Browsing by Subject "Optogenetics"

General anesthesia (GA) can produce analgesia (loss of pain) independent of inducing loss of consciousness, but the underlying mechanisms remain unclear. We hypothesized that GA suppresses pain in part by activating supraspinal analgesic circuits. We discovered a distinct population of GABAergic neurons activated by GA in the mouse central amygdala (CeAGA neurons). In vivo calcium imaging revealed that different GA drugs activate a shared ensemble of CeAGA neurons. CeAGA neurons also possess basal activity that mostly reflect animals’ internal state rather than external stimuli. Optogenetic activation of CeAGA potently suppressed both pain-elicited reflexive and self-recuperating behaviors across sensory modalities, and abolished neuropathic pain-induced mechanical (hyper-)sensitivity. Conversely, inhibition of CeAGA activity exacerbated pain, produced strong aversion, and cancelled the analgesic effect of low-dose ketamine. CeAGA neurons have widespread inhibitory projections to numerous affective pain-processing centers. Our study points to CeAGA as a potential powerful therapeutic target for alleviating chronic pain.

The growing complexity of scientific research demands further development of advanced gene regulation systems. For instance, the ultimate goal of tissue engineering is to develop constructs that functionally and morphologically resemble the native tissue they are expected to replace. This requires patterning of gene expression and control of cellular phenotype within the tissue engineered construct. In the field of synthetic biology, gene circuits are engineered to elucidate mechanisms of gene regulation and predict the behavior of more complex systems. Such systems require robust gene switches that can quickly turn gene expression on or off. Similarly, basic science requires precise genetic control to perturb genetic pathways or understand gene function. Additionally, gene therapy strives to replace or repair genes that are responsible for disease. The safety and efficacy of such therapies require control of when and where the delivered gene is expressed in vivo.

Unfortunately, these fields are limited by the lack of gene regulation systems that enable both robust and flexible cellular control. Most current gene regulation systems do not allow for the manipulation of gene expression that is spatially defined, temporally controlled, reversible, and repeatable. Rather, they provide incomplete control that forces the user to choose to control gene expression in either space or time, and whether the system will be reversible or irreversible.

The recent emergence of the field of optogenetics--the ability to control gene expression using light--has made it possible to regulate gene expression with spatial, temporal, and dynamic control. Light-inducible systems provide the tools necessary to overcome the limitations of other gene regulation systems, which can be slow, imprecise, or cumbersome to work with. However, emerging light-inducible systems require further optimization to increase their efficiency, reliability, and ease of use.

Initially, we engineered a light-inducible gene regulation system that combines zinc finger protein technology and the light-inducible interaction between Arabidopsis thaliana plant proteins GIGANTEA (GI) and the light oxygen voltage (LOV) domain of FKF1. Zinc finger proteins (ZFPs) can be engineered to target almost any DNA sequence through tandem assembly of individual zinc finger domains that recognize a specific three base-pair DNA sequence. Fusion of three different ZFPs to GI (GI-ZFP) successfully targeted the fusion protein to the specific DNA target sequence of the ZFP. Due to the interaction between GI and LOV, co-expression of GI-ZFP with a fusion protein consisting of LOV fused to three copies of the VP16 transactivation domain (LOV-VP16) enabled blue-light dependent recruitment of LOV-VP16 to the ZFP target sequence. We showed that placement of three to nine copies of a ZFP target sequence upstream of a luciferase or eGFP transgene enabled expression of the transgene in response to blue-light. Gene activation was both reversible and tunable based on duration of light exposure, illumination intensity, and the number of ZFP binding sites upstream of the transgene. Gene expression could also be spatially patterned by illuminating the cell culture through photomasks containing various patterns.

Although this system was useful for controlling the expression of a transgene, for many applications it is useful to control the expression of a gene in its natural chromosomal position. Therefore we capitalized on recent advances in programmed gene activation to engineer an optogenetic tool that could easily be targeted to new, endogenous DNA sequences without re-engineering the light inducible proteins. This approach took advantage of CRISPR/Cas9 technology, which uses a gene-specific guide RNA (gRNA) to facilitate Cas9 targeting and binding to a desired sequence, and the light-inducible heterodimerizers CRY2 and CIB1 from Arabidopsis thaliana to engineer a light-activated CRISPR/Cas9 effector (LACE) system. We fused the full-length (FL) CRY2 to the transcriptional activator VP64 (CRY2FL-VP64) and the N-terminal fragment of CIB1 to the N-, C-, or N- and C- terminus of a catalytically inactive Cas9. When CRY2-VP64 and one of the CIBN/dCas9 fusion proteins are expressed with a gRNA, the CIBN/dCas9 fusion protein localizes to the gRNA target. In the presence of blue light, CRY2FL binds to CIBN, which translocates CRY2FL-VP64 to the gene target and activates transcription. Unlike other optogenetic systems, the LACE system can be targeted to new endogenous loci by solely manipulating the specificity of the gRNA without having to re-engineer the light-inducible proteins. We achieved light-dependent activation of the IL1RN, HBG1/2, or ASCL1 genes by delivery of the LACE system and four gene-specific gRNAs per promoter region. For some gene targets, we achieved equivalent activation levels to cells that were transfected with the same gRNAs and the synthetic transcription factor dCas9-VP64. Gene activation was also shown to be reversible and repeatable through modulation of the duration of blue light exposure, and spatial patterning of gene expression was achieved using an eGFP reporter and a photomask.

