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Comprehensive Dual- and Triple-Feature Intersectional Single-Vector Delivery of Diverse Functional Payloads to Cells of Behaving Mammals. Neuron Fenno, L. E., Ramakrishnan, C. n., Kim, Y. S., Evans, K. E., Lo, M. n., Vesuna, S. n., Inoue, M. n., Cheung, K. Y., Yuen, E. n., Pichamoorthy, N. n., Hong, A. S., Deisseroth, K. n. 2020
Abstract

The resolution and dimensionality with which biologists can characterize cell types have expanded dramatically in recent years, and intersectional consideration of such features (e.g., multiple gene expression and anatomical parameters) is increasingly understood to be essential. At the same time, genetically targeted technology for writing in and reading out activity patterns for cells in living organisms has enabled causal investigation in physiology and behavior; however, cell-type-specific delivery of these tools (including microbial opsins for optogenetics and genetically encoded Ca2+ indicators) has thus far fallen short of versatile targeting to cells jointly defined by many individually selected features. Here, we develop a comprehensive intersectional targeting toolbox including 39 novel vectors for joint-feature-targeted delivery of 13 molecular payloads (including opsins, indicators, and fluorophores), systematic approaches for development and optimization of new intersectional tools, hardware for in�vivo monitoring of expression dynamics, and the first versatile single-virus tools (Triplesect) that enable targeting of triply defined cell types.

View details for DOI 10.1016/j.neuron.2020.06.003

View details for PubMedID 32574559
Targeting cells with single vectors using multiple-feature Boolean logic NATURE METHODS Fenno, L. E., Mattis, J., Ramakrishnan, C., Hyun, M., Lee, S. Y., He, M., Tucciarone, J., Selimbeyoglu, A., Berndt, A., Grosenick, L., Zalocusky, K. A., Bernstein, H., Swanson, H., Perry, C., Diester, I., Boyce, F. M., Bass, C. E., Neve, R., Huang, Z. J., Deisseroth, K. 2014; 11 (7): 763-U116
Abstract

Precisely defining the roles of specific cell types is an intriguing frontier in the study of intact biological systems and has stimulated the rapid development of genetically encoded tools for observation and control. However, targeting these tools with adequate specificity remains challenging: most cell types are best defined by the intersection of two or more features such as active promoter elements, location and connectivity. Here we have combined engineered introns with specific recombinases to achieve expression of genetically encoded tools that is conditional upon multiple cell-type features, using Boolean logical operations all governed by a single versatile vector. We used this approach to target intersectionally specified populations of inhibitory interneurons in mammalian hippocampus and neurons of the ventral tegmental area defined by both genetic and wiring properties. This flexible and modular approach may expand the application of genetically encoded interventional and observational tools for intact-systems biology.

View details for DOI 10.1038/NMETH.2996

View details for Web of Science ID 000338321400022

View details for PubMedCentralID PMC4085277
Neocortical excitation/inhibition balance in information processing and social dysfunction NATURE Yizhar, O., Fenno, L. E., Prigge, M., Schneider, F., Davidson, T. J., O'Shea, D. J., Sohal, V. S., Goshen, I., Finkelstein, J., Paz, J. T., Stehfest, K., Fudim, R., Ramakrishnan, C., Huguenard, J. R., Hegemann, P., Deisseroth, K. 2011; 477 (7363): 171-178
Abstract

Severe behavioural deficits in psychiatric diseases such as autism and schizophrenia have been hypothesized to arise from elevations in the cellular balance of excitation and inhibition (E/I balance) within neural microcircuitry. This hypothesis could unify diverse streams of pathophysiological and genetic evidence, but has not been susceptible to direct testing. Here we design and use several novel optogenetic tools to causally investigate the cellular E/I balance hypothesis in freely moving mammals, and explore the associated circuit physiology. Elevation, but not reduction, of cellular E/I balance within the mouse medial prefrontal cortex was found to elicit a profound impairment in cellular information processing, associated with specific behavioural impairments and increased high-frequency power in the 30-80?Hz range, which have both been observed in clinical conditions in humans. Consistent with the E/I balance hypothesis, compensatory elevation of inhibitory cell excitability partially rescued social deficits caused by E/I balance elevation. These results provide support for the elevated cellular E/I balance hypothesis of severe neuropsychiatric disease-related symptoms.

View details for DOI 10.1038/nature10360

View details for Web of Science ID 000294603900027

View details for PubMedID 21796121
The Development and Application of Optogenetics ANNUAL REVIEW OF NEUROSCIENCE, VOL 34 Fenno, L., Yizhar, O., Deisseroth, K. 2011; 34: 389-412
Abstract

Genetically encoded, single-component optogenetic tools have made a significant impact on neuroscience, enabling specific modulation of selected cells within complex neural tissues. As the optogenetic toolbox contents grow and diversify, the opportunities for neuroscience continue to grow. In this review, we outline the development of currently available single-component optogenetic tools and summarize the application of various optogenetic tools in diverse model organisms.

View details for DOI 10.1146/annurev-neuro-061010-113817

View details for Web of Science ID 000293772100017

View details for PubMedID 21692661
A Molecular Calcium Integrator Reveals a Striatal Cell Type Driving Aversion. Cell Kim, C. K., Sanchez, M. I., Hoerbelt, P., Fenno, L. E., Malenka, R. C., Deisseroth, K., Ting, A. Y. 2020
Abstract

The ability to record transient cellular events in the DNA or RNA of cells would enable precise, large-scale analysis, selection, and reprogramming of heterogeneous cell populations. Here, we report a molecular technology for stable genetic tagging of cells that exhibit activity-related increases in intracellular calcium concentration (FLiCRE). We used FLiCRE to transcriptionally label activated neural ensembles in the nucleus accumbens of the mouse brain during brief stimulation of aversive inputs. Using single-cell RNA sequencing, we detected FLiCRE transcripts among the endogenous transcriptome, providing simultaneous readout of both cell-type and calcium activation history. We identified a cell type in the nucleus accumbens activated downstream of long-range excitatory projections. Taking advantage of FLiCRE's modular design, we expressed an optogenetic channel selectively in this cell type and showed that direct recruitment of this otherwise genetically inaccessible population elicits behavioral aversion. The specificity and minute resolution of FLiCRE enables molecularly informed characterization, manipulation, and reprogramming of activated cellular ensembles.

