Study On The Association Of HTR1A And Theta Oscillations In Anterior Cingulate Cortex Of Visceral Hypersensitive Rats Induced By Psychological Stress

Part Ⅰ Association between HTR1A and theta oscillations in anterior cingulate cortex of visceral hypersensitive rats induced by psychological stressObjectiveVisceral hypersensitivity induced by psychological stress is involved in the formation and aggravation of disorders of gut-brain interaction diseases such as irritable bowel syndrome symptoms.Anterior cingulate cortex（ACC）sensitization mediated by serotonin 1A receptor（HTR1A）down-regulation is an important mechanism in this process,and the enhanced theta oscillations（4-10 Hz）are characteristic of local field potential in ACC sensitization.In this part of the study,we applied the agonists/antagonists of HTR1A to ACC slices of rats and observed the changes in theta oscillations,aiming to explore the association between the down-regulated HTR1A and the enhanced theta oscillations in ACC of visceral hypersensitive rats.Methods1.Adult twenty-four male Wistar rats were randomly divided into normal control（NC,n=8）group,chronic water avoidance stress（WAS,n=8）group and sham water avoidance stress（Sham-WAS,n=8）group.The anxiety-like behaviors of rats were evaluated by the open field test and the elevated plus maze test.The visceral sensitivity of rats was evaluated by the visceral motor response（VMR）to 60 mm Hg colorectal distention.The theta oscillations induced by carbachol and kainic acid were recorded in ACC slices of rats.2.Adult twenty-four male Wistar rats were randomly divided into normal control combined with HTR1A agonist（NC+8-OH-DPAT,n=6）group,normal control combined with HTR1A antagonist（NC+WAY100135,n=6）group,chronic water avoidance stress combined with HTR1A agonist（WAS+8-OH-DPAT,n=6）group and chronic water avoidance stress combined with HTR1A antagonist（WAS+WAY100135,n=6）group.20μM 8-OH-DPAT or 10μM WAY100135 were applied to ACC slices of NC and WAS rats.The changes of theta oscillations induced by carbachol and kainic acid were observed and recorded in ACC slices of rats.Results1.Compared with the NC and Sham-WAS groups,the amplitude of VMR to 60mm Hg colorectal distention in WAS group were increased（P<0.001;P<0.05）.In the open field test,the WAS group showed fewer number of central entries（P<0.01;P<0.05）and more raring number than that of NC and Sham-WAS groups（P<0.01;P<0.05）.In the elevated plus maze test,the WAS group spent less percentage of time in open arms than NC and Sham-WAS groups（P<0.001;P<0.01）.During modeling,the WAS group showed fewer body weight gain（P<0.01;P<0.05）and increased number of fecal pellets than NC and Sham-WAS groups（P<0.001;P<0.05）.Moreover,the integrated power of theta oscillations（4-10Hz）in ACC of WAS group were higher than that of NC and Sham-WAS groups（P<0.05;P<0.05）.2.For normal rats,compared with the NC+WAY100135 group,the integrated power of theta oscillations in ACC of NC group and NC+8-OH-DPAT group were decreased（P<0.05;P<0.05）.For rats subjected to chronic water avoidance stress,compared with the WAS+8-OH-DPAT group,the integrated power of theta oscillations in ACC of WAS group and WAS+WAY100135 group were increased（P<0.05;P<0.05）.Conclusion1.Chronic water avoidance stress may induce visceral hypersensitivity and anxiety-like behaviors in rats and act`ivate ACC,which is manifested as the enhancement of theta oscillations.2.HTR1A can regulate theta oscillations in ACC.The enhanced theta oscillations in ACC of water avoidance stress rats were related to the decreased activity of HTR1A.The enhanced theta oscillation mediated by the down-regulated HTR1A in ACC of rats may contribute to the formation of visceral hypersensitivity induced by psychological stress.Part Ⅱ Study on the mechanism of HTR1A regulating theta oscillations in anterior cingulate cortex of visceral hypersensitive rats induced by psychological stressObjectiveThe generation of theta oscillations is related to the firing of neurons.Activation of the HTR1A in the ACC can increase the potassium conductance,thereby hyperpolarizing the membrane potential and reducing the excitability of neurons.This process is