Finally, we engineered a light-activated genetic "on" switch (LAGOS) that provides permanent gene expression in response to an initial dose of blue light illumination. LAGOS is a lentiviral vector that expresses a transgene only upon Cre recombinase-mediated DNA recombination. We showed that this vector, when used in conjunction with a light-inducible Cre recombinase system,1 could be used to express MyoD or the synthetic transcription factor VP64-MyoD2 in response to light in multiple mammalian cell lines, including primary mouse embryonic fibroblasts. We achieved light-mediated upregulation of downstream myogenic markers myogenin, desmin, troponin T, and myosin heavy chains I and II as well as fusion of C3H10T½ cells into myotubes that resembled a skeletal muscle cell phenotype. We also demonstrated LAGOS functionality in vivo by engineering the vector to express human VEGF165 and human ANG1 in response to light. HEK 293T cells stably expressing the LAGOS vector and transiently expressing the light-inducible Cre recombinase proteins were implanted into mouse dorsal window chambers. Mice that were illuminated with blue light had increased microvessel density compared to mice that were not illuminated. Analysis of human VEGF and human ANG1 levels by enzyme-linked immunosorbent assay (ELISA) revealed statistically higher levels of VEGF and ANG1 in illuminated mice compared to non-illuminated mice.

In summary, the objective of this work was to engineer robust light-inducible gene regulation systems that can control genes and cellular fate in a spatial and temporal manner. These studies combine the rapid advances in gene targeting and activation technology with natural light-inducible plant protein interactions. Collectively, this thesis presents several optogenetic systems that are expected to facilitate the development of multicellular cell and tissue constructs for use in tissue engineering, synthetic biology, gene therapy, and basic science both in vitro and in vivo.

Targeted stimulation of the brain has the potential to treat mental illnesses. The objective of this work is to develop methodology that enables scientists to design stimulation methods based on the electrophysiological dynamics. We first develop several factor models that characterize aspects of the dynamics relevant to these illnesses. Using a novel approach, we can then find a single predictive factor of the trait of interest. To improve the quality of the associated loadings, we develop a method for removing concomitant variables that can dominate the observed dynamics. We also develop a novel inference technique that increases the relevance of the predictive loadings. Finally, we demonstrate the efficacy of our methodology by finding a single factor responsible for social behavior. This factor is stimulated in new subjects and modifies behavior in the new individuals. These results indicate that our methodology has high potential in developing future cures of mental illness.

Chronic pain is a major public health issue, affecting over 100 million people in costing over $600 million annually in the United States. The lack of effective therapies for chronic pain have directly contributed to the ongoing epidemic of opioid abuse and addiction. Deeper understanding the pathogenesis of chronic pain is a prerequisite for remedying the status quo. To that end, in this dissertation, I have undertaken two projects that aim to elucidate the key cellular elements of mechanical pain in the periphery and spinal cord.

Mechanical allodynia is a cardinal feature of pathological pain in which innocuous mechanical stimulation such as light touch produces a painful sensation. Recent work has demonstrated the necessity of cutaneous Aβ low-threshold mechanoreceptors (Aβ-LTMRs) for mechanical allodynia-like behaviors in mice, but its remains unclear whether activation of these neurons alone is sufficient to produce pain behaviors in pathological settings. To address this question, in the first part of this dissertation, I generated and characterized a transgenic mouse line that expresses the optogenetic actuator channelrhodopsin-2 (ChR2) conditionally in Vesicular Glutamate Transporter 1 (Vglut1)-expressing sensory neurons(Vglut1-ChR2). I show that the Vglut1-ChR2 comprises a heterogeneous population of Neurofilament 200-positive, large-sized sensory neurons with cutaneous projections that terminate in Merkel Cell-Neurite Complexes, Meissner Corpuscles and Hair Follicles and with spinal projections that terminate in the deep dorsal horn (Lamina IIi-V) and ventral horn in the spinal cord. In naive Vglut1-ChR2 mice, acute transdermal photostimulation of the plantar hindpaw with blue (470nm) light produced paw withdrawal behaviors in an intensity- and frequency-dependent manner that were abolished by selective pharmacological A-fiber blockade. light-evoked nocifensive behaviors such as licking, biting, jumping and vocalization were virtually absent in Vglut1-ChR2, even at the highest stimulation intensity and frequency. Plantar photostimulation of Vglut1-ChR2 mice in a Real-Time Place-Escape/Avoidance (RT-PEA) assay did not produce aversion, in contrast to the strong aversion elicited in mice that conditionally express ChR2 in Nav1.8-positive and Npy2r-positive nociceptors. Surprisingly, in the Spared Nerve Injury model of neuropathic pain, Vglut1-ChR2 mice did not show significant differences in light-evoked withdrawal behaviors or real-time aversion despite hypersensitivity to natural mechanical stimuli. Thus, I conclude that optogenetic activation of Vglut1-ChR2 neurons alone is not sufficient to produce pain-like behaviors in neuropathic mice.

