Linlin Fan

All Publications

• All-optical physiology resolves a synaptic basis for behavioral timescale plasticity. Cell Fan, L. Z., Kim, D. K., Jennings, J. H., Tian, H., Wang, P. Y., Ramakrishnan, C., Randles, S., Sun, Y., Thadhani, E., Kim, Y. S., Quirin, S., Giocomo, L., Cohen, A. E., Deisseroth, K. 2023

  Abstract

  Learning has been associated with modifications of synaptic and circuit properties, but the precise changes storing information in mammals have remained largely unclear. We combined genetically targeted voltage imaging with targeted optogenetic activation and silencing of pre- and post-synaptic neurons to study the mechanisms underlying hippocampal behavioral timescale plasticity. In mice navigating a virtual-reality environment, targeted optogenetic activation of individual CA1 cells at specific places induced stable representations of these places in the targeted cells. Optical elicitation, recording, and modulation of synaptic transmission in behaving mice revealed that activity in presynaptic CA2/3 cells was required for the induction of plasticity in CA1 and, furthermore, that during induction of these place fields in single CA1 cells, synaptic input from CA2/3 onto these same cells was potentiated. These results reveal synaptic implementation of hippocampal behavioral timescale plasticity and define a methodology to resolve synaptic plasticity during learning and memory in behaving mammals.

  View details for DOI 10.1016/j.cell.2022.12.035

  View details for PubMedID 36669484

• Video-based pooled screening yields improved far-red genetically encoded voltage indicators. Nature methods Tian, H., Davis, H. C., Wong-Campos, J. D., Park, P., Fan, L. Z., Gmeiner, B., Begum, S., Werley, C. A., Borja, G. B., Upadhyay, H., Shah, H., Jacques, J., Qi, Y., Parot, V., Deisseroth, K., Cohen, A. E. 2023

  Abstract

  Video-based screening of pooled libraries is a powerful approach for directed evolution of biosensors because it enables selection along multiple dimensions simultaneously from large libraries. Here we develop a screening platform, Photopick, which achieves precise phenotype-activated photoselection over a large field of view (2.3 × 2.3 mm, containing >103 cells, per shot). We used the Photopick platform to evolve archaerhodopsin-derived genetically encoded voltage indicators (GEVIs) with improved signal-to-noise ratio (QuasAr6a) and kinetics (QuasAr6b). These GEVIs gave improved signals in cultured neurons and in live mouse brains. By combining targeted in vivo optogenetic stimulation with high-precision voltage imaging, we characterized inhibitory synaptic coupling between individual cortical NDNF (neuron-derived neurotrophic factor) interneurons, and excitatory electrical synapses between individual hippocampal parvalbumin neurons. The QuasAr6 GEVIs are powerful tools for all-optical electrophysiology and the Photopick approach could be adapted to evolve a broad range of biosensors.

  View details for DOI 10.1038/s41592-022-01743-5

  View details for PubMedID 36624211

• High-fidelity estimates of spikes and subthreshold waveforms from 1-photon voltage imaging in vivo. Cell reports Xie, M. E., Adam, Y. n., Fan, L. Z., Böhm, U. L., Kinsella, I. n., Zhou, D. n., Rozsa, M. n., Singh, A. n., Svoboda, K. n., Paninski, L. n., Cohen, A. E. 2021; 35 (1): 108954

  Abstract

  The ability to probe the membrane potential of multiple genetically defined neurons simultaneously would have a profound impact on neuroscience research. Genetically encoded voltage indicators are a promising tool for this purpose, and recent developments have achieved a high signal-to-noise ratio in vivo with 1-photon fluorescence imaging. However, these recordings exhibit several sources of noise and signal extraction remains a challenge. We present an improved signal extraction pipeline, spike-guided penalized matrix decomposition-nonnegative matrix factorization (SGPMD-NMF), which resolves supra- and subthreshold voltages in vivo. The method incorporates biophysical and optical constraints. We validate the pipeline with simultaneous patch-clamp and optical recordings from mouse layer 1 in vivo and with simulated and composite datasets with realistic noise. We demonstrate applications to mouse hippocampus expressing paQuasAr3-s or SomArchon1, mouse cortex expressing SomArchon1 or Voltron, and zebrafish spines expressing zArchon1.