View details for DOI 10.1016/j.cell.2020.11.015

View details for PubMedID 33308478
Distinct Signaling by Ventral Tegmental Area Glutamate, GABA, and Combinatorial Glutamate-GABA Neurons in Motivated Behavior. Cell reports Root, D. H., Barker, D. J., Estrin, D. J., Miranda-Barrientos, J. A., Liu, B., Zhang, S., Wang, H., Vautier, F., Ramakrishnan, C., Kim, Y. S., Fenno, L., Deisseroth, K., Morales, M. 2020; 32 (9): 108094
Abstract

Ventral tegmental area (VTA) neurons play roles in reward and aversion. We recently discovered that the VTA has neurons that co-transmit glutamate and GABA (glutamate-GABA co-transmitting neurons), transmit glutamate without GABA (glutamate-transmitting neurons), or transmit GABA without glutamate (GABA-transmitting neurons). However, the functions of these VTA cell types in motivated behavior are unclear. To identify the functions of these VTA cell types, we combine recombinase mouse lines with INTRSECT2.0 vectors to selectively target these neurons. We find that VTA cell types have unique signaling patterns for reward, aversion, and learned cues. Whereas VTA glutamate-transmitting neurons signal cues predicting reward, VTA GABA-transmitting neurons signal cues predicting the absence of reward, and glutamate-GABA co-transmitting neurons signal rewarding and aversive outcomes without signaling learned cues related to those outcomes. Thus, we demonstrate that genetically defined subclasses of VTA glutamate and GABA neurons signal different aspects of motivated behavior.

View details for DOI 10.1016/j.celrep.2020.108094

View details for PubMedID 32877676
Genetically targeted chemical assembly of functional materials in living cells, tissues, and animals. Science (New York, N.Y.) Liu, J. n., Kim, Y. S., Richardson, C. E., Tom, A. n., Ramakrishnan, C. n., Birey, F. n., Katsumata, T. n., Chen, S. n., Wang, C. n., Wang, X. n., Joubert, L. M., Jiang, Y. n., Wang, H. n., Fenno, L. E., Tok, J. B., Pa?ca, S. P., Shen, K. n., Bao, Z. n., Deisseroth, K. n. 2020; 367 (6484): 1372?76
Abstract

The structural and functional complexity of multicellular biological systems, such as the brain, are beyond the reach of human design or assembly capabilities. Cells in living organisms may be recruited to construct synthetic materials or structures if treated as anatomically defined compartments for specific chemistry, harnessing biology for the assembly of complex functional structures. By integrating engineered-enzyme targeting and polymer chemistry, we genetically instructed specific living neurons to guide chemical synthesis of electrically functional (conductive or insulating) polymers at the plasma membrane. Electrophysiological and behavioral analyses confirmed that rationally designed, genetically targeted assembly of functional polymers not only preserved neuronal viability but also achieved remodeling of membrane properties and modulated cell type-specific behaviors in freely moving animals. This approach may enable the creation of diverse, complex, and functional structures and materials within living systems.

View details for DOI 10.1126/science.aay4866

View details for PubMedID 32193327
Excitation of diverse classes of cholecystokinin interneurons in the basolateral amygdala facilitates fear extinction. eNeuro Rovira-Esteban, L., Gunduz-Cinar, O., Bukalo, O., Limoges, A., Brockway, E., Muller, K., Fenno, L., Kim, Y. S., Ramakrishnan, C., Andrasi, T., Deisseroth, K., Holmes, A., Hajos, N. 2019
Abstract

There is growing evidence that interneurons orchestrate neural activity and plasticity in corticoamygdala circuits to regulate fear behaviors. However, defining the precise role of cholecystokinin-expressing interneurons (CCK INs) remains elusive due to the technical challenge of parsing this population from CCK-expressing principal neurons (CCK PNs). Here we used an intersectional genetic strategy in CCK-Cre;Dlx5/6-Flpe double-transgenic mice to study the anatomical, molecular and electrophysiological properties of CCK INs in the basal amygdala (BA) and optogenetically manipulate these cells in fear extinction. Electrophysiological recordings confirmed that this strategy targeted GABAergic cells and that a significant proportion expressed functional cannabinoid CB1 receptors; a defining characteristic of CCK-expressing basket cells. However, immunostaining showed that subsets of the genetically-targeted cells expressed either neuropeptide Y (NPY) (29%) or parvalbumin (PV) (17%), but not somatostatin (SOM) or CaMKII-alpha. Further morphological and electrophysiological analyses showed that four interneuron types could be identified among the EYFP-expressing cells: CCK/CB1R-expressing basket cells, neurogliaform cells, PV+ basket and PV+ axo-axonic cells. At the behavioral level, in vivo optogenetic photostimulation of the targeted population during extinction acquisition led to reduced freezing on a light-free extinction retrieval test, indicating extinction memory facilitation; whereas photosilencing was without effect. Conversely, non-selective (i.e., inclusive of INs and PNs) photostimulation or photosilencing of CCK-targeted cells, using CCK-Cre single-transgenic mice, impaired extinction. These data reveal an unexpectedly high degree of phenotypic complexity in a unique population of extinction-modulating BA INs.Significance statement Distinct types of interneurons in the basolateral amygdala (BA) are known to control principal cell activity, allowing complex behaviors. Despite their importance, the role of cholecystokinin (CCK)-expressing inhibitory cells remains unknown. In this work, we could specifically alter the function of CCK-expressing interneurons in the BA by using an INTRSECT viral strategy. Using a combination of anatomical and electrophysiological methods, we found that CCK+ interneurons in the BA are comprised of CB1R-expressing basket cells, neurogliaform cells, parvalbumin-expressing basket as well as axo-axonic cells. Importantly, we provided the first direct evidence that CCK-expressing interneurons in the BA can modulate fear extinction learning. Our data thus show that CCK is expressed in functionally diverse interneuron populations, positioned to impact amygdala operation.

