Production of Viral Constructs for Neuroanatomy, Calcium Imaging, and Optogenetics

Chen, Shih‐Heng; Haam, Juhee; Walker, Mitzie; Scappini, Erica; Naughton, John; Martin, Negin P.

doi:10.1002/cpns.66

Cited by 19 publications

(13 citation statements)

References 36 publications

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“…SARS-CoV-2 pseudotyped lentivirus-based transduction fluorescence inhibition assay. All lentiviruses were propagated in HEK293T/17 cells (ATCC # CRL-11268) according to published Current Protocols in Neuroscience 41 . Briefly, 293 T cells were transiently transfected with plasmids expressing SARS-CoV-2 spike protein (GenScript MC_0101081, human codon optimized, ER retention signal removed), psPAX2 (Addgene #12260), and a lentiviral transfer vector CD512-EF1a-RFP (System Biosciences CD512B-1) using Lipofectamine 2000.…”

Section: Discussionmentioning

confidence: 99%

High affinity nanobodies block SARS-CoV-2 spike receptor binding domain interaction with human angiotensin converting enzyme

Esparza

Martin

Anderson

et al. 2020

Sci Rep

Self Cite

101

121

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There are currently few approved effective treatments for SARS-CoV-2, the virus responsible for the COVID-19 pandemic. Nanobodies are 12–15 kDa single-domain antibody fragments that can be delivered by inhalation and are amenable to relatively inexpensive large scale production compared to other biologicals. We have isolated nanobodies that bind to the SARS-CoV-2 spike protein receptor binding domain and block spike protein interaction with the angiotensin converting enzyme 2 (ACE2) with 1–5 nM affinity. The lead nanobody candidate, NIH-CoVnb-112, blocks SARS-CoV-2 spike pseudotyped lentivirus infection of HEK293 cells expressing human ACE2 with an EC50 of 0.3 µg/mL. NIH-CoVnb-112 retains structural integrity and potency after nebulization. Furthermore, NIH-CoVnb-112 blocks interaction between ACE2 and several high affinity variant forms of the spike protein. These nanobodies and their derivatives have therapeutic, preventative, and diagnostic potential.

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Section: Discussionmentioning

confidence: 99%

High affinity nanobodies block SARS-CoV-2 spike receptor binding domain interaction with human angiotensin converting enzyme

Esparza

Martin

Anderson

et al. 2020

Sci Rep

Self Cite

101

121

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“…Cocaine and morphine were obtained through Boynton Health Pharmacy at the University of Minnesota. All adeno-associated viruses (AAVs) were packaged in AAV8 serotype by the University of Minnesota Viral Vector and Cloning Core (Minneapolis, MN) following standard packaging procedures ( Chen et al, 2019 ), titers were between 0.2 and 4 × 10 14 genocopies/ml. The packaging plasmids (pRC8 and pHelper) were obtained from the University of Pennsylvania Vector Core.…”

Section: Methodsmentioning

confidence: 99%

Differential Impact of Inhibitory G-Protein Signaling Pathways in Ventral Tegmental Area Dopamine Neurons on Behavioral Sensitivity to Cocaine and Morphine

DeBaker

Velasco

McCall

et al. 2021

eNeuro

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Drugs of abuse engage overlapping but distinct molecular and cellular mechanisms to enhance dopamine (DA) signaling in the mesocorticolimbic circuitry. DA neurons of the ventral tegmental area (VTA) are key substrates of drugs of abuse and have been implicated in addiction-related behaviors. Enhanced VTA DA neurotransmission evoked by drugs of abuse can engage inhibitory G protein-dependent feedback pathways, mediated by GABA B receptors (GABA B Rs) and D 2 DA receptors (D 2 Rs). Chemogenetic inhibition of VTA DA neurons potently suppressed baseline motor activity, as well as the motor-stimulatory effect of cocaine and morphine, confirming the critical influence of VTA DA neurons and inhibitory G protein signaling in these neurons on this addictionrelated behavior. To resolve the relative influence of GABA B R-and D 2 R-dependent signaling pathways in VTA DA neurons on behavioral sensitivity to drugs of abuse, we developed a neuron-specific viral CRISPR/Cas9 approach to ablate D 2 R and GABA B R in VTA DA neurons. Ablation of GABA B R or D 2 R did not impact baseline physiological properties or excitability of VTA DA neurons, but it did preclude the direct somatodendritic inhibitory influence of GABA B R or D 2 R activation. D 2 R ablation potentiated the motor-stimulatory effect of cocaine in male and female mice, whereas GABA B R ablation selectively potentiated cocaine-induced activity in male subjects only. Neither D 2 R nor GABA B R ablation impacted morphine-induced motor activity. Collectively, our data show that cocaine and morphine differ in the extent to which they engage inhibitory G protein-dependent feedback pathways in VTA DA neurons and highlight key sex differences that may impact susceptibility to various facets of addiction. SIGNIFICANCE STATEMENT Although inhibitory G protein-dependent signaling involving the GABA B (GABA B R) and D 2 dopamine receptor (D 2 R) in VTA DA neurons is thought to limit DA neurotransmission evoked by drugs of abuse, their relative impact on behavioral sensitivity to such drugs is unclear. Using a neuron-specific viral CRISPR/Cas9 approach, we show that that loss of D 2 R in VTA DA neurons enhances behavioral sensitivity to systemic administration of cocaine in male and female mice, whereas loss of GABA B R enhances cocaine sensitivity only in males. Neither GABA B R nor D 2 R ablation impacted behavioral sensitivity to morphine. Thus, differential engagement of inhibitory feedback pathways in VTA DA neurons likely contributes to drug-specific neurophysiological and behavioral effects and may underlie sex differences associated 84 with some facets of addiction.

