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1.
J Neurosci Methods ; 284: 1-14, 2017 Jun 01.
Article in English | MEDLINE | ID: mdl-28380331

ABSTRACT

BACKGROUND: The use of genetically-encoded fluorescent reporters is essential for the identification and observation of cells that express transgenic modulatory proteins. Near-infrared (NIR) fluorescent proteins have superior light penetration through biological tissue, but are not yet widely adopted. NEW METHOD: Using the near-infrared fluorescent protein, iRFP713, improves the imaging resolution in thick tissue sections or the intact brain due to the reduced light-scattering at the longer, NIR wavelengths used to image the protein. Additionally, iRFP713 can be used to identify transgenic cells without photobleaching other fluorescent reporters or affecting opsin function. We have generated a set of adeno-associated vectors in which iRFP713 has been fused to optogenetic channels, and can be expressed constitutively or Cre-dependently. RESULTS: iRFP713 is detectable when expressed in neurons both in vitro and in vivo without exogenously supplied chromophore biliverdin. Neuronally-expressed iRFP713 has similar properties to GFP-like fluorescent proteins, including the ability to be translationally fused to channelrhodopsin or halorhodopsin, however, it shows superior photostability compared to EYFP. Furthermore, electrophysiological recordings from iRFP713-labeled cells compared to cells labeled with mCherry suggest that iRFP713 cells are healthier and therefore more stable and reliable in an ex vivo preparation. Lastly, we have generated a transgenic rat that expresses iRFP713 in a Cre-dependent manner. CONCLUSIONS: Overall, we have demonstrated that iRFP713 can be used as a reporter in neurons without the use of exogenous biliverdin, with minimal impact on viability and function thereby making it feasible to extend the capabilities for imaging genetically-tagged neurons in slices and in vivo.


Subject(s)
Genes, Reporter/genetics , Green Fluorescent Proteins/metabolism , Microscopy, Fluorescence/methods , Neurons/metabolism , Optogenetics/methods , Spectroscopy, Near-Infrared/methods , Voltage-Sensitive Dye Imaging/methods , Animals , Cells, Cultured , Genetic Vectors/genetics , Green Fluorescent Proteins/genetics , Luminescent Proteins , Molecular Imaging/methods , Neurons/cytology , Rats , Rats, Sprague-Dawley , Rats, Transgenic , Reproducibility of Results , Sensitivity and Specificity
2.
Dev Cell ; 26(5): 511-24, 2013 Sep 16.
Article in English | MEDLINE | ID: mdl-23993788

ABSTRACT

Phagocytosis of large extracellular particles such as apoptotic bodies requires delivery of the intracellular endosomal and lysosomal membranes to form plasmalemmal pseudopods. Here, we identified mucolipin TRP channel 1 (TRPML1) as the key lysosomal Ca2+ channel regulating focal exocytosis and phagosome biogenesis. Both particle ingestion and lysosomal exocytosis are inhibited by synthetic TRPML1 blockers and are defective in macrophages isolated from TRPML1 knockout mice. Furthermore, TRPML1 overexpression and TRPML1 agonists facilitate both lysosomal exocytosis and particle uptake. Using time-lapse confocal imaging and direct patch clamping of phagosomal membranes, we found that particle binding induces lysosomal PI(3,5)P2 elevation to trigger TRPML1-mediated lysosomal Ca2+ release specifically at the site of uptake, rapidly delivering TRPML1-resident lysosomal membranes to nascent phagosomes via lysosomal exocytosis. Thus phagocytic ingestion of large particles activates a phosphoinositide- and Ca2+-dependent exocytosis pathway to provide membranes necessary for pseudopod extension, leading to clearance of senescent and apoptotic cells in vivo.


Subject(s)
Lysosomes/genetics , Phagocytosis/genetics , Transient Receptor Potential Channels/genetics , Transient Receptor Potential Channels/metabolism , Aging/genetics , Animals , Calcium/metabolism , Exocytosis/genetics , Gene Expression Regulation , Mice , Particle Size , Phosphatidylinositol Phosphates/metabolism , Transient Receptor Potential Channels/agonists , Transient Receptor Potential Channels/antagonists & inhibitors
3.
J Neurosci ; 28(35): 8709-23, 2008 Aug 27.
Article in English | MEDLINE | ID: mdl-18753372

ABSTRACT

The COMT (catechol-O-methyltransferase) gene has been linked to a spectrum of human phenotypes, including cognition, anxiety, pain sensitivity and psychosis. Doubts about its clinical impact exist, however, because of the complexity of human COMT polymorphism and clinical variability. We generated transgenic mice overexpressing a human COMT-Val polymorphism (Val-tg), and compared them with mice containing a null COMT mutation. Increased COMT enzyme activity in Val-tg mice resulted in disrupted attentional set-shifting abilities, and impaired working and recognition memory, but blunted stress responses and pain sensitivity. Conversely, COMT disruption improved working memory, but increased stress responses and pain sensitivity. Amphetamine ameliorated recognition memory deficits in COMT-Val-tg mice but disrupted it in wild types, illustrating COMT modulation of the inverted-U relationship between cognition and dopamine. COMT-Val-tg mice showed increased prefrontal cortex (PFC) calcium/calmodulin-dependent protein kinase II (CaMKII) levels, whereas COMT deficiency decreased PFC CaMKII but increased PFC CaMKKbeta and CaMKIV levels, suggesting the involvement of PFC CaMK pathways in COMT-regulated cognitive function and adaptive stress responses. Our data indicate a critical role for the COMT gene in an apparent evolutionary trade-off between cognitive and affective functions.


Subject(s)
Catechol O-Methyltransferase/genetics , Cognition/physiology , Exploratory Behavior/physiology , Mutation/genetics , Stress, Psychological/genetics , Acoustic Stimulation/methods , Amphetamine/pharmacology , Analysis of Variance , Animals , Attention/physiology , Behavior, Animal , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Dopamine/metabolism , Dopamine Agents/pharmacology , Humans , Hyperthermia, Induced/methods , Inhibition, Psychological , Maze Learning/physiology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Motor Activity/genetics , Pain Measurement/methods , Pain Threshold/physiology , Reaction Time/genetics , Reflex, Startle/genetics , Stress, Psychological/enzymology
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