| Sensing temperature changes in vivo and in vitro is an innate ability of organisms,which can effectively regulate and maintain body temperature.Temperature-sensitive TRP?Transient receptor potential,TRP?channels have been identified as the main cellular thermoreceptors,but the molecular and cellular integration mechanisms related to temperature-sensitive behavior in humans and mammals are still unclear.This paper explores the critical period of development of cold-sensitive response in early postnatal rats and mice,to determine the correlation between cold-sensitive behavior and TRPA1channel expression,and to screen the endogenous expression regulation mechanism of TRPA1 channel and cold-sensitive response development,so as to enrich the targeted research on the temperature-sensitive mechanism of TRPA1 channel.Therefore this article detects postnatal 1day?D?-6week?W?cold-sensitive behavior of rats and TRPA1-/-and TRPA1+/+?WT?mice,qRT-PCR detect the cold-sensitive TRP channel mRNA of rats dorsal root ganglion neurons?DRG?,the current expression of rats DRG TRPA1 channel recorded by patch clamp,and the effect of Tenascins on the current of transfected mTRPA1 channel were detected.The main results are as follows:1.Developmental changes of cold sensitivity in rats?1?3W-4W rats have the fastest weight gain,which is the critical period of weight development in rats.?2?The threshold of cold stimulation and contraction in rats at 9?and 13?was the shortest in the 3W incubation period,and the transition at 4W was rises quickly for the development of cold-sensitive behavior in rats.The cold-sensitive behavior of rats changes regularly with weeks,weeks-dependent,and the lower the stimulation temperature,the more sensitive the rats is.2.Developmental changes of DRG cold-sensitive TRP channel mRNA expressionThe expression of TRPC5 mRNA in each week was significantly lower than that in1D rats.The expression level of TRPM8 mRNA was significantly up-regulated at1W-3W,and 4W-6W was significantly down-regulated.The expression of TRPA1mRNA expression was significantly up-regulated at 2W-6W,4W is relatively fast with respect to 3W.The mRNA expression of cold-sensitive TRP channels?TRPC5,TRPM8,TRPA1?in rats DRG cells was weeks-dependent and suggesting that 3W-4W is the expression of TRPA1 and TRPM8 mRNA critical period.3.Developmental changes of DRG TRPA1 currentThe electrophysiological patch clamp was used to record the changes of DRG TRPA1 channel current in 3D-6W rats after birth.The results showed that with the increase of week,the current of TRPA1 increased gradually from 3D-4W except 3W,and the current of 5W-6W decreased gradually,of which 2W-4W is significantly increased.It is indicated that the TRPA1 current is weeks-dependent,3W-4W TRPA1 current rises rapidly,which may be the critical period of TRPA1 current development.4.Changes in weight development of TRPA1-/-and TRPA1+/+mice?1?3W-4W two kinds of mice,have the fastest weight gain and were critical for weight development.?2?The weight of 1D-4W TRPA1-/-mice was higher than TRPA1+/+mice,indicating TRPA1 knockout may increase the weight of 1D-4W mice.?3?When stimulated continuous on 13°C,the weight of TRPA1-/-mice was extremely significant and the increase period was delayed to 2W,indicating 13°C stimulation maybe delayed the weight development of TRPA1-/-mice,and there was no effect on continuous 17°C stimulation.?4?13°C and 17°C continuous stimulation,the weight of TRPA1+/+mice gained a significant increase in weight was advanced to 1W.indicating 13°C and 17°C stimulation maybe accelerated the weight development of TRPA1+/+mice.?5?Continuous 13°C stimulation reduced TRPA1-/-mice?no significant difference?and increased the weight of TRPA1+/+mice,and increased significantly at 4W.It was suggested that continuous 13°C stimulation increased mice weight,and may be related to the TRPA1 gene.5.Cold-sensitive behavioral development on TRPA1-/-and TRPA1+/+mice?1?In a single 13°C cold stimulation of TRPA1-/-mice,The threshold of cold stimulation and contraction rise quickly at 4W,and a significant difference at 4W,indicating that 3W-4W may be cold sensitive to TRPA1-/-mice Critical period of behavioral development.There was a significant difference in cold-sensitive behavior in TRPA1+/+mice early in life.?2?In the early postnatal period,the cold-sensitive behavior of TRPA1-/-mice was weaker than that of TRPA1+/+mice of 1W-6W,and there was a significant difference at4W,indicating that TRPA1 gene deletion may affect mice to perception normal change of low temperature.?3?Compared continuous 13°C with a single 13°C stimulation,the cold-sensitive behavior relatively insensitivity of TRPA1-/-mice was advanced to 3W and extended to5W,and the temperature response of TRPA1+/+mice became insignificant of 3W-6W.It is suggested that continuous low temperature stimulation delays the development of temperature perception in mice,and this effect maybe related to the TRPA1 gene.6.Effect of extracellular matrix on mTRPA1 channel current?1?The current and activation rate induced by TRPA1 voltage in mTRPA1 channel decreased under the conditions of 0.1?g,0.2?g and 0.4?g Tenascin-C,and the effect of0.1?g was the most obvious.The induced current of mTRPA1 channel activator decreased,and decreased significantly at 0.4?g.?2?The conditions for the existence of 2.5?g Tenascin-R in extracellular matrix:the current induced by mTRPA1 channel voltage increases and activates faster,while the current induced by activator decreases.In conclusion,the cold-sensing behavior of rats,expression of TRPA1 mRNA and TRPA1 channel current in rats DRG were weeks-dependent and both change quickly at3W-4W,indicating that 3W-4W maybe is a critical period for the development of TRPA1channel expression.The knockout mice were further used to explores the relationship between cold-sensitive behavior and TRPA1 channel after the critical phase of rats TRPA1 development.Continuous temperature stimulation and TRPA1 genes affect cold-sensitive behavior and weight development in mice.Tenascin-C and Tenascin-R have different effects on the mTRPA1 channel current.and the effects on the currents induced by different induction modes are also different.The molecular and cellular integration mechanisms involved in Tenascins and TRPA1-mediated cold-sensitive developmental changes are awaiting subsequent work. |
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