  View details for DOI 10.1016/j.celrep.2021.108954

  View details for PubMedID 33826882

• All-Optical Electrophysiology Reveals the Role of Lateral Inhibition in Sensory Processing in Cortical Layer 1 CELL Fan, L. Z., Kheifets, S., Bohm, U. L., Wu, H., Piatkevich, K. D., Xie, M. E., Parot, V., Ha, Y., Evans, K. E., Boyden, E. S., Takesian, A. E., Cohen, A. E. 2020; 180 (3): 521-+

  Abstract

  Cortical layer 1 (L1) interneurons have been proposed as a hub for attentional modulation of underlying cortex, but the transformations that this circuit implements are not known. We combined genetically targeted voltage imaging with optogenetic activation and silencing to study the mechanisms underlying sensory processing in mouse barrel cortex L1. Whisker stimuli evoked precisely timed single spikes in L1 interneurons, followed by strong lateral inhibition. A mild aversive stimulus activated cholinergic inputs and evoked a bimodal distribution of spiking responses in L1. A simple conductance-based model that only contained lateral inhibition within L1 recapitulated the sensory responses and the winner-takes-all cholinergic responses, and the model correctly predicted that the network would function as a spatial and temporal high-pass filter for excitatory inputs. Our results demonstrate that all-optical electrophysiology can reveal basic principles of neural circuit function in vivo and suggest an intuitive picture for how L1 transforms sensory and modulatory inputs. VIDEO ABSTRACT.

  View details for DOI 10.1016/j.cell.2020.01.001

  View details for Web of Science ID 000512977500009

  View details for PubMedID 31978320

• Compressed Hadamard microscopy for high-speed optically sectioned neuronal activity recordings JOURNAL OF PHYSICS D-APPLIED PHYSICS Parot, V. J., Sing-Long, C., Adam, Y., Bohm, U. L., Fan, L. Z., Farhi, S. L., Cohen, A. E. 2019; 52 (14)

  View details for DOI 10.1088/1361-6463/aafe88

  View details for Web of Science ID 000457719300001

• All-optical synaptic electrophysiology probes mechanism of ketamine-induced disinhibition NATURE METHODS Fan, L. Z., Nehme, R., Adam, Y., Jung, E., Wu, H., Eggan, K., Arnold, D. B., Cohen, A. E. 2018; 15 (10): 823-+

  Abstract

  Optical assays of synaptic strength could facilitate studies of neuronal transmission and its dysregulation in disease. Here we introduce a genetic toolbox for all-optical interrogation of synaptic electrophysiology (synOptopatch) via mutually exclusive expression of a channelrhodopsin actuator and an archaerhodopsin-derived voltage indicator. Optically induced activity in the channelrhodopsin-expressing neurons generated excitatory and inhibitory postsynaptic potentials that we optically resolved in reporter-expressing neurons. We further developed a yellow spine-targeted Ca2+ indicator to localize optogenetically triggered synaptic inputs. We demonstrated synOptopatch recordings in cultured rodent neurons and in acute rodent brain slice. In synOptopatch measurements of primary rodent cultures, acute ketamine administration suppressed disynaptic inhibitory feedbacks, mimicking the effect of this drug on network function in both rodents and humans. We localized this action of ketamine to excitatory synapses onto interneurons. These results establish an in vitro all-optical model of disynaptic disinhibition, a synaptic defect hypothesized in schizophrenia-associated psychosis.

  View details for DOI 10.1038/s41592-018-0142-8

  View details for Web of Science ID 000448820100031

  View details for PubMedID 30275587

  View details for PubMedCentralID PMC6204345

• Optical control of cell signaling by single-chain photoswitchable kinases. Science Zhou, X. X., Fan, L. Z., Li, P., Shen, K., Lin, M. Z. 2017; 355 (6327): 836-842

  Abstract

  Protein kinases transduce signals to regulate a wide array of cellular functions in eukaryotes. A generalizable method for optical control of kinases would enable fine spatiotemporal interrogation or manipulation of these various functions. We report the design and application of single-chain cofactor-free kinases with photoswitchable activity. We engineered a dimeric protein, pdDronpa, that dissociates in cyan light and reassociates in violet light. Attaching two pdDronpa domains at rationally selected locations in the kinase domain, we created the photoswitchable kinases psRaf1, psMEK1, psMEK2, and psCDK5. Using these photoswitchable kinases, we established an all-optical cell-based assay for screening inhibitors, uncovered a direct and rapid inhibitory feedback loop from ERK to MEK1, and mediated developmental changes and synaptic vesicle transport in vivo using light.

