Activity-dependent Neuronal Control Of Intercellular Communication In DRG Neurons And Fibroblasts

Section 1 Propagation of Ca2+ oscillation in DRG neurons: the involvement of extracellular ATP and P2Y receptor activationRecently emerging evidence implicates a number of neuroactive substances and their receptors in mediating complex cell-to-cell communications in the ganglia. In the present study, we have characterized the chemical coupling mediated by extracellular ATP in cultured dorsal root ganglion (DRG) neurons by using a real time-imaging of ATP, whole cell patch clamping in conjunction with confocal calcium imaging. Sustained depolarizations of single DRG neurons evoked intracellular Ca2+ concentration ([Ca2+]i) oscillations together with TTX-sensitive persistent Na+ influx, and subsequent ATP-dependent propagation [Ca2+]i oscillations to surrounding passive neighbors. [Ca2+]i oscillations were suppressed by both the Na+ channel blocker TTX and inositol-1,4,5-trisphosphate (IP3) receptor antagonist 2-APB, but not ryanodine. The propagation of [Ca2+]i oscillations was prevented in the presence of the ATP-degrading enzyme, apyrases, and inhibited by suramin, a nonselective P2Y receptor antagonist. In parallel, depolarizing stimuli induced ATP release and diffusion, which was abolished by inhibition of PLC- IP3 pathway or pretreatment with thapsigargin to deplete internal Ca2+ stores. Moreover, exogenous application of ATP to DRG neurons elicited the Ca2+ oscillations involving PLC-IP3 pathway. Taken together, these data demonstrated that sustained membrane depolarization elicited ATP release, acting through a highly sensitive P2Y receptors /IP3-mediated signaling pathway to mediate the propagation of intercellular Ca2+ signaling, which suggest a novel signaling pathway for neuronal communication in DRG.Section 2 Activity-dependent neuronal control of gap-junctional communication in fibroblastsA typical feature of fibroblasts is their high degree of intercellular communication through gap junction channels. Abundant between fibroblasts, gap junction coupling plays important roles in fibroblasts physiology. Here we report that activity-dependent neuronal control of gap junctional communication (GJC) in the rat dermal fibroblasts in vitro is associated to NMDA glutamate receptors-mediated intracellular Ca2+ elevations. Not only excited dorsal root ganglion (DRG) neurons by using depolarizing pulses, which exhibited remarkable action potentials and inward sodium currents, tremendously reduced fibroblast GJC in Neuron/Fibroblast (N/F) cocultures, but also resting DRG neurons, which exhibited frequent spontaneous action potentials and inward sodium currents or so, exerted significant roles on fibroblast GJC and CX43 protein. Both fibroblast GJC and CX43 downregulation were prevented by the blockade of NMDA receptors and decrease of intra- and intercellular Ca2+. Immunocytochemistry showed that the NR1 subunit of the NMDA receptor was colocated at plasma membrane of fibroblasts with CX43. Moreover, glutamate applied to fibroblasts triggered NMDA receptor-dependent intracellular Ca2+ elevations and decline of GJC. These data demonstrate that NMDA receptor activity in fibroblasts contributed to the effect of DRG neurons on fibroblast coupling via downregulation of GJC. Since fibroblasts have been shown to facilitate DRG neurite growth and survival, our findings suggest that neuronal and fibroblasts networks interact actively.Section 3 Real-time whole-body optical imaging of electrical acupuncture responses in intact nude miceWhole-body optical imaging of small animals has emerged as a powerful, user friendly, and high-throughput tool for assaying molecular and cellular processes as they occur in vivo. Here we used a whole-body fluorescence imaging system for the first time in a nude mice model to explore the characteristic responses induced by electrical acupuncture (EA). Nude mice were injected with fluorescent dye at identifiable points similar to the acupoint in Traditional Chinese Medicine for animals. Serial measurements of fluorescence diffusion were performed using in vivo fluorescence macroscopic. EA applied to the acupoint injected with low molecule weight dye evoked a fluorescence channel over a long distance of the fore limb to be coincident with the course of classical meridian. Stimulation applied to the acupoint injected with high molecule weight dyes did not evoke similar fluorescence dye activities. And the activity of low molecular mass dye could be inhibited by gap junction inhibitors including acetic acid-Sodium acetate, halothane, and octanol. This result indicates that the fluorescence dye activity induced by EA is correlated with gap junction communication. Therefore, this study may illustrate objective investigation of EA responses in small animals using a real time whole-body imaging, and evaluate the functional importance on mediating or transmitting EA effectiveness of gap junctions and calcium.