The Role Of TRPC1/4/5 Channels In The Anterior Cingulate Cortex On The Anxiety-like Behavior Associated With Chronic Inflammatory Pain

Pain is an unpleasant sensory and emotional experience and a great challenge for public health.Pain-induced unpleasant emotional experiences protect the body from harmful stimuli,but pain can cause many negative effects.According to statistics,the incidence of chronic pain in the world has been more than 30%,and many accompanied by serious anxiety,which in turn can aggravate the pain perception of patients.A large number of previous studies have revealed the detailed mechanism underlying pain processing at the spinal cord level.However,how the cerebral cortex translates this signal from the spinal cord into an emotional experience remains unclear.Cingulate cortex is the gyrus between the cingulate sulcus in the medial hemisphere of the brain and the corpus callosum,which belongs to the limbic system and is one of the components of the medial pain system.The anterior cingulate cortex(ACC)receives fiber projections from the thalamus and brainstem,processing the harmful information ascending from the neurons in the spinal dorsal horn.Meanwhile,ACC is also considered as a higher brain functional region,which is involved in the regulation of collective memory,cognition,emotion and other functions.Emerging studies have shown that ACC plays an important role in mediating the aversive emotions such as pain-induced anxiety,but the underlying mechanism remains unclear.Therefore,it is of great significance to explore the mechanism of ACC in the anxiety induced by chronic pain and to provide potential targets for the treatment of chronic pain and related anxiety.Canonical transient receptor potential channels(TRPC),the first subfamily of TRP channel superfamily to be cloned,are Ca2+ permeable non-selective cation channel on the cell membrane and play an important role in various physiological and pathological processes.However,the function of TRPC channels is still unclear due to the lack of specific and selective pharmacological drugs.Currently,seven members of TRPC subfamily have been identified,i.e.TRPC1-TRPC7.Given the amino acid sequence homology and functional similarities,TRPC1,TRPC4 and TRPC5,are often assembled together to form homologous or heterologous channels.TRPC1/4/5 channels have been implicated in a variety of biological functions.In our previous study,we found that TRPC channels are widely expressed in the dorsal root ganglion,and superficial spinal dorsal horn neurons in the pain pathway,indicating that TRPC channels are involved in the pain processing.Studies have reported that TRPC1/4/5 channels not only regulate pain perception in neuropathic pain,but also regulates anxiety,depression and fear.Whether TRPC1,TRPC4 and TRPC5 channels in ACC mediate the negative emotions such as anxiety associated with chronic pain has not been reported so far.Therefore,by utilization of TRPC 1/4/5 TKO(triple knock out)mice as an animal tool,this study bears two aims in mind.One is to reveal the functional significance of TRPC 1/4/5 channels in the development of chronic pain and pain-related aversive emotion.Second is to understand the underlying mechanism by which TRPC 1/4/5 channels regulate chronic pain and its related aversive emotion.This helps to open up a new research direction for the pathologic mechanism of chronic pain progression,and to provide a new way for the treatment of chronic pain.Part Ⅰ:To investigate the relationship between anxiety-like behavior induced by chronic inflammatory pain and the expression changes of TRPC1/4/5 channels in ACC Objective:To observe whether pain sensitization and anxiety-like behaviors occur and to detect changes of mRNA level,protein level and expression distribution of TRPC1/4/5 channel in ACC in mice under the condition of chronic inflammatory pain.Methods:Wild type(WT)mice were used at age of 8 week with a body weight of 24-28 g.The experimental group was injected with 20 μl complete Freund’s adjuvant into the intraplantar surface of left hindpaw of mice to establish a model of chronic inflammatory pain,.The control group was injected with the same amount of saline on the left hindpaw.The changes of mechanical and thermal sensitivity were detected in mice in the basal state and 1 d and 7 d following CFA injection,and the open-field and elevated plus maze tests were conducted in mice in the basal state and 7 days after CFA injection.And the samples were collected on the 7th day after CFA inflammation,assays of qRT-PCR,Western Blot and fluorescence in situ hybridization(FISH)were performed to determine the expression changes and distribution of mRNA and protein levels of TRPC1,TRPC4 and TRPC5Results:At 7d following intraplantar injection of CFA,mice displayed dramatic mechanical and