The Regulatory Mechanism Of L-type Calcium Channel In The Amygdala On Abnormal Fear Of PTSD Rat

Objective:Posttraumatic stress disorder?PTSD?is a disorder that develops in some people who have experienced huge disasters or unusual threats,such as earthquakes,tsunamis,floods and major diseases.The core symptoms are hyperarousal?fear,anxiety?,re-experience?flashback,repeated traumatic experience?,persistent avoidance,emotional numbness,and loss of confidence in the future.The exact pathogenesis of PTSD has not been fully elucidated yet.Therefore,it is necessary to explore the pathogenesis of PTSD.Stress response can be reflected by the level of stress hormone-corticosterone,which plays an important role in regulating fear memory.The amygdala is the key region to regulate fear and anxiety.Acoustic startle response?ASR?is a typical index to detect fear response.The amygdala regulates the startling reflex and modulates emotions.Therefore,we used different decibels of voice stimulation to evaluate the behavior of PTSD rats.Inhibitory gating?IG?is an adaptive mechanism.The evidence showed that stress can change the state of IG.At present,the IG ability of amygdala is not clear.It is impossible to detect the patients'IG response through the damaged neuro-electrophysiology.Therefore,we used in vivo to explore the degree of amygdala response in PTSD.Ca²⁺is one of the most important intracellular messengers.The nerve conduction function will be affected when the intracellular calcium concentration and the steady-state of membrane current were destroyed.L-type Ca²⁺channels?LTCCs?is the main way of Ca²⁺influx.It strictly controls the process of Ca²⁺entering cells and play an important role in maintaining calcium homeostasis.The?1 subunit is the main functional subunit of LTCC and is the voltage sensor of membrane potential.At present,it has been proved that there are four subtypes of?1 subunit,namely?1s?Cav1.1?,?1C?Cav1.2?,?1D?Cav1.3?and?1F?Cav1.4?.Cav1.2 and Cav1.3 were distributed in central nervous system.They are involved in the regulation of calcium homeostasis,hormone secretion,gene expression and other functions.CACNA1C is the gene encoding Cav1.2 and CACNA1D is the gene encoding Cav1.3.One of the hypotheses about the pathogenesis of PTSD is that calcium overload leads to gene dysfunction.The intracellular Ca²⁺concentration was measured in the previous study.It was found that the concentration of Ca²⁺increased after PTSD,and the expression of calmodulin gene combined with Ca²⁺increased.In addition to calmodulin,Ca²⁺also can bind to and activate PKC,AC and some other signal transduction genes,and then regulate the downstream signal transduction pathways of Ras-Raf-ERK1/2-CREB and cAMP-PKA-CREB,and regulate fear,anxiety finally.Method:1.236 wistar male rats were used for single prolonged stress?SPS?paradigm,which were divided into normal control group and SPS group.2.To evaluate the rat status;to detect the rats plasma corticosterone level with Elisa kit;to detect the auditory fear of rats with ASR and electrophysiological experiments.3.The calcium current density of amygdala was measured by whole cell patch clamp.4.The expression of CACNA1C,CACNA1D,p-PKC,Ras,Raf1,ERK1/2,p-ERK1/2,?-arrestin2,PDE4 and CREB were detected by immunofluorescence,Western Blot,immunohistochemistry,and RT-PCR.5.Elisa was used to detect the activity of cAMP.Results:1.Compared with normal rats,the level of corticosterone in SPS rats increased at9:00-10:00 on day 1 and 7 after exposure;and decreased at 18:00-19:00 on day 1 and 7after exposure.That means corticosterone secretion in SPS rats was disordered.After SPS,the rats gain body weight and drinking water were decreased,that indicated the general state of rats was affected after stress.2.During training,with the increase of decibel,the amplitude was increased,peaked at 115dB.The latency decreased at 115 dB and duration reached the longest time at 115 dB.On SPS day 7,the amplitude increased in each decibel,and has statistically significant at 115dB after SPS;no difference of latency after SPS;the duration has significant increased at115dB after SPS.3.Both control animals and SPS animals have IG effect in the amygdala.The IG ratio of SPS rats was higher than control rats at 25 ms?N25?after the test sound started,the difference has statistically significant;at 40 ms?P40?,the T/C value of SPS rats was tendency higher than the control rats,but the difference was no statistically significant.4.The whole cell membrane patch clamp record showed that the calcium current density of SPS rats was lower than control rats at 10 mV,the difference has statistically significant.5.CACNA1C was mainly expressed in the cytoplasm,the intensity was increased after SPS exposure.The CACNA1D was mainly expressed in the nucleus,the intensity was decreased after SPS;the mRNA expression of CACNA1C in SPS rats was higher than the control rats,the difference has statistically significant.There was no difference in the expression of CACNA1D mRNA between two groups.6.The optical density of HRas in SPS rats has lower trend than control rats,and the optical density of Raf1 and ERK1/2 was increased,the difference has statistically significant;there was no difference of phosphorylated ERK1/2 mRNA between two group.There was no difference in the mRNA expression of ERK1 between two groups,and the mRNA level of ERK2 in SPS rats was higher than control rats.7.?-arrestin2 was mainly distributed in the cytoplasm of BLA neurons.After SPS exposure,the expression of?-arrestin2 was significantly lower than the control group.In addition,on the 7th day after SPS,part of?-arrestin2 positive products were mainly distributed near the cell membrane.It is suggested that?-arrestin2 positive products may transfer from cytoplasm to cell membrane after SPS.PDE4 positive products were mainly distributed in the nucleus and cytoplasm of BLA.In the normal control group,the neurons in BLA showed strong positive reaction and heavy staining.Compared with the normal control group,?-arrestin2 and PDE4 in BLA of rats on the 7th day of SPS were significantly reduced.The results of Western Blot showed that the optical density of?-arrestin2 and PDE4 decreased significantly,and the ratio of?-arrestin2 and PDE4 decreased to the lowest level on the day 7 after SPS.RT-PCR results were consistent with this.The expression of cAMP in amygdala increased significantly on the day 1,7 and 14 after SPS exposure.The level of cAMP began to rise on day 1 after SPS,peaked on the day 7 after SPS,and then recovered.PKA level increased on the day 1 after SPS and peaked on the SPS 7d.There was a significant difference between two group and decreased on SPS 14d.The results of RT-PCR were consistent with those of Western Blot.The results of CREB immunohistochemistry showed that CREB positive was mainly expressed in the nucleus,the immunoreactivity of SPS was up-regulated on day 1,and peaked on day 7 after SPS,and decreased on day 14,which was statistically significant compared with control group.The results of RT-PCR were consistent with immunohistochemistry results.Conclusion:1.The dysfunction of corticosterone secretion and fear enhanced in SPS rats.2.The L-type calcium channel function of amygdaloid neurons in SPS rats was decreased,fear may be enhanced through the dysregulation of Ras-Raf-ERK1/2 signal transduction pathway.3.The interaction of?-arrestin2 and PDE4 in BLA was weakened after SPS which result in dysfunction of PKA-cAMP-CREB signal transduction pathway.That may be the regulatory mechanism for fear enhancement.