View details for DOI 10.1523/ENEURO.0220-19.2019

View details for PubMedID 31636080
Mapping Brain-Wide Afferent Inputs of Parvalbumin-Expressing GABAergic Neurons in Barrel Cortex Reveals Local and Long-Range Circuit Motifs. Cell reports Hafner, G., Witte, M., Guy, J., Subhashini, N., Fenno, L. E., Ramakrishnan, C., Kim, Y. S., Deisseroth, K., Callaway, E. M., Oberhuber, M., Conzelmann, K., Staiger, J. F. 2019; 28 (13): 3450
Abstract

Parvalbumin (PV)-expressing GABAergic neurons are the largest class of inhibitory neocortical cells. We visualize brain-wide, monosynaptic inputs to PV neurons in mouse barrel cortex. We develop intersectional rabies virus tracing to specifically target GABAergic PV cells and exclude a small fraction of excitatory PV cells from our starter population. Local inputs are mainly from layer (L) IV and excitatory cells. A small number of inhibitory inputs originate from LI neurons, which connect to LII/III PV neurons. Long-range inputs originate mainly from other sensory cortices and the thalamus. In visual cortex, most transsynaptically labeled neurons are located in LIV, which contains a molecularly mixed population of projection neurons with putative functional similarity to LIII neurons. This study expands our knowledge of the brain-wide circuits in which PV neurons are embedded and introduces intersectional rabies virus tracing as an applicable tool to dissect the circuitry of more clearly defined cell types.

View details for DOI 10.1016/j.celrep.2019.08.064

View details for PubMedID 31553913
A hypothalamus-habenula circuit controls aversion MOLECULAR PSYCHIATRY Lazaridis, I., Tzortzi, O., Weglage, M., Martin, A., Xuan, Y., Parent, M., Johansson, Y., Fuzik, J., Furth, D., Fenno, L. E., Ramakrishnan, C., Silberberg, G., Deisseroth, K., Carlen, M., Meletis, K. 2019; 24 (9): 1351?68
View details for DOI 10.1038/s41380-019-0369-5

View details for Web of Science ID 000482211800008
A hypothalamus-habenula circuit controls aversion. Molecular psychiatry Lazaridis, I., Tzortzi, O., Weglage, M., Martin, A., Xuan, Y., Parent, M., Johansson, Y., Fuzik, J., Furth, D., Fenno, L. E., Ramakrishnan, C., Silberberg, G., Deisseroth, K., Carlen, M., Meletis, K. 2019
Abstract

Encoding and predicting aversive events are critical functions of circuits that support survival and emotional well-being. Maladaptive circuit changes in emotional valence processing can underlie the pathophysiology of affective disorders. The lateral habenula (LHb) has been linked to aversion and mood regulation through modulation of the dopamine and serotonin systems. We have defined the identity and function of glutamatergic (Vglut2) control of the LHb, comparing the role of inputs originating in the globus pallidus internal segment (GPi), and lateral hypothalamic area (LHA), respectively. We found that LHb-projecting LHA neurons, and not the proposed GABA/glutamate co-releasing GPi neurons, are responsible for encoding negative value. Monosynaptic rabies tracing of the presynaptic organization revealed a predominantly limbic input onto LHA Vglut2 neurons, while sensorimotor inputs were more prominent onto GABA/glutamate co-releasing GPi neurons. We further recorded the activity of LHA Vglut2 neurons, by imaging calcium dynamics in response to appetitive versus aversive events in conditioning paradigms. LHA Vglut2 neurons formed activity clusters representing distinct reward or aversion signals, including a population that responded to mild foot shocks and predicted aversive events. We found that the LHb-projecting LHA Vglut2 neurons encode negative valence and rapidly develop a prediction signal for negative events. These findings establish the glutamatergic LHA-LHb circuit as a critical node in value processing.

View details for PubMedID 30755721
Sono-optogenetics facilitated by a circulation-delivered rechargeable light source for minimally invasive optogenetics. Proceedings of the National Academy of Sciences of the United States of America Wu, X. n., Zhu, X. n., Chong, P. n., Liu, J. n., Andre, L. N., Ong, K. S., Brinson, K. n., Mahdi, A. I., Li, J. n., Fenno, L. E., Wang, H. n., Hong, G. n. 2019
Abstract

Optogenetics, which uses visible light to control the cells genetically modified with light-gated ion channels, is a powerful tool for precise deconstruction of neural circuitry with neuron-subtype specificity. However, due to limited tissue penetration of visible light, invasive craniotomy and intracranial implantation of tethered optical fibers are usually required for in vivo optogenetic modulation. Here we report mechanoluminescent nanoparticles that can act as local light sources in the brain when triggered by brain-penetrant focused ultrasound (FUS) through intact scalp and skull. Mechanoluminescent nanoparticles can be delivered into the blood circulation via i.v. injection, recharged by 400-nm photoexcitation light in superficial blood vessels during circulation, and turned on by FUS to emit 470-nm light repetitively in the intact brain for optogenetic stimulation. Unlike the conventional "outside-in" approaches of optogenetics with fiber implantation, our method provides an "inside-out" approach to deliver nanoscopic light emitters via the intrinsic circulatory system and switch them on and off at any time and location of interest in the brain without extravasation through a minimally invasive ultrasound interface.

View details for DOI 10.1073/pnas.1914387116

View details for PubMedID 31811026
Structural mechanisms of selectivity and gating in anion channelrhodopsins. Nature Kato, H. E., Kim, Y. S., Paggi, J. M., Evans, K. E., Allen, W. E., Richardson, C., Inoue, K., Ito, S., Ramakrishnan, C., Fenno, L. E., Yamashita, K., Hilger, D., Lee, S. Y., Berndt, A., Shen, K., Kandori, H., Dror, R. O., Kobilka, B. K., Deisseroth, K. 2018
Abstract

Both designed and natural anion-conducting channelrhodopsins (dACRs and nACRs, respectively) have been widely applied in optogenetics (enabling selective inhibition of target-cell activity during animal behaviour studies), but each class exhibits performance limitations, underscoring trade-offs in channel structure-function relationships. Therefore, molecular and structural insights into dACRs and nACRs will be critical not only for understanding the fundamental mechanisms of these light-gated anionchannels, but also to create next-generation optogenetic tools. Here we report crystal structures of the dACR iC++, along with spectroscopic, electrophysiological and computational analyses that provide unexpected insights into pH dependence, substrate recognition, channel gating and ion selectivity of both dACRs and nACRs. These results enabled us to create an anion-conducting channelrhodopsin integrating the key features of large photocurrent and fast kinetics alongside exclusive anion selectivity.