In the second part of this dissertation, I investigated the cellular determinants of mechanical pain processing in the spinal dorsal horn (SDH), which is comprised of distinct neuronal populations that process different somatosensory modalities. Somatostatin (SST)-expressing interneurons in the SDH have been implicated specifically in mediating mechanical pain. Identifying the transcriptomic profile of SST neurons could elucidate the unique genetic features of this population and enable selective analgesic targeting. To that end, I combined the Isolation of Nuclei Tagged in Specific Cell Types (INTACT) method and Fluorescence Activated Nuclei Sorting (FANS) to capture tagged SST nuclei in the SDH of adult male mice. Using RNA-sequencing (RNA-seq), I uncovered more than 13,000 genes. Differential gene expression analysis revealed more than 900 genes with at least 2-fold enrichment. In addition to many known dorsal horn genes, I identified and validated several novel transcripts from pharmacologically tractable functional classes: Carbonic Anhydrase 12 (Car12), Phosphodiesterase 11A (Pde11a), and Protease-Activated Receptor 3 (F2rl2). In situ hybridization of these novel genes showed differential expression patterns in the SDH, demonstrating the presence of transcriptionally distinct subpopulations within the SST population. Overall, my findings provide new insights into the gene repertoire of SST dorsal horn neurons and reveal several novel targets for pharmacological modulation of this pain-mediating population and pathological pain.

The brain supports numerous complex processes ranging from signal processing and motor control to learning and memory. These processes rely on signal transduction between interconnected networks of neurons that form neural circuits. Understanding how neural circuits function requires non-invasive, genetically specific technologies to both record and manipulate neural activity. Recording neural activity establishes a correlative relationship between the activity and cognitive function, while manipulating neural activity establishes a causal relationship between the activity and behavioral or physiological processes. Genetically encoded protein tools facilitate neuroscience research in both experimental paradigms. Genetically encoded sensors enable optical recording of neural activity across a wide spatiotemporal range. These indicators detect diverse forms of neural activity, including calcium ion flux, membrane voltage potential, and neurotransmitter concentration. Conversely, optogenetic actuators enable targeted, optical excitation or inhibition of neurons upon activation with a specific wavelength of light.

Advancement of genetically encoded tools will allow researchers to access new experimental regimes of neuroscience. Enhancing the fluorescence response and temporal fidelity of genetically encoded sensors improves signal detection fidelity, enabling neuroscientists to access more neurons at once and more precisely analyze neural circuits. Expanding the spectral diversity of genetically encoded tools makes it possible to record from multiple neural populations simultaneously or to optogenetically excite one population with a specific wavelength of light while recording the activity of another in a distinct optical channel. Such multi-channel experiments enable neuroscientists to investigate the influence of the activity of an ensemble of neurons on the activity of another ensemble downstream in a neural circuit or feedback between neural circuits. However, expanding the palette of protein sensors and actuators for such multi-channel experiments has been challenging. Most state-of-the-art genetically encoded sensors fuse cyan-light-sensitive green fluorescent protein to a sensing domain, so the dual channel experiments described above require a complementary sensor or actuator that is sensitive to a spectrally distinct wavelength of light. However, the performance of red fluorescent genetically encoded tools typically lags relative to their green counterparts, and using cyan-light-activated sensors in conjunction with green-light-activated actuators introduces high optical crosstalk. Additionally, the dynamic properties and context-dependent performance of genetically encoded sensors make high-throughput screens of this class of tools labor intensive and time consuming. This constraint on the throughput of screens has limited development efforts to a miniscule fraction of the possible variants of each sensor.

In this dissertation, I expanded the spectral diversity of the tools described above and developed a novel strategy for high throughput evolution of genetically encoded sensors. First, I developed a red fluorescent genetically encoded voltage sensor by engineering the fluorescence resonance energy transfer (FRET) efficiency between a voltage sensitive domain and a red fluorescent protein. This red fluorescent sensor enabled high fidelity recordings of neural activity with sub-millisecond temporal resolution, dual-channel recordings in parallel with green fluorescent sensors, and simultaneous optogenetic excitation and voltage imaging with minimal optical crosstalk. Second, I developed an optogenetic actuator with a blue-shifted activation spectrum by employing this same FRET mechanism. I demonstrated that the activation spectrum of optogenetic tools could be tuned by engineering FRET efficiency between the actuator domain and a compatible fluorescent protein. This straightforward strategy represents a technical step forward for engineering the spectra of optogenetic actuators, which has been difficult to achieve without compromising functionality. Third, I developed a screening method that enabled pooled, high-throughput screens of diverse libraries containing genetically encoded sensor mutants. This method employed both experimental and computational advancements. I used in situ optical mRNA sequencing to determine the sequence of each screened protein variant and machine learning to predict the function of unscreened variants. I expanded the coverage of the possible sequence space by over an order of magnitude compared to traditional directed evolution.