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“…Adeno-associated viruses (AAV) were produced by the Viral Vector and Cloning Core facility at the University of Minnesota following standard packaging procedures (Chen et al, 2019). pRC packaging plasmids for AAV1, 2, 5, 6, 9 and rh10 were obtained from the University of Pennsylvania Vector Core.…”

Section: Methodsmentioning

confidence: 99%

Automated Live-Cell Imaging of Synapses in Rat and Human Neuronal Cultures

Green

Pengo

Raybuck

et al. 2019

Front. Cell. Neurosci.

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Synapse loss and dendritic damage correlate with cognitive decline in many neurodegenerative diseases, underlie neurodevelopmental disorders, and are associated with environmental and drug-induced CNS toxicities. However, screening assays designed to measure loss of synaptic connections between live cells are lacking. Here, we describe the design and validation of automated synaptic imaging assay (ASIA), an efficient approach to label, image, and analyze synapses between live neurons. Using viral transduction to express fluorescent proteins that label synapses and an automated computer-controlled microscope, we developed a method to identify agents that regulate synapse number. ASIA is compatible with both confocal and wide-field microscopy; wide-field image acquisition is faster but requires a deconvolution step in the analysis. Both types of images feed into batch processing analysis software that can be run on ImageJ, CellProfiler, and MetaMorph platforms. Primary analysis endpoints are the number of structural synapses and cell viability. Thus, overt cell death is differentiated from subtle changes in synapse density, an important distinction when studying neurodegenerative processes. In rat hippocampal cultures treated for 24 h with 100 μM 2-bromopalmitic acid (2-BP), a compound that prevents clustering of postsynaptic density 95 (PSD95), ASIA reliably detected loss of postsynaptic density 95-enhanced green fluorescent protein (PSD95-eGFP)-labeled synapses in the absence of cell death. In contrast, treatment with 100 μM glutamate produced synapse loss and significant cell death, determined from morphological changes in a binary image created from co-expressed mCherry. Treatment with 3 mM lithium for 24 h significantly increased the number of fluorescent puncta, showing that ASIA also detects synaptogenesis. Proof of concept studies show that cell-specific promoters enable the selective study of inhibitory or principal neurons and that alternative reporter constructs enable quantification of GABAergic or glutamatergic synapses. ASIA can also be used to study synapse loss between human induced pluripotent stem cell (iPSC)-derived cortical neurons. Significant synapse loss in the absence of cell death was detected in the iPSC-derived neuronal cultures treated with either 100 μM 2-BP or 100 μM glutamate for 24 h, while 300 μM glutamate produced synapse loss and cell death. ASIA shows promise for identifying agents that evoke synaptic toxicities and screening for compounds that prevent or reverse synapse loss.

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Production of Viral Constructs for Neuroanatomy, Calcium Imaging, and Optogenetics

Cited by 19 publications

References 36 publications

High affinity nanobodies block SARS-CoV-2 spike receptor binding domain interaction with human angiotensin converting enzyme

High affinity nanobodies block SARS-CoV-2 spike receptor binding domain interaction with human angiotensin converting enzyme

Differential Impact of Inhibitory G-Protein Signaling Pathways in Ventral Tegmental Area Dopamine Neurons on Behavioral Sensitivity to Cocaine and Morphine

Automated Live-Cell Imaging of Synapses in Rat and Human Neuronal Cultures

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