Aiko Hori

Cold sensitivity of recombinant TRPA1 channels

TRPM8 and TRPA1, members of the transient receptor potential (TRP) channel family, are candidates for cooling-activated receptors. It is accepted that TRPM8 responds to moderate cooling, although it is controversial whether TRPA1 responds to deep cooling. Here, using Ca(2+) imaging and/or patch-clamp recordings, we examined the thermal sensitivity of primary cultured dorsal root ganglion (DRG) neurons and mouse TRPA1-expressing human embryonic kidney (HEK) 293 cells. In a subset of cultured mouse DRG neurons, deep cooling (5-18 degrees C) and allyl isothiocyanate (AITC, agonist of TRPA1) induced increases in intracellular Ca(2+) level. Most AITC-sensitive (TRPA1-expressing) neurons responded to deep cooling. In TRPA1-expressing HEK293 cells, deep cooling and AITC-induced Ca(2+) responses and whole-cell currents. In inside-out patches excised from TRPA1-expressing HEK293 cells, deep cooling, and AITC activated the same channels, which were inhibited by camphor (antagonist for TRPA1). When temperature was decreased below 18 degrees C, unit conductance of the channel decreased but open probability of it increased. Deep cooling-induced increase of the open probability of TRPA1 may underlie the increase in whole-cell currents induced by deep cooling. It is concluded that TRPA1 is a deep cooling-activated channel, which supports the previous findings that TRPA1 responds to deep cooling.

Noxious heat receptors present in cold-sensory cells in rats

Noxious heat above approximately 45 degrees C applied on cold spots evokes a paradoxical cold sensation by activating cold fibers. It remains unresolved whether cold receptors respond to heat as well, or whether noxious-heat receptors and cold receptors coexist in the same fiber. Recently, noxious heat receptors (TRPV1) and cold receptors (TRPM8) have been cloned. It is controversial, however, whether TRPV1 and TRPM8 coexist in the same sensory neuron. Here, we investigate colocalization of these receptors in dorsal root ganglion (DRG) of rats. TRPV1 was expressed in 29% of TRPM8-positive cells in DRG sections. In Ca2+ imaging, noxious heat excited many of the cold-sensitive cells in culture. In a whole-cell current-clamp mode, noxious heat, capsaicin, cooling and menthol all evoked receptor potentials and impulses in a subset of DRG neurons. This colocalization of TRPV1 and TRPM8 in a DRG neuron may be the basis for the paradoxical cold sensation.

Warming-activated channels of warm-sensitive neurons in rat hypothalamic slices

In mammals, multiple thermostats in several brain areas, e.g. the preoptic area and anterior hypothalamus (POAH), may regulate core temperature. In these areas, warm-sensitive (WS) and cold-sensitive (CS) neurons have been recorded. In our previous study, we proposed that primary WS and CS neurons act as multiple thermostats having threshold temperatures as target temperatures of regulation. Here we investigated ionic basis of WS neurons in rat POAH slices with patch-clamp technique. In whole-cell current-clamp recordings, warming above threshold temperatures induced depolarization (receptor potential), leading to spike generation. Whole-cell voltage-clamp and cell-attached patch recordings indicated that warming-activated non-selective cation channels underlie the receptor potential and spike generation. We conclude that the warming-activated channel in primary WS neurons is a key factor of thermostats.

Heat-activated currents in neurons cultured from rat dorsal root ganglia

Nociceptive heat-activated non-selective cation current has been reported in neurons of rat dorsal root wglia (DRG). However. non-nociceptive heat-activated current has not been clarified. Here. we investigated ionic mechanism of heat-activated current in neurons cultured from DRG of l-2 weeks-old rats by patch-clamp whole-cell recording. In voltage-clamp (-60 mV) recording warming above a threshold temperature induced inward current. Threshold temperature distributed widely from 30 to 45 oC. The heat-activated current was not inhibited by extracellular application of ruthenium red. indicating that the conductance was different from that of capsaicin receptors. GDPpS added in pipette solution did not block the heat-activated current. suggesting that the current activation was not mediated by G-protein. Moreover, EGTA added in pipette solution did not change the heat-activated current, suggesting that it was not modulated by intracellular free Ca ions. We clarified properties of non-nociceptive heatactivated conductance in a subset of DRG neurons.