Transgenic mouse lines for non-invasive ratiometric monitoring of intracellular chloride.

Chloride is the most abundant physiological anion and participates in a variety of cellular processes including trans-epithelial transport, cell volume regulation, and regulation of electrical excitability. The development of tools to monitor intracellular chloride concentration ([Cli]) is therefore important for the evaluation of cellular function in normal and pathological conditions. Recently, several Cl-sensitive genetically encoded probes have been described which allow for non-invasive monitoring of [Cli]. Here we describe two mouse lines expressing a CFP-YFP-based Cl probe called Cl-Sensor. First, we generated transgenic mice expressing Cl-Sensor under the control of the mouse Thy1 mini promoter. Cl-Sensor exhibited good expression from postnatal day two (P2) in neurons of the hippocampus and cortex, and its level increased strongly during development. Using simultaneous whole-cell monitoring of ionic currents and Cl-dependent fluorescence, we determined that the apparent EC 50 for Cli was 46 mM, indicating that this line is appropriate for measuring neuronal [Cli] in postnatal mice. We also describe a transgenic mouse reporter line for Cre-dependent conditional expression of Cl-Sensor, which was targeted to the Rosa26 locus and by incorporating a strong exogenous promoter induced robust expression upon Cre-mediated recombination. We demonstrate high levels of tissue-specific expression in two different Cre-driver lines targeting cells of the myeloid lineage and peripheral sensory neurons. Using these mice the apparent EC 50 for Cli was estimated to be 61 and 54 mM in macrophages and DRG, respectively. Our data suggest that these mouse lines will be useful models for ratiometric monitoring of Cli in specific cell types in vivo.

Residues in the pore region of Drosophila transient receptor potential A1 dictate sensitivity to thermal stimuli.

The capacity to sense temperature is essential for the survival of all animals. At the molecular level, ion channels belonging to the transient receptor potential (TRP) family of channels function as temperature sensors in animals across several phyla. TRP channels are opened directly by changes in temperature and show pronounced sensitivity at their activation range. To determine how temperature activates these channels, we analysed channels belonging to the TRPA family, which detect heat in insects and cold in mammals. By constructing chimeric proteins consisting of human and Drosophila TRPA1 channels, we mapped regions that regulate thermal activation and identified residues in the pore helix that invert temperature sensitivity of TRPA1. From analysis of individual channels we defined the gating reaction of Drosophila TRPA1 and determined how mutagenesis alters the energy landscape for channel opening. Our results reveal specific molecular requirements for thermal activation of TRPA1 and provide mechanistic insight into this process.

Generation and characterization of an Advillin-Cre driver mouse line.

Progress in the somatosensory field has been restricted by the limited number of genetic tools available to study gene function in peripheral sensory neurons. Here we generated a Cre-driver mouse line that expresses Cre-recombinase from the locus of the sensory neuron specific gene Advillin. These mice displayed almost exclusive Cre-mediated recombination in all peripheral sensory neurons. As such, the Advillin-Cre-driver line will be a powerful tool for targeting peripheral neurons in future investigations.

The tyrosine phosphatase Shp2 (PTPN11) directs Neuregulin-1/ErbB signaling throughout Schwann cell development.

The nonreceptor tyrosine phosphatase Shp2 (PTPN11) has been implicated in tyrosine kinase, cytokine, and integrin receptor signaling. We show here that conditional mutation of Shp2 in neural crest cells and in myelinating Schwann cells resulted in deficits in glial development that are remarkably similar to those observed in mice mutant for Neuregulin-1 (Nrg1) or the Nrg1 receptors, ErbB2 and ErbB3. In cultured Shp2 mutant Schwann cells, Nrg1-evoked cellular responses like proliferation and migration were virtually abolished, and Nrg1-dependent intracellular signaling was altered. Pharmacological inhibition of Src family kinases mimicked all cellular and biochemical effects of the Shp2 mutation, implicating Src as a primary Shp2 target during Nrg1 signaling. Together, our genetic and biochemical analyses demonstrate that Shp2 is an essential component in the transduction of Nrg1/ErbB signals.

The contribution of TRPM8 and TRPA1 channels to cold allodynia and neuropathic pain.

Cold allodynia is a common feature of neuropathic pain however the underlying mechanisms of this enhanced sensitivity to cold are not known. Recently the transient receptor potential (TRP) channels TRPM8 and TRPA1 have been identified and proposed to be molecular sensors for cold. Here we have investigated the expression of TRPM8 and TRPA1 mRNA in the dorsal root ganglia (DRG) and examined the cold sensitivity of peripheral sensory neurons in the chronic construction injury (CCI) model of neuropathic pain in mice.In behavioral experiments, chronic constriction injury (CCI) of the sciatic nerve induced a hypersensitivity to both cold and the TRPM8 agonist menthol that developed 2 days post injury and remained stable for at least 2 weeks. Using quantitative RT-PCR and in situ hybridization we examined the expression of TRPM8 and TRPA1 in DRG. Both channels displayed significantly reduced expression levels after injury with no change in their distribution pattern in identified neuronal subpopulations. Furthermore, in calcium imaging experiments, we detected no alterations in the number of cold or menthol responsive neurons in the DRG, or in the functional properties of cold transduction following injury. Intriguingly however, responses to the TRPA1 agonist mustard oil were strongly reduced.Our results indicate that injured sensory neurons do not develop abnormal cold sensitivity after chronic constriction injury and that alterations in the expression of TRPM8 and TRPA1 are unlikely to contribute directly to the pathogenesis of cold allodynia in this neuropathic pain model.

Direct activation of the ion channel TRPA1 by Ca2+.

TRPA1 is an ion channel expressed by nociceptors and activated by irritant compounds such as mustard oil. The endogenous function of TRPA1 has remained unclear, a fact highlighted by ongoing debate over its potential role as a sensor of noxious cold. Here we show that intracellular Ca(2+) activates human TRPA1 via an EF-hand domain and that cold sensitivity occurs indirectly (and nonphysiologically) through increased [Ca(2+)](i) during cooling in heterologous systems.