Overexpression of Channelrhodopsin 2 interferes with the GABAb receptor-mediated depression of GABA release from the somatostatin-containing interneurons of the prefrontal cortex

Liu, Lei; Ito, Wataru; Morozov, Alexei

doi:10.1101/168245

Cited by 3 publications

(3 citation statements)

References 17 publications

(17 reference statements)

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“…These accumulations were not considered somatic staining and therefore not included in the quantification. A similar phenomenon has been observed by other groups following viral overexpression of ChR2-eYFP ( Figure 1 B in 60 ).…”

Section: Methodssupporting

confidence: 89%

Circuit-selective cell-autonomous regulation of inhibition in pyramidal neurons by Ste20-like kinase

et al. 2022

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Section: Methodssupporting

confidence: 89%

Circuit-selective cell-autonomous regulation of inhibition in pyramidal neurons by Ste20-like kinase

et al. 2022

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“…Several reports indicated that expressing heterologous proteins, for example Channelrhodopsin (ChR2) 12 , may change network homeostasis 45 . To test whether we can observe this in our system, we compared development changes in neuronal activity in neurons expressing the microbial opsin CatCh to those that did not express any heterologous protein.…”

Section: Resultsmentioning

confidence: 99%

Efficient mesoscale phenotypic screening in cultured primary neuron culture

Nedrud

Schmidt

2019

Preprint

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14A longstanding question in neuroscience is how the activity of ion channels shapes neuronal 15 activity and, as a result, computation in circuits and networks. Optogenetic reagents are tools to 16 answer this question by enabling precise and dynamic perturbation of cellular states. However, 17 development of these reagents can be hampered by low-throughput assays in non-physiological 18 contexts. Here, we develop an all optical phenotypic screen in cultured primary hippocampal 19 neurons that enables the functional assessment of large libraries of genetically encoded 20 optogenetic actuators. Combining real-time analysis and data reduction methods allows for 21 continuous observation of several thousand neurons for several days without onerous data 22 storage overhead. This screening system may be useful in a diversity of research questions that 23 can be coupled to optical perturbation and sensing. 24 of constellation pharmacology is that differences in cell-specific channel and receptor expression 59 -collectively called a constellation-create distinct functional phenotypes [21][22][23][24] . It is therefore ion 60 channel diversity itself within populations of cells that provides screen-able content, i.e. 61 distinguishable and characteristic responses after the addition of a pharmacological agent that 62 perturb their function. Functional calcium imaging provides the means to collect this content in a 63 high-throughput manner. However, several technical limitations stand in the way, including the 64 need for manual manipulation (e.g., addition of the pharmacological agent). 65 66 Here, we develop an all optical phenotypic screen in cultured primary hippocampal neurons. We 67 introduce real-time analysis and data reduction methods, which allow for continuous observation 68 of several thousand neurons for several days without onerous data storage overhead. Our 69 analysis of calcium imaging data in neurons after the addition of free peptide toxins suggests that 70 toxin effects can be categorized according to 'fingerprints'. When we train a classification model 71 on these toxin fingerprints, we can detect those neurons -among many transfected with a library 72 of linker and toxin shuffled lumitoxins-that most closely resemble a desired phenotype. In this 73 way we identify a voltage-dependent Na + channel-specific lumitoxin that recapitulates the effects 74 of its free peptide toxin analog in a genetically encoded package. Our phenotypic toxin screening 75 assay sets the stage for linking genotype and phenotype in future studies with more complex 76 library designs. How this assay can be adapted to other research questions that can be coupled 77 to an optical readout is discussed. 78 79Results 80 81 We used cultured hippocampal neurons, which are known to spike spontaneously after several 82 days in culture [25][26][27] . Much of their ion channel and receptor complement expression over time 83 has been extensively studied [28][29][30][31][32] . For an all-optical screen, both sensor and actuator need to ...

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“…A note of caution that we feel compelled to point out is that our investigations rely heavily on an overexpression system, which has been shown in published studies to yield artifactual results [26][27][28][29][30][31] and likely also observed in many unpublished works. We used the same lentiviral vector to overexpress other genes in KPL cells, including a catalytically dead Cas9 (dCas9), and found that regardless of the gene, overexpression appeared to increase tumor burden in vivo, compared to vector control (data not shown).…”

Section: Markosyan Et Al Observed a Decrease In Tumor Cell Proliferation And In Vivo Tumor Burden Withmentioning

confidence: 98%

Tumor-specific EphA2 receptor tyrosine kinase inhibits anti-tumor immunity by recruiting suppressive myeloid populations in NSCLC

Shiuan

Song

Wang

et al. 2020

Preprint

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Given the success of both targeted and immunotherapies against cancer, there is increasing utility for identifying targeted agents that also promote anti-tumor immunity. EphA2 is a receptor tyrosine kinase that contributes to tumor growth and metastasis and has been identified as a viable target for many solid cancers. Investigating EphA2's impact on the host immune system may advance our understanding of tumor immune evasion and the consequences of targeting EphA2 on the tumor microenvironment. Here, we examine how tumor-specific EphA2 affects the activation and infiltration of immune cell populations and the cytokine and chemokine milieu in non-small cell lung cancer (NSCLC). Effects of EphA2 overexpression in murine NSCLC cells were evaluated in both in vitro cell viability assays and in vivo tumor models. Tumor immune infiltrate was assessed by flow cytometry. Cytokine and chemokine expression was evaluated using NanoString technology and qRT-PCR. Although EphA2 overexpression in NSCLC cells did not display proliferative advantage in vitro, it conferred a growth advantage in vivo. Analysis of lung tumor infiltrate revealed decreased natural killer and T cells in the EphA2-overexpressing tumors, as well as increased myeloid populations, including tumor-associated macrophages (TAMs). T cell activation, particularly in CD8 T cells, was decreased, while PD-1 expression was increased. These changes were accompanied by increased monocyte-attracting chemokines, specifically CCL2, CCL7, CCL8, and CCL12, and immunosuppressive proteins TGF-β and arginase 1. Our studies suggest EphA2 on tumor cells recruits monocytes and promotes their differentiation into TAMs that likely inhibit activation and infiltration of cytotoxic lymphocytes, promoting tumor immune escape.Further studies are needed to determine the molecular mechanisms by which EphA2 affects the recruitment of these cell types and to test the function of these myeloid cells.

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Overexpression of Channelrhodopsin 2 interferes with the GABAb receptor-mediated depression of GABA release from the somatostatin-containing interneurons of the prefrontal cortex

Cited by 3 publications

References 17 publications

Circuit-selective cell-autonomous regulation of inhibition in pyramidal neurons by Ste20-like kinase

Circuit-selective cell-autonomous regulation of inhibition in pyramidal neurons by Ste20-like kinase

Efficient mesoscale phenotypic screening in cultured primary neuron culture

Tumor-specific EphA2 receptor tyrosine kinase inhibits anti-tumor immunity by recruiting suppressive myeloid populations in NSCLC

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