View details for PubMedID 30158697
Crystal structure of the natural anion-conducting channelrhodopsin GtACR1. Nature Kim, Y. S., Kato, H. E., Yamashita, K., Ito, S., Inoue, K., Ramakrishnan, C., Fenno, L. E., Evans, K. E., Paggi, J. M., Dror, R. O., Kandori, H., Kobilka, B. K., Deisseroth, K. 2018
Abstract

The naturally occurring channelrhodopsin variant anion channelrhodopsin-1 (ACR1), discovered in the cryptophyte algae Guillardia theta, exhibits large light-gated anionconductance and high anionselectivity when expressed in heterologous settings, properties that support its use as an optogenetic tool to inhibit neuronal firing with light. However, molecular insight into ACR1 is lacking owing to the absence of structural information underlying light-gated anion conductance. Here we present the crystal structure of G. theta ACR1 at 2.9A resolution. The structure reveals unusual architectural features that span the extracellular domain, retinal-binding pocket, Schiff-base region, and anion-conduction pathway. Together with electrophysiological and spectroscopic analyses, these findings reveal the fundamental molecular basis of naturally occurring light-gated anion conductance, and provide a framework for designing the next generation of optogenetic tools.

View details for PubMedID 30158696
The central amygdala controls learning in the lateral amygdala NATURE NEUROSCIENCE Yu, K., Ahrens, S., Zhang, X., Schiff, H., Ramakrishnan, C., Fenno, L., Deisseroth, K., Zhao, F., Luo, M., Gong, L., He, M., Zhou, P., Paninski, L., Li, B. 2017; 20 (12): 1680-+
Abstract

Experience-driven synaptic plasticity in the lateral amygdala is thought to underlie the formation of associations between sensory stimuli and an ensuing threat. However, how the central amygdala participates in such a learning process remains unclear. Here we show that PKC-?-expressing central amygdala neurons are essential for the synaptic plasticity underlying learning in the lateral amygdala, as they convey information about the unconditioned stimulus to lateral amygdala neurons during fear conditioning.

View details for PubMedID 29184202

View details for PubMedCentralID PMC5755715
Distinct Thalamic Reticular Cell Types Differentially Modulate Normal and Pathological Cortical Rhythms. Cell reports Clemente-Perez, A., Makinson, S. R., Higashikubo, B., Brovarney, S., Cho, F. S., Urry, A., Holden, S. S., Wimer, M., D�vid, C., Fenno, L. E., Acs�dy, L., Deisseroth, K., Paz, J. T. 2017; 19 (10): 2130-2142
Abstract

Integrative brain functions depend on widely distributed, rhythmically coordinated computations. Through its long-ranging connections with cortex and most senses, the thalamus orchestrates the flow of cognitive and sensory information. Essential in this process, the nucleus reticularis thalami (nRT) gates different information streams through its extensive inhibition onto other thalamic nuclei, however, we lack an understanding of how different inhibitory neuron subpopulations in nRT function as�gatekeepers. We dissociated the connectivity, physiology, and circuit functions of neurons within rodent nRT, based on parvalbumin (PV) and somatostatin (SOM) expression, and validated the existence of such populations in human nRT. We found that PV, but not SOM, cells are rhythmogenic, and that PV and SOM neurons are connected to and�modulate distinct thalamocortical circuits. Notably, PV, but not SOM, neurons modulate somatosensory behavior and disrupt seizures. These results provide a conceptual framework for how nRT may gate incoming information to modulate brain-wide rhythms.

View details for DOI 10.1016/j.celrep.2017.05.044

View details for PubMedID 28591583
Next-generation probes, particles, and proteins for neural interfacing SCIENCE ADVANCES Rivnay, J., Wang, H., Fenno, L., Deisseroth, K., Malliaras, G. G. 2017; 3 (6): e1601649
Abstract

Bidirectional interfacing with the nervous system enables neuroscience research, diagnosis, and therapy. This two-way communication allows us to monitor the state of the brain and its composite networks and cells as well as to influence them to treat disease or repair/restore sensory or motor function. To provide the most stable and effective interface, the tools of the trade must bridge the soft, ion-rich, and evolving nature of neural tissue with the largely rigid, static realm of microelectronics and medical instruments that allow for readout, analysis, and/or control. In this Review, we describe how the understanding of neural signaling and material-tissue interactions has fueled the expansion of the available tool set. New probe architectures and materials, nanoparticles, dyes, and designer genetically encoded proteins push the limits of recording and stimulation lifetime, localization, and specificity, blurring the boundary between living tissue and engineered tools. Understanding these approaches, their modality, and the role of cross-disciplinary development will support new neurotherapies and prostheses and provide neuroscientists and neurologists with unprecedented access to the brain.

View details for PubMedID 28630894
Thirst-associated preoptic neurons encode an aversive motivational drive. Science (New York, N.Y.) Allen, W. E., DeNardo, L. A., Chen, M. Z., Liu, C. D., Loh, K. M., Fenno, L. E., Ramakrishnan, C. n., Deisseroth, K. n., Luo, L. n. 2017; 357 (6356): 1149?55
Abstract

Water deprivation produces a drive to seek and consume water. How neural activity creates this motivation remains poorly understood. We used activity-dependent genetic labeling to characterize neurons activated by water deprivation in the hypothalamic median preoptic nucleus (MnPO). Single-cell transcriptional profiling revealed that dehydration-activated MnPO neurons consist of a single excitatory cell type. After optogenetic activation of these neurons, mice drank water and performed an operant lever-pressing task for water reward with rates that scaled with stimulation frequency. This stimulation was aversive, and instrumentally pausing stimulation could reinforce lever-pressing. Activity of these neurons gradually decreased over the course of an operant session. Thus, the activity of dehydration-activated MnPO neurons establishes a scalable, persistent, and aversive internal state that dynamically controls thirst-motivated behavior.

