1. Double electrode oocyte voltage-clamp technique to study cloned heterologously expressed receptors
2. Giga-seal patch clamp technique (whole-cell and single-channel current recordings from cultured and dissociated neurons and from spinal cord slices)
3. 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
4. Fast solution exchange in outside-out patches (surrogate synapse)
5. Detailed kinetic analysis of single channel currents using Hidden Markov Model based techniques (I played a part in its development, Chung et al., 1990)
6. Fluorescence imaging for detecting changes in intracellular Ca2+ and free radicals using fluorescence and confocal microscopy
7. 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
8. Radiochemical assay to detect TRPV1 levels (3HRTX) and phosphorylated protein (32P) labeling to determine changes in receptor expression
9. Western blots to detect TRPV1 (His-tagged TRPV1) and TRPM8 (FLAG-tagged) and to detect phosphorylated forms
10. Real-time RT-PCR to determine the expression of TRP channels in different regions of the CNS
11. 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
12. Heterologous expression in HEK293 and CHO cells with GFP-tagged TRPV1 to determine the levels of expression, translocation and internalization using confocal microscopy
13. Site directed mutagenesis to study molecular determinants
14. Organ bath experiments to study changes in vascular tone
15. 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)
16. Streptozotocin and alloxon induced diabetic mice
17. Nocifensive behavior testing using hot plate, cold plate and intraplantar injection of algesic and analgesic substances
18. Intrathecal injection of drugs to test whether nocifensive behavior could be blocked at the level of the spinal cord
19. Animal models of inflammatory (carrageenen) and chronic (sciatic nerve ligation) pain
20. Chronic focal infusion of drugs using osmotic mini pump
21. Diode laser fiber type selective stimulation (DLss) for selective activation of Ad or C fibers