 1 (Hap1) is expressed more abundantly in the hypothalamus than in other brain regions, and lack of Hap1 in mice leads to early postnatal death. Hap1 is also involved in intracellular trafficking of the GABAA receptor. Here, we report that fasting upregulates the expression of Hap1 in the rodent hypothalamus, whereas intracerebroventricular administration of insulin downregulates Hap1 by increasing its degradation through ubiquitination. Decreasing the expression of mouse hypothalamic Hap1 by siRNA reduces the level and activity of hypothalamic GABAA receptors and causes a decrease in food intake and body weight. These findings provide evidence linking hypothalamic Hap1 to GABA in the stimulation of feeding and suggest that this mechanism is involved in the feeding-inhibitory actions of insulin in the brain. [ABSTRACT FROM AUTHOR]

Optogenetic techniques provide genetically targeted, spatially and temporally precise approaches to correlate cellular activities and physiological outcomes. In the nervous system, G protein-coupled receptors (GPCRs) have essential neuromodulatory functions through binding extracellular ligands to induce intracellular signaling cascades. In this work, we develop and validate an optogenetic tool that disrupts Gα q signaling through membrane recruitment of a minimal regulator of G protein signaling (RGS) domain. This approach, Photo-induced Gα Modulator-Inhibition of Gα q (PiGM-Iq), exhibited potent and selective inhibition of Gα q signaling. Using PiGM-Iq we alter the behavior of Caenorhabditis elegans and Drosophila with outcomes consistent with GPCR-Gα q disruption. PiGM-Iq changes axon guidance in cultured dorsal root ganglia neurons in response to serotonin. PiGM-Iq activation leads to developmental deficits in zebrafish embryos and larvae resulting in altered neuronal wiring and behavior. Furthermore, by altering the minimal RGS domain, we show that this approach is amenable to Gα i signaling. Our unique and robust optogenetic Gα inhibiting approaches complement existing neurobiological tools and can be used to investigate the functional effects neuromodulators that signal through GPCR and trimeric G proteins., Competing Interests: Competing interests statement:The authors declare no competing interest.

Recent discovery of the light-activated ion channel, channelrhodopsin (ChR), has provided researchers a powerful and convenient tool to manipulate the membrane potential of specific cells with light. With genetic targeting of these channels and illumination of light to a specific location, the experimenter can selectively activate the voltage-gated ion channels (VGICs) of ChR-expressing cells, initiating electrical signaling in temporally and spatially precise manners. In neuroscience research, this can be used to study electrical signal processing within one neuron at the cellular level, or the synaptic connectivity between neurons at the circuitry level. To conduct experiments with ChRs, these exogenous channels need to be introduced into the cells of interest, commonly through a viral approach. This chapter provides an overview of the design, production, and validation of recombinant adeno-associated virus (rAAV) for ChR expression that can be used in vitro or in vivo to infect neurons. The virus produced can be used to conduct "optogenetic" experiments in behaving animals, in vitro preparations and cultured cells, and can be used to study signal transduction and processing at a cellular or circuitry level.

Classically, temporally precise excitation of membrane potential in neurons within intact tissue can be achieved by direct electrical stimulation or indirect electrical stimulation induced by changing magnetic fields. Both of these approaches have a predetermined selectivity based on the biophysical properties of the nervous tissue and membrane in the region of the stimulation. A recent advance in selective excitation of neurons is the "optogenetic" approach utilizing channelrhodopsins (ChRs). By expressing the light-responsive ChR in neurons using cell-type selective promoters or other methods, specific neurons can be depolarized by light in a temporally precise manner with millisecond resolution even if their membrane biophysical properties are less favorable for electrical stimulation. In addition, ChRs can be used to depolarize nonneuronal cells in the nervous tissue, and to sustain depolarization over a prolonged period of time, both of which cannot be achieved with electrical or magnetic stimulations. To conduct an experiment with ChR, experimenters need to make the correct choices on the three main components to such an experiment: the expression system, the illumination source, and the ChR variant used. This chapter aims to provide some discussions on the current developments of these aspects of the experiments. To express ChR in neurons, the common expression systems include viral vectors, in utero electroporation, and transgenic animals, each with their advantages and limitations regarding the cost, expression pattern, and the required effort. In terms of the instrumentation, an illumination source that is capable of providing the desired wavelength with high intensity is crucial for the success of the experiment. The important factors regarding the light source used include the cost, light density output, efficiency for fiber coupling for in vivo rodent experiments, and the available methods to control light intensity and onset/termination. The third component of the experiment is the choice of the appropriate variants of ChR. Many novel ChR variants with unique properties have been engineered, and it can be difficult for the experimenters to choose the right variant with the desired properties for their experiments, as some information necessary for the experimenter to make the right selection is often incomplete or unavailable. Currently, the available variants for neuroscientific research are wild-type ChR2, ChR2+H134R, ChETA, VChR1, SFO, ChD, ChEF, ChIEF, ChRGR, CatCh, and TC. The features and limitations of these different variants are presented here. Lastly, this chapter will provide some suggestion for the future development of the light source, expression system, and the development of the "next" generation of ChRs., (Copyright © 2012 Elsevier B.V. All rights reserved.)