View details for PubMedID 28912243
A Guide to Creating and Testing New INTRSECT Constructs. Current protocols in neuroscience Fenno, L. E., Mattis, J. n., Ramakrishnan, C. n., Deisseroth, K. n. 2017; 80: 4.39.1?4.39.24
Abstract

As the power of genetically encoded interventional and observational tools for neuroscience expands, the boundaries of experimental design are increasingly defined by limits in selectively expressing these tools in relevant cell types. Single-recombinase-dependent expression systems have been widely used as a means to restrict gene expression based on single features by combining recombinase-dependent viruses with recombinase-expressing transgenic animals. This protocol details how to create INTRSECT constructs and use multiple recombinases to achieve targeting of a desired gene to subsets of neurons that are defined by multiple genetic and/or topological features. This method includes the design and utilization of both viruses and transgenic animals: these tools are inherently flexible and modular and may be used in different combinations to achieve the desired gene expression pattern. � 2017 by John Wiley & Sons, Inc.

View details for PubMedID 28678399
Modulation of prefrontal cortex excitation/inhibition balance rescues social behavior in CNTNAP2-deficient mice. Science translational medicine Selimbeyoglu, A. n., Kim, C. K., Inoue, M. n., Lee, S. Y., Hong, A. S., Kauvar, I. n., Ramakrishnan, C. n., Fenno, L. E., Davidson, T. J., Wright, M. n., Deisseroth, K. n. 2017; 9 (401)
Abstract

Alterations in the balance between neuronal excitation and inhibition (E:I balance) have been implicated in the neural circuit activity-based processes that contribute to autism phenotypes. We investigated whether acutely reducing E:I balance in mouse brain could correct deficits in social behavior. We used mice lacking the CNTNAP2 gene, which has been implicated in autism, and achieved a temporally precise reduction in E:I balance in the medial prefrontal cortex (mPFC) either by optogenetically increasing the excitability of inhibitory parvalbumin (PV) neurons or decreasing the excitability of excitatory pyramidal neurons. Surprisingly, both of these distinct, real-time, and reversible optogenetic modulations acutely rescued deficits in social behavior and hyperactivity in adult mice lacking CNTNAP2 Using fiber photometry, we discovered that native mPFC PV neuronal activity differed between CNTNAP2 knockout and wild-type mice. During social interactions with other mice, PV neuron activity increased in wild-type mice compared to interactions with a novel object, whereas this difference was not observed in CNTNAP2 knockout mice. Together, these results suggest that real-time modulation of E:I balance in the mouse prefrontal cortex can rescue social behavior deficits reminiscent of autism phenotypes.

View details for PubMedID 28768803
Midbrain circuits for defensive behaviour NATURE Tovote, P., Esposito, M. S., Botta, P., Haudun, F. C., Fadok, J. P., Markovic, M., Wolff, S. B., Ramakrishnan, C., Fenno, L., Deisseroth, K., Herry, C., Arber, S., Luthi, A. 2016; 534 (7606): 206-?
Abstract

Survival in threatening situations depends on the selection and rapid execution of an appropriate active or passive defensive response, yet the underlying brain circuitry is not understood. Here we use circuit-based optogenetic, in vivo and in vitro electrophysiological, and neuroanatomical tracing methods to define midbrain periaqueductal grey circuits for specific defensive behaviours. We identify an inhibitory pathway from the central nucleus of the amygdala to the ventrolateral periaqueductal grey that produces freezing by disinhibition of ventrolateral periaqueductal grey excitatory outputs to pre-motor targets in the magnocellular nucleus of the medulla. In addition, we provide evidence for anatomical and functional interaction of this freezing pathway with long-range and local circuits mediating flight. Our data define the neuronal circuitry underlying the execution of freezing, an evolutionarily conserved defensive behaviour, which is expressed by many species including fish, rodents and primates. In humans, dysregulation of this 'survival circuit' has been implicated in anxiety-related disorders.

View details for DOI 10.1038/nature17996

View details for Web of Science ID 000377475100032

View details for PubMedID 27279213
Chronic Optogenetic Activation Augments A beta Pathology in a Mouse Model of Alzheimer Disease CELL REPORTS Yamamoto, K., Tanei, Z., Hashimoto, T., Wakabayashi, T., Okuno, H., Naka, Y., Yizhar, O., Fenno, L. E., Fukayama, M., Bito, H., Cirrito, J. R., Holtzman, D. M., Deisseroth, K., Iwatsubo, T. 2015; 11 (6): 859-865
Abstract

In�vivo experimental evidence indicates that acute neuronal activation increases A? release from presynaptic terminals, whereas long-term effects of�chronic synaptic activation on A? 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 A?42 levels immediately after acute light activation. Five months of chronic optogenetic stimulation increased A? 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 A? pathology. These findings implicate functional abnormalities of specific neuronal circuitry in A? pathology and Alzheimer disease.

View details for DOI 10.1016/j.celrep.2015.04.017

View details for Web of Science ID 000354406900002

View details for PubMedID 25937280
Mapping Anatomy to Behavior in Thy1:18 ChR2-YFP Transgenic Mice Using Optogenetics. Cold Spring Harbor protocols Fenno, L. E., Gunaydin, L. A., Deisseroth, K. 2015; 2015 (6): pdb prot075598-?
Abstract

Linking the activity of defined neural populations with behavior is a key goal of neuroscience. In the context of controlling behavior, electrical stimulation affords researchers precision in the temporal domain with gross regional specificity, whereas pharmacology allows for more specific manipulation of cell types, but in the absence of temporal precision. The use of microbial opsins-light activated, genetically encoded ion channels and pumps-to control mammalian neurons now allows researchers to "sensitize" genetically and/or topologically defined populations of neurons to light to induce either depolarization or hyperpolarization in both a cell-type-specific and temporally precise manner not achievable with previous techniques. Here, we describe the use of transgenic mice expressing the blue-light gated cation channel Channelrhodopsin-2 (ChR2) under control of the Thy1 promoter for the purpose of linking neuronal activity to behavior through restricted delivery of light to an anatomic region of interest. The surgical procedure for implanting a fiber-optic light delivery guide into the mouse brain, the process of optically stimulating the brain in a behaving animal, and post hoc evaluation are given, along with necessary reagents and discussion of common technical problems and their solutions.