mediated by G protein-coupled inwardly rectifying potassium（GIRK）channels.In this part of the study,we used genetic intervention technology to label the neurons expressing HTR1A in the ACC of rats,and observed the effect of HTR1A and GIRK on the synchrony of neurons’firing and theta oscillations,aiming to further elucidate the mechanism of HTR1A regulating theta oscillations in ACC of visceral hypersensitivity rats induced by psychological stress.Methods1.The adeno-associated virus（r AAV-HTR1A-m Cherry-WPRE-p A）that containing the promoter of HTR1A was constructed.Then it was microinjected into the bilateral ACC of six adult male Wistar rats.The frozen section was used for immunofluorescence staining to evaluate the transfection effect and specificity of the virus.2.Twelve adult male Wistar rats were randomly divided into normal control(NC_HTR1A,n=6)group and chronic water avoidance stress(WAS_HTR1A,n=6)group.Before modeling,the rats were microinjected with the HTR1A virus in the bilateral ACC.The local field potentials and the firing of HTR1A-expressing pyramidal neurons in ACC slices were recorded simultaneously,and the synchrony of the two was analyzed.The synchrony of neuron firing was quantified by the distribution of spike phase in the theta oscillation（4-10Hz）cycle,and the mean vectors r andφrepresented the magnitude and direction of circular distribution,respectively.3.Adult twelve male Wistar rats were randomly divided into chronic water avoidance stress combined with HTR1A agonist(WAS_HTR1A+8-OH-DPAT,n=6)group,chronic water avoidance stress combined with HTR1A agonist and GIRK inhibiter(WAS_HTR1A+8-OH-DPAT+Ba²⁺,n=6)group.Before modeling,the rats were microinjected with the HTR1A virus in the bilateral ACC.Applied 20μM8-OH-DPAT,200μM Ba²⁺to ACC slices and recorded the local field potential as well as the firing of HTR1A-expressing pyramidal neurons in ACC,then analyzed the synchrony of the two.Results1.The red fluorescent signal of HTR1A virus can be observed in the Cg1 region of ACC,and it is widely co-labeled with the green fluorescent signal of HTR1A immunofluorescence labeling.2.In ACC slices,compared with the NC_HTR1Agroup,the WAS_HTR1Agroup showed an increased r-value（P<0.05）and increased firing rate of HTR1A-expressing pyramidal neurons（P<0.05）,while the angleφwas not statistically different between the two groups（P>0.05）.3.Compared with the WAS_HTR1Agroup and the WAS_HTR1A+8-OH-DPAT+Ba²⁺group,the ACC slices of WAS_HTR1A+8-OH-DPAT group showed decreased r-value（P<0.01;P<0.05）and decreased firing rate of HTR1A-expressing pyramidal neurons（P<0.05;P<0.05）,while the angleφwas not statistically different between the three groups（P>0.05）.Conclusion1.The adeno-associated virus r AAV-HTR1A-m Cherry-WPRE-p A efficiently labels HTR1A-expressing neurons in ACC of rats.2.In visceral hypersensitivity rats induced by psychological stress,the enhanced theta oscillations in ACC is related to the increased firing rate and synchrony of HTR1A-expressing pyramidal neurons.3.HTR1A may regulate the firing rate and synchrony of pyramidal neurons in the ACC through GIRK,affect theta oscillations and participate in the formation of visceral hypersensitivity.Part Ⅲ The central mechanism of tandospirone,an HTR1A partial agonist,preventing visceral hypersensitivity and anxiety-like behavior in psychological stress ratsObjectiveIBS patients are often accompanied by mental and psychological comorbidities such as anxiety and depression.Previous studies have found that the anti-anxiety drug tandospirone can also relieve the gastrointestinal symptoms of IBS patients with anxiety while treating their anxiety symptoms.The mechanism involved may be related to the activation of HTR1A in ACC by tandospirone,thereby improving visceral hypersensitivity.To explore the central mechanism of tandospirone preventing visceral hypersensitivity and anxiety-like behavior in psychological stress rats,the effects of tandospirone on the theta oscillations and the firing of HTR1A-expressing pyramidal neurons in ACC were studied in vivo and in vitro.Methods1.Adult eighteen male Wistar rats were randomly divided into normal control combined with