thermal hypersensitivity as well as pain-related anxiety-like behavior During the process of chronic inflammatory pain,we used FISH technique to further verify that the expression of TRPC1,TRPC4 and TRPC5 was markedly upregulated in ACC under chronic inflammatory pain states.To further confirm the involvement of TRPC channels in the process of chronic inflammatory pain,mRNA and protein expression levels of TRPC1,TRPC4 and TRPC5 channels in ACC of CFA group were significantly higher than those in saline control group by qRT-PCR and Western Blot analysis.Part Ⅱ:To investigate the role of TRPC1/4/5 channels in the development of anxietylike behavior associated with chronic inflammatory painin miceObjective:To observe the effect of TRPC1/4/5 on anxiety-like behavior in mice during chronic inflammatory pain.Methods:TRPC1/4/5 TKO and WT mice were used to establish the model of chronic inflammatory pain,and the control group was set up as before(injection of the same amount of saline).Open field and elevated plus maze tests were conducted on the 7th day to observe the effect of TRPC1/4/5 on anxiety-like behavior of mice in the process of chronic inflammatory pain by comparing anxiety-like behavior indicators of saline group and CFA group.Results:There was no significant difference in anxiety-like behavior between TRPC1/4/5 TKO and WT mice in the open field and elevated plus maze experiment under physiological conditions(before CFA injection).However,in states of inflammatory pain,the absence of TRPC1/4/5 inhibited the anxiety-like behavior induced by CFA in mice,manifesting as the reduced travelling distance in the center area in the open field test and decreased entry times in the open arm in elevated plus maze paradigm.These results suggest that TRPC1/4/5 channel may be involved in the anxiety-like behavior induced by chronic inflammatory pain in mice.Part Ⅲ:To investigate the effect of TRPC1/4/5 on the neuronal excitability and synapticplasticity in ACC during the anxiety-like behavior induced by chronic inflammatory painObjective:To observe whether TRPC1/4/5 could change the level of intracellular Ca2+ in AAC neurons,which in turn participate in the changes of neuronal excitability and functional synaptic plasticity and hence result in the occurrence of anxiety-like behavior induced by chronic inflammatory pain.Methods:WT mice and TRPC1/4/5 TKO mice(4-5 week-old)underwent CFA and saline injection,respectively..Seven days after injection,ACC slices were made and whole-cell patch-clamp recording of ACC neurons was performed.Pyramidal neurons in Layer Ⅱ-Ⅲof ACC were recorded and neuronal excitability was analyzed.For recording LTP,evoked EPSCs were recorded in layer Ⅱ-Ⅲ neurons in response to conditioning stimulation of layer V For calcium imaging,Targeted injection of rAAV2/9-CaMKII-GcaMP6s was performed in ACC of WT and TRPC1/4/5 TKO mice.The ceramic fiber was inserted above the virus injection point and fixed.After 21 days of viral infection,mechanical stimulation ws applied to the intraplantar surface of hindpaw via von Frey filaments with strength of 0.07g,1.40g and 4.00g.Photometry calcium imaging in ACC was conducted in in awaken mice,the changes of Ca2+ signal were recorded under physiological states and CFA-inflamed states.Results:When the same intensity of depolarizing step current injection was given,the firing frequency of ACC pyramidal neurons in TRPC1/4/5 TKO mice was lower than that in WT mice,suggesting that TRPC1/4/5 were involved in the excitability of ACC pyramidal neurons.In WT mice,pyramidal neurons in layer Ⅱ-Ⅲ of ACC displayed long-term potentiaon(LTP)in response to conditioning stimulus,which was completely abolished by the deletion of TRPC1,TRPC4 and TRPC5.These results suggest that TRPC1/4/5 channels are responsible for the excitability of pyramidal neurons and induction of LTP in ACC.Further mechanistic analysis with photometry calcium imaging revealed that TRPC1/4/5 channels facilitate ACC neuronal excitability and synaptic plasticity via influx of Ca2+through TRPC1/4/5 channels.Main conclusions:1.By utilizing TRPC1/4/5TKO mouse model,combined with molecular biology techniques and animal behavioral methods,we demonstrated that TRPC 1/4/5 channels in the ACC play an important role in mediating chronic pain-induced anxiety-like behaviors.2.Applying the whole-cell patch-clamp techinque,we found that TRPC1/4/5 channels mediate the molecular mechanisms of chronic pain-induced pain affect by enhancing the excitability and synaptic plasticity of layer Ⅱ-Ⅲ pyramidal neurons in the ACC.3.Application of optical fiber calcium signal recording technolog,we confirmed that TRPC1/4/5 channels facilitate the excitability of neurons and LTP in chronic inflammatory pain.These effects are attributed to TRPC1/4/5-mediated Ca2+ elevation in the ACC neurons.