View details for DOI 10.1101/pdb.prot075598

View details for PubMedID 26034299
Optogenetic neuronal stimulation promotes functional recovery after stroke PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cheng, M. Y., Wang, E. H., Woodson, W. J., Wang, S., Sun, G., Lee, A. G., Arac, A., Fenno, L. E., Deisseroth, K., Steinberg, G. K. 2014; 111 (35): 12913-12918
Abstract

Clinical and research efforts have focused on promoting functional recovery after stroke. Brain stimulation strategies are particularly promising because they allow direct manipulation of the target area's excitability. However, elucidating the cell type and mechanisms mediating recovery has been difficult because existing stimulation techniques nonspecifically target all cell types near the stimulated site. To circumvent these barriers, we used optogenetics to selectively activate neurons that express channelrhodopsin 2 and demonstrated that selective neuronal stimulations in the ipsilesional primary motor cortex (iM1) can promote functional recovery. Stroke mice that received repeated neuronal stimulations exhibited significant improvement in cerebral blood flow and the neurovascular coupling response, as well as increased expression of activity-dependent neurotrophins in the contralesional cortex, including brain-derived neurotrophic factor, nerve growth factor, and neurotrophin 3. Western analysis also indicated that stimulated mice exhibited a significant increase in the expression of a plasticity marker growth-associated protein 43. Moreover, iM1 neuronal stimulations promoted functional recovery, as stimulated stroke mice showed faster weight gain and performed significantly better in sensory-motor behavior tests. Interestingly, stimulations in normal nonstroke mice did not alter motor behavior or neurotrophin expression, suggesting that the prorecovery effect of selective neuronal stimulations is dependent on the poststroke environment. These results demonstrate that stimulation of neurons in the stroke hemisphere is sufficient to promote recovery.

View details for DOI 10.1073/pnas.1404109111

View details for Web of Science ID 000341230800084

View details for PubMedCentralID PMC4156770
Optogenetic neuronal stimulation promotes functional recovery after stroke. Proceedings of the National Academy of Sciences of the United States of America Cheng, M. Y., Wang, E. H., Woodson, W. J., Wang, S., Sun, G., Lee, A. G., Arac, A., Fenno, L. E., Deisseroth, K., Steinberg, G. K. 2014; 111 (35): 12913-12918
Abstract

Clinical and research efforts have focused on promoting functional recovery after stroke. Brain stimulation strategies are particularly promising because they allow direct manipulation of the target area's excitability. However, elucidating the cell type and mechanisms mediating recovery has been difficult because existing stimulation techniques nonspecifically target all cell types near the stimulated site. To circumvent these barriers, we used optogenetics to selectively activate neurons that express channelrhodopsin 2 and demonstrated that selective neuronal stimulations in the ipsilesional primary motor cortex (iM1) can promote functional recovery. Stroke mice that received repeated neuronal stimulations exhibited significant improvement in cerebral blood flow and the neurovascular coupling response, as well as increased expression of activity-dependent neurotrophins in the contralesional cortex, including brain-derived neurotrophic factor, nerve growth factor, and neurotrophin 3. Western analysis also indicated that stimulated mice exhibited a significant increase in the expression of a plasticity marker growth-associated protein 43. Moreover, iM1 neuronal stimulations promoted functional recovery, as stimulated stroke mice showed faster weight gain and performed significantly better in sensory-motor behavior tests. Interestingly, stimulations in normal nonstroke mice did not alter motor behavior or neurotrophin expression, suggesting that the prorecovery effect of selective neuronal stimulations is dependent on the poststroke environment. These results demonstrate that stimulation of neurons in the stroke hemisphere is sufficient to promote recovery.

View details for DOI 10.1073/pnas.1404109111

View details for PubMedID 25136109
Targeting cells with single vectors using multiple-feature Boolean logic. Nature methods Fenno, L. E., Mattis, J., Ramakrishnan, C., Hyun, M., Lee, S. Y., He, M., Tucciarone, J., Selimbeyoglu, A., Berndt, A., Grosenick, L., Zalocusky, K. A., Bernstein, H., Swanson, H., Perry, C., Diester, I., Boyce, F. M., Bass, C. E., Neve, R., Huang, Z. J., Deisseroth, K. 2014; 11 (7): 763-772
Abstract

Precisely defining the roles of specific cell types is an intriguing frontier in the study of intact biological systems and has stimulated the rapid development of genetically encoded tools for observation and control. However, targeting these tools with adequate specificity remains challenging: most cell types are best defined by the intersection of two or more features such as active promoter elements, location and connectivity. Here we have combined engineered introns with specific recombinases to achieve expression of genetically encoded tools that is conditional upon multiple cell-type features, using Boolean logical operations all governed by a single versatile vector. We used this approach to target intersectionally specified populations of inhibitory interneurons in mammalian hippocampus and neurons of the ventral tegmental area defined by both genetic and wiring properties. This flexible and modular approach may expand the application of genetically encoded interventional and observational tools for intact-systems biology.