  View details for DOI 10.1126/science.aah3605

  View details for PubMedID 28232577

• Molecular Mechanism of Disease-Associated Mutations in the Pre-M1 Helix of NMDA Receptors and Potential Rescue Pharmacology PLOS GENETICS Ogden, K. K., Chen, W., Swanger, S. A., McDaniel, M. J., Fan, L. Z., Hu, C., Tankovic, A., Kusumoto, H., Kosobucki, G. J., Schulien, A. J., Su, Z., Pecha, J., Bhattacharya, S., Petrovski, S., Cohen, A. E., Aizenman, E., Traynelis, S. F., Yuan, H. 2017; 13 (1): e1006536

  Abstract

  N-methyl-D-aspartate receptors (NMDARs), ligand-gated ionotropic glutamate receptors, play key roles in normal brain development and various neurological disorders. Here we use standing variation data from the human population to assess which protein domains within NMDAR GluN1, GluN2A and GluN2B subunits show the strongest signal for being depleted of missense variants. We find that this includes the GluN2 pre-M1 helix and linker between the agonist-binding domain (ABD) and first transmembrane domain (M1). We then evaluate the functional changes of multiple missense mutations in the NMDAR pre-M1 helix found in children with epilepsy and developmental delay. We find mutant GluN1/GluN2A receptors exhibit prolonged glutamate response time course for channels containing 1 or 2 GluN2A-P552R subunits, and a slow rise time only for receptors with 2 mutant subunits, suggesting rearrangement of one GluN2A pre-M1 helix is sufficient for rapid activation. GluN2A-P552R and analogous mutations in other GluN subunits increased the agonist potency and slowed response time course, suggesting a functionally conserved role for this residue. Although there is no detectable change in surface expression or open probability for GluN2A-P552R, the prolonged response time course for receptors that contained GluN2A-P552R increased charge transfer for synaptic-like activation, which should promote excitotoxic damage. Transfection of cultured neurons with GluN2A-P552R prolonged EPSPs, and triggered pronounced dendritic swelling in addition to excitotoxicity, which were both attenuated by memantine. These data implicate the pre-M1 region in gating, provide insight into how different subunits contribute to gating, and suggest that mutations in the pre-M1 helix can compromise neuronal health. Evaluation of FDA-approved NMDAR inhibitors on the mutant NMDAR-mediated current response and neuronal damage provides a potential clinical path to treat individuals harboring similar mutations in NMDARs.

  View details for DOI 10.1371/journal.pgen.1006536

  View details for Web of Science ID 000394147700014

  View details for PubMedID 28095420

  View details for PubMedCentralID PMC5240934

• Optical control of biological processes by light-switchable proteins WILEY INTERDISCIPLINARY REVIEWS-DEVELOPMENTAL BIOLOGY Fan, L. Z., Lin, M. Z. 2015; 4 (5): 545-554

  Abstract

  Cellular processes such as proliferation, differentiation, or migration depend on precise spatiotemporal coordination of protein activities. Correspondingly, reaching a quantitative understanding of cellular behavior requires experimental approaches that enable spatial and temporal modulation of protein activity. Recently, a variety of light-sensitive protein domains have been engineered as optogenetic actuators to spatiotemporally control protein activity. In the present review, we discuss the principle of these optical control methods and examples of their applications in modulating signaling pathways. By controlling protein activity with spatiotemporal specificity, tunable dynamics, and quantitative control, light-controllable proteins promise to accelerate our understanding of cellular and organismal biology. WIREs Dev Biol 2015, 4:545-554. doi: 10.1002/wdev.188 For further resources related to this article, please visit the WIREs website.

  View details for DOI 10.1002/wdev.188

  View details for Web of Science ID 000359429900006

  View details for PubMedID 25858669

  View details for PubMedCentralID PMC4529752