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In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain

Ouzounov DG, Wang T, Wang M, Feng D, Horton NG, Cruz Hernández JC, Cheng Y, Reimer J, Tolias A, Nishimura N, Xu C

Nature Methods (2017)

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High-resolution optical imaging is critical to understanding brain function. We demonstrate that three-photon microscopy at 1,300-nm excitation enables functional imaging of GCaMP6s-labeled neurons beyond the depth limit of two-photon microscopy. We record spontaneous activity from up to 150 neurons in the hippocampal stratum pyramidale at ~1-mm depth within an intact mouse brain. Our method creates opportunities for noninvasive recording of neuronal activity with high spatial and temporal resolution deep within scattering brain tissues.

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Simultaneous Optical and Electrical In Vivo Analysis of the Enteric Nervous System

Rakhilin N, Barth B, Choi J, Munoz N, Kulkarni S, LaVinka C, Dong X, Spencer M, Pasricha P, Nishimura N, Jones J, Small D, Cheng YT, Cao Y, Kan E, Shen X

Nat Commun (2016)

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The enteric nervous system (ENS) is a major division of the nervous system and vital to the gastrointestinal (GI) tract and its communication with the rest of the body. Unlike the brain and spinal cord, relatively little is known about the ENS in part because of the inability to directly monitor its activity in live animals. Here, we integrate a transparent graphene sensor with a customized abdominal window for simultaneous optical and electrical recording of the ENS in vivo. The implanted device captures ENS responses to neurotransmitters, drugs and optogenetic manipulation in real time.

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A Mathematical Model Relating Cortical Oxygenated And Deoxygenated Hemoglobin Flows And Volumes To Neural Activity

Nathan Cornelius, Nozomi Nishimura, Minah Suh, Theodore Schwartz, and Peter Doerschuk

Journal of Neural Engineering (2015)

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Objective. To describe a toolkit of components for mathematical models of the relationship between cortical neural activity and space-resolved and time-resolved flows and volumes of oxygenated and deoxygenated hemoglobin motivated by optical intrinsic signal imaging (OISI). Approach. Both blood flow and blood volume and both oxygenated and deoxygenated hemoglobin and their interconversion are accounted for. Flow and volume are described by including analogies to both resistive and capacitive electrical circuit elements. Oxygenated and deoxygenated hemoglobin and their interconversion are described by generalization of Kirchhoff's laws based on well-mixed compartments. Main results. Mathematical models built from this toolkit are able to reproduce experimental single-stimulus OISI results that are described in papers from other research groups and are able to describe the response to multiple-stimuli experiments as a sublinear superposition of responses to the individual stimuli. Significance. The same assembly of tools from the toolkit but with different parameter values is able to describe effects that are considered distinctive, such as the presence or absence of an initial decrease in oxygenated hemoglobin concentration, indicating that the differences might be due to unique parameter values in a subject rather than different fundamental mechanisms.

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Comprehensive models of human primary and metastatic colorectal tumors in immunodeficient and immunocompetent mice by chemokine targeting

Chen HJ, Sun J, Huang Z, Hou H Jr, Arcilla M, Rakhilin N, Joe DJ, Choi J, Gadamsetty P, Milsom J, Nandakumar G, Longman R, Zhou XK, Edwards R, Chen J, Chen KY, Bu P, Wang L, Xu Y, Munroe R, Abratte C, Miller AD, Gümüş ZH, Shuler M, Nishimura N, Edelmann W, Shen X, Lipkin SM.

Nature Biotechnology (2015)

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Current orthotopic xenograft models of human colorectal cancer (CRC) require surgery and do not robustly form metastases in the liver, the most common site clinically. CCR9 traffics lymphocytes to intestine and colorectum. We engineered use of the chemokine receptor CCR9 in CRC cell lines and patient-derived cells to create primary gastrointestinal (GI) tumors in immunodeficient mice by tail-vein injection rather than surgery. The tumors metastasize inducibly and robustly to the liver. Metastases have higher DKK4 and NOTCH signaling levels and are more chemoresistant than paired subcutaneous xenografts. Using this approach, we generated 17 chemokine-targeted mouse models (CTMMs) that recapitulate the majority of common human somatic CRC mutations. We also show that primary tumors can be modeled in immunocompetent mice by microinjecting CCR9-expressing cancer cell lines into early-stage mouse blastocysts, which induces central immune tolerance. We expect that CTMMs will facilitate investigation of the biology of CRC metastasis and drug screening.

