Machine learning ensemble directed engineering of genetically encoded fluorescent calcium indicators
Wait S, Expòsit M*, Lin S*, Rappleye M, Lee JD, Torp L, Ascensio A, Smith N, Regnier M, Moussavi-Harami F, Baker D, Kim CK, and Berndt A. Nature Computational Science (2024).

“In this study, we focused on the transformative potential of machine learning in the engineering of genetically encoded fluorescent indicators (GEFIs), protein-based sensing tools that are critical for real-time monitoring of biological activity. We applied an alternative approach using machine learning to predict the outcomes of sensor mutagenesis by analyzing established libraries that link sensor sequences to functions. By leveraging the learning capabilities of our ensemble, we were able to accelerate the identification of promising mutations and reduce the experimental burden associated with trial-and-error mutagenesis. Overall, these findings have significant implications for optimizing GEFIs and other protein-based tools, demonstrating the utility of machine learning as a powerful asset in protein engineering.”

Rapid, biochemical tagging of cellular activity history in vivo
Zhang R, Anguiano M, Aarrestad IK, Lin S, Chandra J, Vadde SS, Olson DE, and Kim CK. bioRxiv (2023).

“Intracellular calcium (Ca2+) is ubiquitous to cell signaling across all biology. While existing fluorescent sensors and reporters can detect activated cells with elevated Ca2+ levels, these approaches require implants to deliver light to deep tissue, precluding their noninvasive use in freely-behaving animals. Here we engineered an enzyme-catalyzed approach that rapidly and biochemically tags cells with elevated Ca2+ in vivo. Ca2+-activated Split-TurboID (CaST) labels activated cells within 10 minutes with an exogenously-delivered biotin molecule. The enzymatic signal increases with Ca2+ concentration and biotin labeling time, demonstrating that CaST is a time-gated integrator of total Ca2+ activity. Furthermore, the CaST read-out can be performed immediately after activity labeling, in contrast to transcriptional reporters that require hours to produce signal. These capabilities allowed us to apply CaST in a new context to tag prefrontal cortex neurons activated by psilocybin, and to correlate the CaST signal with psilocybin-induced head-twitch responses in untethered mice.”

Tagging neurons with light: Molecular circuits for activity-guided optogenetics [Science & PINS Prize runner-up essay]
Kim, CK. Science (2023).

“Optogenetics, which enables cell-type specific neuromodulation using light-activated opsins, is a powerful methodology for manipulating neuronal activity and behavior in animal models. However, one challenge is targeting the relevant subpopulation of neurons with light to drive the desired behavioral response. We developed FLiCRE, an engineered light- and calcium-dependent transcription reporter, that can tag activated neuronal circuits within brief time windows of behavior or stimuli (1-10 minutes) in mice. FLiCRE relies on user-delivered blue light and high intracellular calcium to label activated neurons with a red light-sensitive opsin. This allowed us to deliver blue light to mice during a stimulus, tag the relevant activated neurons with an opsin, and then subsequently re-activate the neurons the following day with red light to achieve the desired behavioral result.“

Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells
Li S-Y*, Cheah JS*, Zhao B, Xu C, Roh H, Kim CK, Cho KF, Udeshi ND, Carr SA, Ting AY. Nature Methods (2023).

“The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions and function with light. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the proximity labeling enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. Overall, LOV-Turbo increases the spatial and temporal precision of proximity labeling, expanding the scope of experimental questions that can be addressed with proximity labeling.”

Building synapses: using a synthetic approach to bridge synaptic membranes [commentary]
Kim CK, Kolodkin AL, Kang S, & Stuber GD. Building synapses: using a synthetic approach to bridge synaptic membranes. Faculty Reviews (2022).

:Synapses are specialized cellular junctions essential for communication between neurons. Synapse loss occurs in many neurodegenerative diseases. Harnessing our molecular knowledge of the development and maintenance of synapses, Suzuki et al. present the first comprehensive attempt to use a synthetic protein to bridge the pre- and postsynaptic membranes. They show that this powerful approach can stimulate the formation of pre- and postsynaptic specializations in vitro, rescue synaptic deficits of mutant mice in vivo, and ameliorate synapse loss and behavioral abnormalities in both Alzheimer's disease and spinal cord injury mouse models.”

Correcting for the hemoglobin absorption artifact in fiber photometry data [preview]
Zhang R and Kim CK. Cell Reports Methods (2022).

”Fiber photometry is a widely used fluorescence approach for measuring neuronal activity in freely behaving animals; however, these signals can be contaminated by hemoglobin absorption artifacts. Zhang et al. propose a computational method to quantify and correct hemoglobin photon absorption effects using spectral fiber photometry, resulting in more accurate neuronal activity measurements.”

A molecular calcium integrator reveals a striatal cell type driving aversion
Kim CK*, Sanchez MI*, Hoerbelt P, Fenno LE, Deisseroth K, Ting AY. Cell (2020).

“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). The specificity and minute resolution of FLiCRE enables molecularly informed characterization, manipulation, and reprogramming of activated cellular ensembles.”

Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions
Kim CK, Cho KF, Kim MW, Ting AY. Elife (2019).

“Technologies that convert transient protein-protein interactions (PPIs) into stable expression of a reporter gene are useful for genetic selections, high-throughput screening, and multiplexing with omics technologies. Here we report SPARK2, a transcription factor that is activated by the coincidence of a PPI and a luciferase moiety to control the light-sensitive LOV domain. We apply SPARK2 to high-throughput screening for GPCR agonists and for the detection of trans-cellular contacts, all with versatile transcriptional readout.”

Molecular tools for imaging and recording neuronal activity [review]
Wang W*, Kim CK*, Ting AY. Nature chemical biology (2019).

“To understand how the brain relates to behavior, a large variety of genetically encoded sensors have been developed to monitor and record the series of events following neuronal firing, including action potentials, intracellular calcium rise, neurotransmitter release and immediate early gene expression. In this Review, we discuss the existing genetically encoded tools for detecting and integrating neuronal activity in animals and highlight the remaining challenges and future opportunities for molecular biologists.”

Interacting neural ensembles in orbitofrontal cortex for social and feeding behavior
Jennings JH*, Kim CK*, Marshel JH*, Raffiee M, Ye L, Quirin S, Pak S, Ramakrishnan C, Deisseroth K. Nature (2019).

“Categorically distinct basic drives can exert potent influences on each other; such interactions are likely to have important adaptive consequences and can become maladaptive. Here we coupled genetically encoded activity imaging with optogenetic control of multiple individually defined cells, to both optically monitor and manipulate the activity of many orbitofrontal cortex neurons during rewarding experiences. These results reveal the presence of potent cellular-level subnetworks within the orbitofrontal cortex that can be precisely engaged to bidirectionally control feeding behaviours subject to, for example, social influences.”

A neural circuit mechanism for encoding aversive stimuli in the mesolimbic dopamine system
de Jong JW*, Afjei SA*, Pollock Dorocic I, Peck JR, Liu C, Kim CK, Tian L, Deisseroth K, Lammel S. Neuron (2019). 

“Ventral tegmental area (VTA) dopamine (DA) neurons play a central role in mediating motivated behaviors, but the circuitry through which they signal positive and negative motivational stimuli is incompletely understood. Using in vivo fiber photometry, we simultaneously recorded activity in DA terminals in different nucleus accumbens (NAc) subnuclei during an aversive and reward conditioning task. We find that DA terminals in the ventral NAc medial shell (vNAcMed) are excited by unexpected aversive outcomes and to cues that predict them, whereas DA terminals in other NAc subregions are persistently depressed.”

Molecular and circuit-dynamical identification of top-down neural mechanisms for restraint of reward seeking
Kim CK*, Ye L*, Jennings JH, Pichamoorthy N, Tang DD, Yoo A-CW, Ramakrishnan C, Deisseroth K. Cell (2017).

“Reward-seeking behavior is fundamental to survival, but suppression of this behavior can be essential as well. Here, we show using 2-photon Ca2+ imaging and activity-dependent labeling that a specific subset of superficial mPFC projections to a subfield of NAc neurons naturally encodes the decision to initiate or suppress reward seeking when faced with risk of punishment. This mPFC-to-NAc projection displayed unique molecular-genetic and microcircuit-level features concordant with a conserved role in the regulation of reward-seeking behavior, providing cellular and anatomical identifiers of behavioral and possible therapeutic significance.”

Rabies screen reveals GPe control of cocaine-triggered plasticity
Beier KT, Kim CK, Hoerbelt P, Hung LW, Heifets BD, DeLoach KE, Mosca TJ, Neuner S, Deisseroth K, Luo L, & Malenka RC. Nature (2017). 

“Identification of neural circuit changes that contribute to behavioural plasticity has routinely been conducted on candidate circuits that were preselected on the basis of previous results. Here we present an unbiased rabies-based method for identifying experience-triggered circuit-level changes in neuronal ensembles in mice. We demonstrated that cocaine increased GPe neuron activity, which accounted for the increase in GPe rabies labelling. These results suggest that rabies-based unbiased screening of changes in input populations can identify previously unappreciated circuit elements that critically support behavioural adaptations.”

Modulation of prefrontal cortex excitation/inhibition balance rescues social behavior in CNTNAP2-deficient mice
Selimbeyoglu A, Kim CK, Inoue M, Lee SY, Hong ASO, Ramakrishnan C, Fenno LE, Davidson TJ, Wright M, & Deisseroth K. Science translational medicine (2017).

“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 found that acutely reducing E:I balance in mouse brain could correct deficits in social behavior in mice lacking the CNTNAP2 gene, which has been implicated in autism. Using fiber photometry, we discovered that native mPFC PV neuronal activity differed between CNTNAP2 knockout and wild-type mice. 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.”

Integration of optogenetics with complementary methodologies in systems neuroscience [review]
Kim CK*, Adhikari A*, Deisseroth K. Nature Reviews Neuroscience (2017).

