TECHNIQUES CURRENTLY BEING EMPLOYED IN THE LABORATORY

• Double electrode oocyte voltage-clamp technique to study cloned heterologously expressed receptors

• Giga-seal patch clamp technique (whole-cell and single-channel current recordings from cultured
  and dissociated neurons and from spinal cord slices)

• Evoked and spontaneous/miniature excitatory and inhibitory synaptic currents from DRG
  and DH co-cultures using double patch and patch clamp techniques and from spinal cord slices from
  control and diabetic mice to determine changes in synaptic transmission

• Fast solution exchange in outside-out patches (surrogate synapse)

• Detailed kinetic analysis of single channel currents using Hidden Markov Model based techniques
    (I played a part in its development, Chung et al., 1990)

• Fluorescence imaging for detecting changes in intracellular Ca2+ and free radicals using
  fluorescence and confocal microscopy

• Immunocytochemistry to determine expression of receptor proteins in DRGs, dissociated DRG neurons
  and to determine dendritic arborization in dorsal horn. Tissues will be collected from animals generated
  using chronic pain models and diabetic animals exhibiting hyperalgesic and hypoalgesic phenotypes

• Radiochemical assay to detect TRPV1 levels (3HRTX) and phosphorylated protein (32P) labeling to
  determine changes in receptor expression

• Western blots to detect TRPV1 (His-tagged TRPV1) and TRPM8 (FLAG-tagged)
  and to detect phosphorylated forms

• Determination of membrane translocation of TRPV1 and TRPM8 by detecting levels of protein in the
  membrane fraction. Detect translocation of GFP-tagged TRPV1 and TRPM8 using real time confocal imaging

• Heterologous expression in HEK293 and CHO cells with GFP-tagged TRPV1 to determine the levels of
  expression, translocation and internalization using confocal microscopy

• Site directed mutagenesis to study molecular determinants

• Organ bath experiments to study changes in vascular tone

• Generation of autoimmune diabetic mice (TCR-SFE (T-cell receptor for hemagglutinin)/Ins-HA/B10.D2
  (pancreatic beta cells produce hemagglutinin). The off springs become diabetic within 3-6 weeks of age,
  where diabetes is functionally defined by hyperglycemia (blood glucose >299 mg/dl)

• Streptozotocin and alloxon induced diabetic mice

• Nocifensive behavior testing using hot plate, cold plate and intraplantar injection of
  algesic and analgesic substances

• Intrathecal injection of drugs to test whether nocifensive behavior could be blocked at
  the level of the spinal cord

• Animal models of inflammatory (carrageenen) and chronic (sciatic nerve ligation) pain

• Single unit chronic microwire recording from awake behaving animals

• Focal microinjections in awake behaving animals

• Chronic focal infusion using osmotic mini pump