View details for DOI 10.1038/nmeth.2996

View details for PubMedID 24908100
Natural neural projection dynamics underlying social behavior. Cell Gunaydin, L. A., Grosenick, L., Finkelstein, J. C., Kauvar, I. V., Fenno, L. E., Adhikari, A., Lammel, S., Mirzabekov, J. J., Airan, R. D., Zalocusky, K. A., Tye, K. M., Anikeeva, P., Malenka, R. C., Deisseroth, K. 2014; 157 (7): 1535-1551
Abstract

Social interaction is a complex behavior essential for many species and is impaired in major neuropsychiatric disorders. Pharmacological studies have implicated certain neurotransmitter systems in social behavior, but circuit-level understanding of endogenous neural activity during social interaction is lacking. We therefore developed and applied a new methodology, termed fiber photometry, to optically record natural neural activity in genetically and connectivity-defined projections to elucidate the real-time role of specified pathways in mammalian behavior. Fiber photometry revealed that activity dynamics of a ventral tegmental area (VTA)-to-nucleus�accumbens (NAc) projection could encode and predict key features of social, but not novel object, interaction. Consistent with this observation, optogenetic control of cells specifically contributing to this projection was sufficient to modulate social behavior, which was mediated by type 1 dopamine receptor signaling downstream in the NAc. Direct observation of deep projection-specific activity in this way captures a fundamental and previously inaccessible dimension of mammalian circuit dynamics.

View details for DOI 10.1016/j.cell.2014.05.017

View details for PubMedID 24949967

View details for PubMedCentralID PMC4123133
Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins NATURE METHODS Mattis, J., Tye, K. M., Ferenczi, E. A., Ramakrishnan, C., O'Shea, D. J., Prakash, R., Gunaydin, L. A., Hyun, M., Fenno, L. E., Gradinaru, V., Yizhar, O., Deisseroth, K. 2012; 9 (2): 159-172
View details for DOI 10.1038/NMETH.1808

View details for Web of Science ID 000300029600026
The Microbial Opsin Family of Optogenetic Tools CELL Zhang, F., Vierock, J., Yizhar, O., Fenno, L. E., Tsunoda, S., Kianianmomeni, A., Prigge, M., Berndt, A., Cushman, J., Polle, J., Magnuson, J., Hegemann, P., Deisseroth, K. 2011; 147 (7): 1446-1457
Abstract

The capture and utilization of light is an exquisitely evolved process. The single-component microbial opsins, although more limited than multicomponent cascades in processing, display unparalleled compactness and speed. Recent advances in understanding microbial opsins have been driven by molecular engineering for optogenetics and by comparative genomics. Here we provide a Primer on these light-activated ion channels and pumps, describe a group of opsins bridging prior categories, and explore the convergence of molecular engineering and genomic discovery for the utilization and understanding of these remarkable molecular machines.

View details for DOI 10.1016/j.cell.2011.12.004

View details for Web of Science ID 000298403400011

View details for PubMedID 22196724
SNCA Triplication Parkinson's Patient's iPSC-derived DA Neurons Accumulate alpha-Synuclein and Are Susceptible to Oxidative Stress PLOS ONE Byers, B., Cord, B., Ha Nam Nguyen, H. N., Schuele, B., Fenno, L., Lee, P. C., Deisseroth, K., Langston, J. W., Pera, R. R., Palmer, T. D. 2011; 6 (11)
Abstract

Parkinson's disease (PD) is an incurable age-related neurodegenerative disorder affecting both the central and peripheral nervous systems. Although common, the etiology of PD remains poorly understood. Genetic studies infer that the disease results from a complex interaction between genetics and environment and there is growing evidence that PD may represent a constellation of diseases with overlapping yet distinct underlying mechanisms. Novel clinical approaches will require a better understanding of the mechanisms at work within an individual as well as methods to identify the specific array of mechanisms that have contributed to the disease. Induced pluripotent stem cell (iPSC) strategies provide an opportunity to directly study the affected neuronal subtypes in a given patient. Here we report the generation of iPSC-derived midbrain dopaminergic neurons from a patient with a triplication in the ?-synuclein gene (SNCA). We observed that the iPSCs readily differentiated into functional neurons. Importantly, the PD-affected line exhibited disease-related phenotypes in culture: accumulation of ?-synuclein, inherent overexpression of markers of oxidative stress, and sensitivity to peroxide induced oxidative stress. These findings show that the dominantly-acting PD mutation is intrinsically capable of perturbing normal cell function in culture and confirm that these features reflect, at least in part, a cell autonomous disease process that is independent of exposure to the entire complexity of the diseased brain.

View details for DOI 10.1371/journal.pone.0026159

View details for Web of Science ID 000297555400007

View details for PubMedID 22110584

View details for PubMedCentralID PMC3217921
A new mode of corticothalamic transmission revealed in the Gria4(-/-) model of absence epilepsy NATURE NEUROSCIENCE Paz, J. T., Bryant, A. S., Peng, K., Fenno, L., Yizhar, O., Frankel, W. N., Deisseroth, K., Huguenard, J. R. 2011; 14 (9): 1167-U225
Abstract

Cortico-thalamo-cortical circuits mediate sensation and generate neural network oscillations associated with slow-wave sleep and various epilepsies. Cortical input to sensory thalamus is thought to mainly evoke feed-forward synaptic inhibition of thalamocortical (TC) cells via reticular thalamic nucleus (nRT) neurons, especially during oscillations. This relies on a stronger synaptic strength in the cortico-nRT pathway than in the cortico-TC pathway, allowing the feed-forward inhibition of TC cells to overcome direct cortico-TC excitation. We found a systemic and specific reduction in strength in GluA4-deficient (Gria4(-/-)) mice of one excitatory synapse of the rhythmogenic cortico-thalamo-cortical system, the cortico-nRT projection, and observed that the oscillations could still be initiated by cortical inputs via the cortico-TC-nRT-TC pathway. These results reveal a previously unknown mode of cortico-thalamo-cortical transmission, bypassing direct cortico-nRT excitation, and describe a mechanism for pathological oscillation generation. This mode could be active under other circumstances, representing a previously unknown channel of cortico-thalamo-cortical information processing.

View details for DOI 10.1038/nn.2896

View details for Web of Science ID 000294284900017

View details for PubMedID 21857658

View details for PubMedCentralID PMC3308017
Optogenetics in Neural Systems NEURON Yizhar, O., Fenno, L. E., Davidson, T. J., Mogri, M., Deisseroth, K. 2011; 71 (1): 9-34
Abstract

Both observational and perturbational technologies are essential for advancing the understanding of brain function and dysfunction. But while observational techniques have greatly advanced in the last century, techniques for perturbation that are matched to the speed and heterogeneity of neural systems have lagged behind. The technology of optogenetics represents a step toward addressing this disparity. Reliable and targetable single-component tools (which encompass both light sensation and effector function within a single protein) have enabled versatile new classes of investigation in the study of neural systems. Here we provide a primer on the application of optogenetics in neuroscience, focusing on the single-component tools and highlighting important problems, challenges, and technical considerations.

