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In JoVE (1)
Other Publications (9)
- Journal of Immunology (Baltimore, Md. : 1950)
- The Journal of Biological Chemistry
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Spectrochimica Acta. Part A, Molecular and Biomolecular Spectroscopy
- Science (New York, N.Y.)
- Biology Direct
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- International Immunopharmacology
- Nature Communications
Articles by Limei Ma in JoVE
JoVE Neuroscience
Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor
1Stowers Institute for Medical Research, 2Department of Anatomy and Cell Biology, The University of Kansas School of Medicine
The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.
Other articles by Limei Ma on PubMed
Two New Isotype-specific Switching Activities Detected for Ig Class Switching
Ig class switch recombination (CSR) occurs by an intrachromosomal deletional process between switch (S) regions in B cells. To facilitate the study of CSR, we derived a new B cell line, 1.B4.B6, which is uniquely capable of mu --> gamma3, mu --> epsilon, and mu --> alpha, but not mu --> gamma1 CSR at its endogenous loci. The 1.B4.B6 cell line was used in combination with plasmid-based isotype-specific S substrates in transient transfection assays to test for the presence of trans-acting switching activities. The 1.B4.B6 cell line supports mu --> gamma3, but not mu --> gamma1 recombination, on S substrates. In contrast, normal splenic B cells activated with LPS and IL-4 are capable of plasmid-based mu --> gamma1 CSR and demonstrate that this S plasmid is active. Activation-induced deaminase (AID) was used as a marker to identify existing B cell lines as possible candidates for supporting CSR. The M12 and A20 cell lines were identified as AID positive and, following activation with CD40L and other activators, were found to differentially support mu --> epsilon and mu --> alpha plasmid-based CSR. These studies provide evidence for two new switching activities for mu --> gamma1 and mu --> epsilon CSR, which are distinct from mu --> gamma3 and mu --> alpha switching activities previously described. AID is expressed in all the B cell lines capable of CSR, but cannot account for the isotype specificity defined by the S plasmid assay. These results are consistent with a model in which isotype-specific switching factors are either isotype-specific recombinases or DNA binding proteins with sequence specificity for S DNA.
Transcriptional Regulation of the Mouse Gene Encoding the Alpha-4 Subunit of the GABAA Receptor
The gamma-aminobutyric acid type A receptors (GABAA-Rs) mediate fast inhibitory synaptic transmission in the brain. The alpha4 subunit of the GABAA-R confers distinct pharmacological properties on the receptor and its expression pattern exhibits plasticity in response to physiological and pharmacological stimuli, including withdrawal from progesterone and alcohol. We have analyzed the promoter region of the mouse GABRA4 gene that encodes the alpha4 subunit and found that the promoter has multiple transcriptional initiation sites and lacks a TATA box. The minimal promoter for GABRA4 spans the region between -444 to -19 bp relative to the coding ATG and shows high activity in cultured mouse cortical neurons. Both Sp3 and Sp4 transcription factors can interact with the two Sp1 binding sites within the minimal promoter and are critical for maximal activity of the promoter in neurons.
Alcohol Regulates Gene Expression in Neurons Via Activation of Heat Shock Factor 1
Drinking alcohol causes widespread alterations in gene expression that can result in long-term physiological changes. Although many alcohol-responsive genes (ARGs) have been identified, the mechanisms by which alcohol alters transcription are not well understood. To elucidate these mechanisms, we investigated Gabra4, a neuron-specific gene that is rapidly and robustly activated by alcohol (10-60 mM), both in vitro and in vivo. Here we show that alcohol can activate elements of the heat shock pathway in mouse cortical neurons to enhance the expression of Gabra4 and other ARGs. The activation of Gabra4 by alcohol or high temperature is dependent on the binding of heat shock factor 1 (HSF1) to a short downstream DNA sequence, the alcohol response element (ARE). Alcohol and heat stimulate the translocation of HSF1 from the cytoplasm to the nucleus and the induction of HSF1-dependent genes, Hsp70 and Hsp90, in cultured neurons and in the mouse cerebral cortex in vivo. The reduction of HSF1 levels using small interfering RNA prevented the stimulation of Gabra4 and Hsp70 by alcohol and heat shock. Microarray analysis showed that many ARGs contain ARE-like sequences and that some of these genes are also activated by heat shock. We suggest that alcohol activates phylogenetically conserved pathways that involve intermediates in the heat shock cascade and that sequence elements similar to the ARE may mediate some of the changes in gene expression triggered by alcohol intake, which could be important in a variety of pathophysiological responses to alcohol.
Flow-injection Chemiluminescence Determination of Chrysin and Baicalein Assisted by Theoretical Prediction of Chemiluminescence Behavior of Chrysin and Baicalein
In this paper, the molecular connectivity indices were applied to theoretically predict the direct chemiluminescence (CL) behavior of chrysin and baicalein with our recently proposed discriminant function. Then, combined with flow-injection analysis, a new CL system for determination of chrysin and baicalein was proposed. The method was based on the oxidation of chrysin and baicalein by acidic KMnO(4) in the presence of formaldehyde to produce strong CL emission. The present paper suggested a new model to discover new CL analytical system: first, to theoretical predict the CL behavior, and the second, to suggest analytical system.
Encoding Gender and Individual Information in the Mouse Vomeronasal Organ
The mammalian vomeronasal organ detects complex chemical signals that convey information about gender, strain, and the social and reproductive status of an individual. How these signals are encoded is poorly understood. We developed transgenic mice expressing the calcium indicator G-CaMP2 and analyzed population responses of vomeronasal neurons to urine from individual animals. A substantial portion of cells was activated by either male or female urine, but only a small population of cells responded exclusively to gender-specific cues shared across strains and individuals. Female cues activated more cells and were subject to more complex hormonal regulations than male cues. In contrast to gender, strain and individual information was encoded by the combinatorial activation of neurons such that urine from different individuals activated distinctive cell populations.