PBS（NC+PBS,n=6）group,chronic water avoidance stress combined with PBS（WAS+PBS,n=6）group and chronic water avoidance stress combined with tandospirone（WAS+TDS,n=6）group.Multi-channel electrodes were embedded in the ACC of rats.Intraperitoneal injection of PBS or tandospirone10 mg/kg were conducted before chronic water avoidance stress.The anxiety-like behaviors of rats were evaluated by open field test and advanced plus maze test.The visceral sensitivity of rats was evaluated by the visceral motor response（VMR）to 60mm Hg colorectal distention.The local field potentials of the ACC and electromyography were recorded simultaneously during 60 mm Hg colorectal distention.2.Adult twelve male Wistar rats were randomly divided into chronic water avoidance stress combined with tandospirone and PBS（WAS+TDS+PBS,n=6）group and chronic water avoidance stress combined with tandospirone and HTR1A antagonist（WAS+TDS+WAY100635,n=6）group.A cannula was embedded in the ACC of rats.Intraperitoneal injection of tandospirone 10mg/kg and microinjection of PBS 0.2μl or WAY100635 30nmol/0.2μl PBS into the ACC were conducted before chronic water avoidance stress.The anxiety-like behaviors of rats were evaluated by open field test and advanced plus maze test.The visceral sensitivity of rats was evaluated by the visceral motor response（VMR）to 60 mm Hg colorectal distention.3.Twelve adult male Wistar rats were randomly divided into chronic water avoidance stress combined with tandospirone(WAS_HTR1A+TDS,n=6)group and chronic water avoidance stress combined with tandospirone and HTR1A antagonist(WAS_HTR1A+TDS+WAY100635,n=6)group.Before modeling,the rats were microinjected with the HTR1A virus in the bilateral ACC.Applied 20μM tandospirone,10μM WAY100635 to ACC slices and recorded the local field potential as well as firing of HTR1A-expressing pyramidal neurons in ACC,then analyzed the synchrony of the two.Results1.The results of in vivo experiments showed that,compared with the NC+PBS group,the WAS+PBS group showed a decreased number of central entries（P<0.05）and increased raring number（P<0.05）in the open field test,spent less percentage of time in open arms（P<0.001）in the elevated plus maze test.Moreover,the VMR amplitude（P<0.01）and the integrated power of theta oscillations（4-10Hz）in the ACC（P<0.001）during 60mm Hg colorectal distention of WAS+PBS group were increased synchronously.Compared with the WAS+PBS group,the WAS+TDS group showed increased number of central entries（P<0.05）in the open field test and spent more percentage of time in open arms（P<0.05）in the elevated plus maze test.Besides,the VMR amplitude（P<0.05）and the integrated power of theta oscillations in ACC（P<0.05）of WAS+TDS group were decreased synchronously.Linear regression analysis showed that during colorectal distention,the integrated power of theta oscillations in the ACC was positively correlated with VMR amplitude（R²=0.7255,P<0.0001）.2.Compared with the WAS+TDS+PBS group,the WAS+TDS+WAY100635 group showed decreased number of central entries（P<0.01）in the open field test,showed less percentage of number of open arms entries（P<0.05）and spent less percentage of time（P<0.01）in open arms in the elevated plus maze test.Moreover,the VMR amplitude of WAS+TDS+WAY100635 group were higher than that of WAS+TDS+PBS group（P<0.01）.3.The results of in vitro experiments showed that,compared with the WAS_HTR1Agroup and the WAS_HTR1A+TDS+WAY100635 group,the WAS_HTR1A+TDS group showed decreased integrated power of theta oscillations（4-10Hz）induced by carbachol and kainic acid（P<0.05;P<0.05）,decreased firing rate of HTR1A-expressing pyramidal neurons（P<0.01;P<0.05）,and decreased r-value（P<0.05;P<0.05）,while the angleφwas not statistically different between the three groups（P>0.05）.Conclusion1.Tandospirone prevent the visceral hypersensitivity and anxiety-like behaviors induced by psychological stress in rats.2.Tandospirone may inhibit the firing rate and synchrony of pyramidal neurons in the ACC by activating HTR1A,and inhibit theta oscillations in the ACC,thereby reducing the activity of the ACC,exerting its anxiolytic effects and improving the visceral hypersensitivity induced by psychological stress.