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Mechanistic insight into the TH1-biased immune response to recombinant subunit vaccines delivered by probiotic bacteria-derived outer membrane vesicles

Rosenthal JA, Huang CJ, Doody AM, Leung T, Mineta K, Feng DD, Wayne EC, Nishimura N, Leifer C, DeLisa MP, Mendez S, Putnam D.

PLoS One. (2014)

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Recombinant subunit vaccine engineering increasingly focuses on the development of more effective delivery platforms. However, current recombinant vaccines fail to sufficiently stimulate protective adaptive immunity against a wide range of pathogens while remaining a cost effective solution to global health challenges. Taking an unorthodox approach to this fundamental immunological challenge, we isolated the TLR-targeting capability of the probiotic E. coli Nissle 1917 bacteria (EcN) by engineering bionanoparticlate antigen carriers derived from EcN outer membrane vesicles (OMVs). Exogenous model antigens expressed by these modified bacteria as protein fusions with the bacterial enterotoxin ClyA resulted in their display on the surface of the carrier OMVs. Vaccination with the engineered EcN OMVs in a BALB/c mouse model, and subsequent mechanism of action analysis, established the EcN OMV's ability to induce self-adjuvanted robust and protective humoral and T(H)1-biased cellular immunity to model antigens. This finding appears to be strain-dependent, as OMV antigen carriers similarly engineered from a standard K12 E. coli strain derivative failed to generate a comparably robust antigen-specific TH1 bias. The results demonstrate that unlike traditional subunit vaccines, these biomolecularly engineered "pathogen-like particles" derived from traditionally overlooked, naturally potent immunomodulators have the potential to effectively couple recombinant antigens with meaningful immunity in a broadly applicable fashion.

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Three-photon excited fluorescence imaging of unstained tissue using a GRIN lens endoscope

David M. Huland, Kriti Charan, Dimitre G. Ouzounov, Jason S. Jones, Nozomi Nishimura, and Chris Xu

Biomedical Optics Express 4, 651 (2013)

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We present a compact and portable three-photon gradient index (GRIN) lens endoscope system suitable for imaging of unstained tissues, potentially deep within the body, using a GRIN lens system of 1 mm diameter and 8 cm length. The lateral and axial resolution in water is 1.0 μm and 9.5 μm, respectively. The ~200 μm diameter field of view is imaged at 2 frames/s using a fiber-based excitation source at 1040 nm. Ex vivo imaging is demonstrated with unstained mouse lung at 5.9 mW average power. These results demonstrate the feasibility of three-photon GRIN lens endoscopy for optical biopsy.

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Suppressed neuronal activity and concurrent arteriolar vasoconstriction may explain negative blood oxygenation level-dependent signal

28. Devor A, Tian P, Nishimura N, Teng IC, Hillman EM, Narayanan SN, Ulbert I, Boas DA, Kleinfeld D, Dale AM.

Journal of Neuroscience (2007)

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Synaptic transmission initiates a cascade of signal transduction events that couple neuronal activity to local changes in blood flow and oxygenation. Although a number of vasoactive molecules and specific cell types have been implicated, the transformation of stimulus-induced activation of neuronal circuits to hemodynamic changes is still unclear. We use somatosensory stimulation and a suite of in vivo imaging tools to study neurovascular coupling in rat primary somatosensory cortex. Our stimulus evoked a central region of net neuronal depolarization surrounded by net hyperpolarization. Hemodynamic measurements revealed that predominant depolarization corresponded to an increase in oxygenation, whereas predominant hyperpolarization corresponded to a decrease in oxygenation. On the microscopic level of single surface arterioles, the response was composed of a combination of dilatory and constrictive phases. Critically, the relative strength of vasoconstriction covaried with the relative strength of oxygenation decrease and neuronal hyperpolarization. These results suggest that a neuronal inhibition and concurrent arteriolar vasoconstriction correspond to a decrease in blood oxygenation, which would be consistent with a negative blood oxygenation level-dependent functional magnetic resonance imaging signal.

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