A Family of GFP-like Proteins with Different Spectral Properties in Lancelet Branchiostoma Floridae
Members of the green fluorescent protein (GFP) family share sequence similarity and the 11-stranded beta-barrel fold. Fluorescence or bright coloration, observed in many members of this family, is enabled by the intrinsic properties of the polypeptide chain itself, without the requirement for cofactors. Amino acid sequence of fluorescent proteins can be altered by genetic engineering to produce variants with different spectral properties, suitable for direct visualization of molecular and cellular processes. Naturally occurring GFP-like proteins include fluorescent proteins from cnidarians of the Hydrozoa and Anthozoa classes, and from copepods of the Pontellidae family, as well as non-fluorescent proteins from Anthozoa. Recently, an mRNA encoding a fluorescent GFP-like protein AmphiGFP, related to GFP from Pontellidae, has been isolated from the lancelet Branchiostoma floridae, a cephalochordate (Deheyn et al., Biol Bull, 2007 213:95).
Distinct Signals Conveyed by Pheromone Concentrations to the Mouse Vomeronasal Organ
In mammalian species, detection of pheromone cues by the vomeronasal organ (VNO) at different concentrations can elicit distinct behavioral responses and endocrine changes. It is not well understood how concentration-dependent activation of the VNO impacts innate behaviors. In this study, we find that, when mice investigate the urogenital areas of a conspecific animal, the urinary pheromones can reach the VNO at a concentration of approximately 1% of that in urine. At this level, urinary pheromones elicit responses from a subset of cells that are tuned to sex-specific cues and provide unambiguous identification of the sex and strain of animals. In contrast, low concentrations of urine do not activate these cells. Strikingly, we find a population of neurons that is only activated by low concentrations of urine. The properties of these neurons are not found in neurons responding to putative single-compound pheromones. Additional analyses show that these neurons are masked by high-concentration pheromones. Thus, an antagonistic interaction in natural pheromones results in the activation of distinct populations of cells at different concentrations. The differential activation is likely to trigger different downstream circuitry and underlies the concentration-dependent pheromone perception.
Isoforskolin Pretreatment Attenuates Lipopolysaccharide-induced Acute Lung Injury in Animal Models
Isoforskolin was isolated from Coleus forskohlii native to Yunnan in China. We hypothesize that isoforskolin pretreatment attenuates acute lung injury induced by lipopolysaccharide (endotoxin). Three acute lung injury models were used: situ perfused rat lung, rat and mouse models of endotoxic shock. Additionally, lipopolysaccharide stimulated proinflammatory cytokine production was evaluated in human mononuclear leukocyte. In situ perfused rat lungs, pre-perfusion with isoforskolin (100, and 200 μM) and dexamethasone (65 μM, positive control) inhibited lipopolysaccharide (10 mg/L) induced increases in lung neutrophil adhesion rate, myeloperoxidase activity, lung weight Wet/Dry ratio, permeability-surface area product value, and tumor necrosis factor (TNF)-α levels. In rats, pretreatments with isoforskolin (5, 10, and 20 mg/kg, i.p.) and dexamethasone (5mg/kg, i.p.) markedly reduced lipopolysaccharide (6 mg/kg i.v.) induced increases of karyocyte, neutrophil counts and protein content in bronchoalveolar lavage fluid, and plasma myeloperoxidase activity. Lung histopathology showed that morphologic changes induced by lipopolysaccharide were less pronounced in the isoforskolin and dexamethasone pretreated rats. In mice, 5 mg/kg isoforskolin and dexamethasone caused 100% and 80% survival, respectively, after administration of lipopolysaccharide (62.5mg/kg, i.v., 40% survival if untreated). In human mononuclear leukocyte, isoforskolin (50, 100, and 200 μM) and dexamethasone (10 μM) pre-incubation lowered lipopolysaccharide (2 μg/mL) induced secretion of the cytokine TNF-α, and interleukins (IL)-1β, IL-6, and IL-8. In conclusion, pretreatment with isoforskolin attenuates lipopolysaccharide-induced acute lung injury in several models, and it is involved in down-regulation of inflammatory responses and proinflammatory cytokines TNF-α, IL-1β, IL-6, and IL-8.
Requirement of Calcium-activated Chloride Channels in the Activation of Mouse Vomeronasal Neurons
In terrestrial vertebrates, the vomeronasal organ (VNO) detects and transduces pheromone signals. VNO activation is thought to be mediated by the transient receptor potential C2 (TRPC2) channel. The aberrant behavioural phenotypes observed in TRPC2-/- mice are generally attributed to the lost VNO function. Recently, calcium-activated chloride channels have been shown to contribute to VNO activation. Here we show that CACCs can be activated in VNO slice preparations from the TRPC2-/- mice and this activation is blocked by pharmacological agents that inhibit intracellular Ca(2+) release. Urine-evoked Cl(-) current is sufficient to drive spiking changes in VNO neurons from both wild-type (WT) and TRPC2-/- mice. Moreover, blocking Cl(-) conductance essentially abolishes VNO activation in WT neurons. These results suggest a TRPC2-independent signalling pathway in the VNO and the requirement of calcium-activated chloride channels currents to mediate pheromone activation. Our data further suggest that TRPC2-/- mice retain partial VNO function.