View details for DOI 10.1016/j.neuron.2011.06.004

View details for PubMedID 21745635
Amygdala circuitry mediating reversible and bidirectional control of anxiety NATURE Tye, K. M., Prakash, R., Kim, S., Fenno, L. E., Grosenick, L., Zarabi, H., Thompson, K. R., Gradinaru, V., Ramakrishnan, C., Deisseroth, K. 2011; 471 (7338): 358-362
Abstract

Anxiety--a sustained state of heightened apprehension in the absence of immediate threat--becomes severely debilitating in disease states. Anxiety disorders represent the most common of psychiatric diseases (28% lifetime prevalence) and contribute to the aetiology of major depression and substance abuse. Although it has been proposed that the amygdala, a brain region important for emotional processing, has a role in anxiety, the neural mechanisms that control anxiety remain unclear. Here we explore the neural circuits underlying anxiety-related behaviours by using optogenetics with two-photon microscopy, anxiety assays in freely moving mice, and electrophysiology. With the capability of optogenetics to control not only cell types but also specific connections between cells, we observed that temporally precise optogenetic stimulation of basolateral amygdala (BLA) terminals in the central nucleus of the amygdala (CeA)--achieved by viral transduction of the BLA with a codon-optimized channelrhodopsin followed by restricted illumination in the downstream CeA--exerted an acute, reversible anxiolytic effect. Conversely, selective optogenetic inhibition of the same projection with a third-generation halorhodopsin (eNpHR3.0) increased anxiety-related behaviours. Importantly, these effects were not observed with direct optogenetic control of BLA somata, possibly owing to recruitment of antagonistic downstream structures. Together, these results implicate specific BLA-CeA projections as critical circuit elements for acute anxiety control in the mammalian brain, and demonstrate the importance of optogenetically targeting defined projections, beyond simply targeting cell types, in the study of circuit function relevant to neuropsychiatric disease.

View details for DOI 10.1038/nature09820

View details for Web of Science ID 000288444000041

View details for PubMedID 21389985

View details for PubMedCentralID PMC3154022
An Implantable Optical Stimulation Delivery System for Actuating an Excitable Biosubstrate IEEE JOURNAL OF SOLID-STATE CIRCUITS Paralikar, K., Cong, P., Yizhar, O., Fenno, L. E., Santa, W., Nielsen, C., Dinsmoor, D., Hocken, B., Munns, G. O., Giftakis, J., Deisseroth, K., Denison, T. 2011; 46 (1): 321-332
View details for DOI 10.1109/JSSC.2010.2074110

View details for Web of Science ID 000285839200028
Global and local fMRI signals driven by neurons defined optogenetically by type and wiring NATURE Lee, J. H., Durand, R., Gradinaru, V., Zhang, F., Goshen, I., Kim, D., Fenno, L. E., Ramakrishnan, C., Deisseroth, K. 2010; 465 (7299): 788-792
Abstract

Despite a rapidly-growing scientific and clinical brain imaging literature based on functional magnetic resonance imaging (fMRI) using blood oxygenation level-dependent (BOLD) signals, it remains controversial whether BOLD signals in a particular region can be caused by activation of local excitatory neurons. This difficult question is central to the interpretation and utility of BOLD, with major significance for fMRI studies in basic research and clinical applications. Using a novel integrated technology unifying optogenetic control of inputs with high-field fMRI signal readouts, we show here that specific stimulation of local CaMKIIalpha-expressing excitatory neurons, either in the neocortex or thalamus, elicits positive BOLD signals at the stimulus location with classical kinetics. We also show that optogenetic fMRI (of MRI) allows visualization of the causal effects of specific cell types defined not only by genetic identity and cell body location, but also by axonal projection target. Finally, we show that of MRI within the living and intact mammalian brain reveals BOLD signals in downstream targets distant from the stimulus, indicating that this approach can be used to map the global effects of controlling a local cell population. In this respect, unlike both conventional fMRI studies based on correlations and fMRI with electrical stimulation that will also directly drive afferent and nearby axons, this of MRI approach provides causal information about the global circuits recruited by defined local neuronal activity patterns. Together these findings provide an empirical foundation for the widely-used fMRI BOLD signal, and the features of of MRI define a potent tool that may be suitable for functional circuit analysis as well as global phenotyping of dysfunctional circuitry.

View details for DOI 10.1038/nature09108

View details for Web of Science ID 000278551800047

View details for PubMedID 20473285

View details for PubMedCentralID PMC3177305
Temporally precise in vivo control of intracellular signalling NATURE Airan, R. D., Thompson, K. R., Fenno, L. E., Bernstein, H., Deisseroth, K. 2009; 458 (7241): 1025-1029
Abstract

In the study of complex mammalian behaviours, technological limitations have prevented spatiotemporally precise control over intracellular signalling processes. Here we report the development of a versatile family of genetically encoded optical tools ('optoXRs') that leverage common structure-function relationships among G-protein-coupled receptors (GPCRs) to recruit and control, with high spatiotemporal precision, receptor-initiated biochemical signalling pathways. In particular, we have developed and characterized two optoXRs that selectively recruit distinct, targeted signalling pathways in response to light. The two optoXRs exerted opposing effects on spike firing in nucleus accumbens in vivo, and precisely timed optoXR photostimulation in nucleus accumbens by itself sufficed to drive conditioned place preference in freely moving mice. The optoXR approach allows testing of hypotheses regarding the causal impact of biochemical signalling in behaving mammals, in a targetable and temporally precise manner.

View details for DOI 10.1038/nature07926

View details for Web of Science ID 000265412900042

View details for PubMedID 19295515

Lief Ericsson Fenno

Instructor, Psychiatry and Behavioral Sciences

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