“Modern optogenetics can be tuned to evoke activity that corresponds to naturally occurring local or global activity in timing, magnitude or individual-cell patterning. This outcome has been facilitated by the recent integration of optogenetics with complementary technologies, spanning electrophysiology, activity imaging and anatomical methods for structural and molecular analysis. This integrated approach now supports optogenetic identification of the native, necessary and sufficient causal underpinnings of physiology and behaviour on acute or chronic timescales and across cellular, circuit-level or brain-wide spatial scales.”

Simultaneous fast measurement of circuit dynamics at multiple sites across the mammalian brain
Kim CK*, Yang SJ*, Pichamoorthy N, Young NP, Kauvar I, Jennings JH, Lerner TN, Berndt A, Lee SY, Ramakrishnan C, Davidson TJ, Inoue M, Bito H, Deisseroth K. Nature Methods (2016).

“Real-time activity measurements from multiple specific cell populations and projections are likely to be important for understanding the brain as a dynamical system. Here we developed frame-projected independent-fiber photometry (FIP) to record fluorescence activity signals from many brain regions simultaneously in freely behaving mice. We simultaneously recorded activity along multiple axonal pathways during sensory experience, performed simultaneous two-color activity recording, and applied optical perturbation tuned to elicit dynamics that match naturally occurring patterns observed during behavior.”

Projections from neocortex mediate top-down control of memory retrieval
Rajasethupathy P*, Sankaran S*, Marshel JH*, Kim CK, Berndt A, Lee SY, Jaffe A, Liston C, & Deisseroth K. Nature (2015).

“Top-down prefrontal cortex inputs to the hippocampus have been hypothesized to be important in memory consolidation, retrieval, and the pathophysiology of major psychiatric diseases; however, no such direct projections have been identified and functionally described. Here we report the discovery of a monosynaptic prefrontal cortex (predominantly anterior cingulate) to hippocampus (CA3 to CA1 region) projection in mice, and find that optogenetic manipulation of this projection (here termed AC-CA) is capable of eliciting contextual memory retrieval in virtual-reality environments.”

Extended field-of-view and increased-signal 3D holographic illumination with time-division multiplexing
Yang SJ, Allen WE, Kauvar I, Andalman AS, Young NP, Kim CK, Marshel JH, Wetzstein G, & Deisseroth K. Optics Express (2015). 

“Phase spatial light modulators (SLMs) are widely used for generating multifocal 3D illumination patterns, but these are limited to a FOV constrained by the pixel count or size of the SLM. Further, with 2-photon SLM-based excitation, increasing the number of focal spots penalizes the total signal linearly--requiring more laser power than can be tolerated by the sample. Here we use galvanometer mirrors to time-sequentially reposition multiple 3D holograms, both extending the field of view and increasing the total time-averaged 2-photon signal. We apply our approach to 3D 2-photon in vivo neuronal calcium imaging.”

Prolonged, brain-wide expression of nuclear-localized GCaMP3 for functional circuit mapping
Kim CK, Miri A, Leung L, Berndt A, Mourrain P, Tank DW, & Burdine RD. Frontiers in Neural Circuits (2014).

“Larval zebrafish offer the potential for large-scale optical imaging of neural activity throughout the central nervous system; however, several barriers challenge their utility. Here, we report the creation of transgenic zebrafish strains exhibiting robust, nuclearly targeted expression of GCaMP3 across the brain up to at least 14 dpf. Our methodological approach will facilitate studies of larval zebrafish circuitry by both improving resolution of functional Ca2+ signals and by allowing brain-wide expression of improved GECIs, or potentially any probe, further into development.”

Gating of neural error signals during motor learning
Kimpo RR*, Rinaldi JM*, Kim CK*, Payne HL*, & Raymond JL. eLife (2014).

“Cerebellar climbing fiber activity encodes performance errors during many motor learning tasks, but the role of these error signals in learning has been controversial. We compared two motor learning paradigms that elicited equally robust putative error signals in the same climbing fibers: learned increases and decreases in the gain of the vestibulo-ocular reflex (VOR). Our data suggest that the ability of climbing fibers to induce plasticity can be dynamically gated in vivo, even under conditions where climbing fibers are robustly activated by performance errors.”

Diverging neural pathways assemble a behavioural state from separable features in anxiety
Kim SY*, Adhikari A*, Lee SY, Marshel JH, Kim CK, Mallory CS, Lo M, Pak S, Mattis J, Lim BK, Malenka RC, Warden MR, Neve R, Tye KM, & Deisseroth K. Nature (2013).

“Behavioural states in mammals are characterized by several features that are coordinately regulated by diverse nervous system outputs, ranging from behavioural choice patterns to changes in physiology. Here we investigate if and how defined neural projections arising from a single brain region in mice could mediate diverse features of anxiety. These results demonstrate that distinct BNST subregions exert opposite effects in modulating anxiety, and illustrate circuit mechanisms underlying selection of features for the assembly of the anxious state.”