Genetically Targeted All-Optical Electrophysiology with a Transgenic Cre-Dependent Optopatch Mouse

Lou, Shan; Adam, Yoav; Weinstein, Eli N.; Williams, Eric; Williams, Katherine; Parot, Vicente; Kavokine, Nikita; Liberles, Stephen D.; Madisen, Linda; Zeng, Hongkui; Cohen, Adam E.

doi:10.1523/jneurosci.1582-16.2016

Cited by 78 publications

(90 citation statements)

References 60 publications

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“…Cohen , “All-optical electrophysiology of synaptic transmission and synaptic plasticity” in submission ). QuasAr voltage indicators have been shown to report membrane voltage changes in cultured neurons with good fidelity and linearity [43]. Optical stimulation of the presynaptic cells evoked EPSPs which were detected via fluorescence from the postsynaptic cells.…”

Section: Resultsmentioning

confidence: 99%

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Molecular Mechanism of Disease-Associated Mutations in the Pre-M1 Helix of NMDA Receptors and Potential Rescue Pharmacology

et al. 2017

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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.

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Section: Resultsmentioning

confidence: 99%

“…The immunostaining was following the protocol described in [43]. Inhibitory neurons were counted manually in fluorescence images.…”

Section: Methodsmentioning

confidence: 99%

Molecular Mechanism of Disease-Associated Mutations in the Pre-M1 Helix of NMDA Receptors and Potential Rescue Pharmacology

et al. 2017

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“…In the next several years, we are likely to see the use of this tool broadened to neuromodulator based questions and in vivo drug screening approaches. Developments in voltage indicators such will also allow for fine temporal precision of measuring neuromodulatory effects on membrane potential [85,86]. …”

Section: Reportersmentioning

confidence: 99%

Optogenetic approaches for dissecting neuromodulation and GPCR signaling in neural circuits

Spangler

Bruchas

2017

Current Opinion in Pharmacology

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Optogenetics has revolutionized neuroscience by providing means to control cell signaling with spatiotemporal control in discrete cell types. In this review, we summarize four major classes of optical tools to manipulate neuromodulatory GPCR signaling: opsins (including engineered chimeric receptors); photoactivatable proteins; photopharmacology through caging - photoswitchable molecules; fluorescent protein based reporters and biosensors. Additionally, we highlight technologies to utilize these tools in vitro and in vivo, including Cre dependent viral vector expression and two-photon microscopy. These emerging techniques targeting specific members of the GPCR signaling pathway offer an expansive base for investigating GPCR signaling in behavior and disease states, in addition to paving a path to potential therapeutic developments.

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“…[41] These paired ‘Optopatch’ constructs enabled wide-field all-optical stimulation and readout of neural activity. Optopatch measurements have been used to probe the excitability of primary rodent and human iPSC-derived CNS [41] and sensory DRG [81] neurons. A recent application probed the excitability of human iPSC-derived motor neurons containing a mutation causal for ALS.…”

Section: Optogenetics For Phenotypic Screeningmentioning

confidence: 99%

“…The challenge with passive imaging in acute slice is that under typical conditions, there is little spontaneous activity. Recently, Optopatch measurements of excitability were demonstrated in acute slice, using a transgenic Cre-dependent Optopatch mouse to target Optopatch expression to genetically specified subsets of neurons [81]. It has not yet been possible to perform Optopatch measurements of synaptic transmission in acute slice because postsynaptic potentials in tissue are ∼20-fold smaller than in culture [91].…”

Section: Optogenetics For Phenotypic Screeningmentioning

confidence: 99%

Optogenetic Approaches to Drug Discovery in Neuroscience and Beyond

Zhang

Cohen

2017

Trends in Biotechnology

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Recent advances in optogenetics have opened new routes to drug discovery, particularly in neuroscience. Physiological cellular assays probe functional phenotypes that connect genomic data to patient health. Optogenetic tools, and in particular tools for all-optical electrophysiology, now provide means to probe cellular disease models with unprecedented throughput and information content. These techniques promise to identify functional phenotypes associated with disease states and to identify compounds that improve cellular function regardless of whether the compound acts directly on a target or through a bypass mechanism. This review discusses opportunities and unresolved challenges in applying optogenetic techniques throughout the discovery pipeline, from target identification and validation, to target-based and phenotypic screens, to clinical trials.

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Genetically Targeted All-Optical Electrophysiology with a Transgenic Cre-Dependent Optopatch Mouse

Cited by 78 publications

References 60 publications

Molecular Mechanism of Disease-Associated Mutations in the Pre-M1 Helix of NMDA Receptors and Potential Rescue Pharmacology

Molecular Mechanism of Disease-Associated Mutations in the Pre-M1 Helix of NMDA Receptors and Potential Rescue Pharmacology

Optogenetic approaches for dissecting neuromodulation and GPCR signaling in neural circuits

Optogenetic Approaches to Drug Discovery in Neuroscience and Beyond

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