The Involvement Of TRPC3 And TRPC6 Channels In Nociceptive Information Transmission And Pain Hypersensitivity And Its Underlying Mechanisms

Pain is not only a symptom, but also a kind of disease. The incidence of chronic pain is increasing year by year. More and more people suffer from chronic pain, which severely threatens patients' life quality and physical and mental health. These patients are overwhelmed with chronic pain so that they hardly wish to live. Chronic pain is recurrent and very difficult to treat. In addition to the burden on the individual, chronic pain also exacts a high cost from society. Chronic pain is often accompanied by aversive emotion, such as anxiety, depression, fear and even vicious mood. Although much progress has been made on chronic pain, the treatment of chronic pain in clinic is still not optimistic. There are many limitations to clinical analgesic and the drugs are unspecific.Therefore, to explore the underlying mechanisms of chronic pain and to develop new analgesic therapy is urgently needed and is the basic responsibilities and goals of each pain researcher and clinician.Dorsal root ganglion(DRG) neurons are the first stop of pain signal ascendingpathway from the periphery to the central nervous system(CNS), which plays a very important role in the process of pain signal transmission and processing. Upon inflammation or injury, DRG neurons become hyperexcitable, which serve as a "pacemaker" and "source" of chronic pain signals. These exaggerated pain signals are subsequently conveyed to the spinal cord, and then further to the CNS, leading to pain and hyperalgesia.Accumulating evidence has shown that transient receptor potential(TRP) channels play a crucial role in sensing a variety of external noxious stimuli, such as cold, heat,mechanical and chemical stimuli. These noxious stimuli are transformed into action potentials and transmitted to the spinal cord and then further to the brain which eventually evoke pain. However, due to the lack of specific tools, the exploration of function of TRPC channels are far behind other TRP channels. Recently, TRPC channels, especially TRPC3 and TRPC6 subtypes are reported to be expressed in the CNS and to be involved in various functions, i.e. nerve growth, development, conginition and motor coordination.Besides the expression in the CNS, TRPC3 and TRPC6 are also expressed in the ascending pain pathway, especially in the small to medium-diameter DRG neurons(also called nociceptor). However, which kind of stimuli do TRPC3 and TRPC6 respond to and what role do they play in the transmission of nociceptive information remains elusive.Studies from other biological system have shown that TRPC3 and TRPC6 channels display structural and functional redundancy. Deletion of one gene can lead to the compensation of the other gene. Therefore, to reveal the exact function of TRPC3 and TRPC6 channels in the nociceptive signal transmission and pain hypersensitivity, we introduce the TRPC3 and TRPC6 channel double knockout(TRPC3/6 DKO) mice in which both TRPC3 and TRPC6 are deleted in structure and function. New insights derived from these advances are expected to expediate development of novel analgesics acting at TRPC3 and TRPC6 for the treatment of chronic pain.Part 1:To investigate the function of TRPC3 and TRPC6 channels in sensing external stimuli under physiological and pathological states.Objective:To observe the function of TRPC3/6 channels in sensing external stimuli under physiological and pathological statesMethods : In the normal physiological condition, we detect the difference of reactivity to mechanical and thermal stimulation between knockout mice and wild-type mice. Subcutaneous inject Complete Freund's adjuvant(CFA) into the unilateral planta pedis of adult mice hind paw to establish the pain model. Then detect the difference of reactivity to mechanical and thermal stimulation between knockout mice and wild type mice in multiple time points. After injecting bradykinin into one side of the hind paw, we detect the difference of spontaneous pain reaction time. To verify whether knockout the TRPC3/6 genes can affect the mice's motor coordination ability by rotate rod.Results : The sensitivity of TRPC3/TRPC6 channels to mechanical and thermal stimuli were tested under basal states in adult mice. It was found that deletion of TRPC3/TRPC6 reduced the sensitivity to mechanical and thermal stimuli, manifesting as increased mechanical threshold to Von Frey hairs and prolonged latency to radiant heat stimulation applied to hindpaws in TRPC3/6 DKO mice as compared to wildtype mice.This result suggests that TRPC3 and TRPC6 channels may collaboratively play an important role in sensing peripheral mechanical and thermal stimulus.To further investigate whether TRPC3 and TRPC6 channels are involved in the pain hypersensitivity induced by injury or inflammation, we utilized a chronic inflammatory pain model evoked by unilateral injection of Complete Freund's Adjuvant(CFA) into the intraplantar surface of mice hindpaw. Following inflammation, wildtype mice demonstrated the characteristic left-ward and upward shift in the stimulus-response curve over basal curves reflecting mechanical hypersensitivity. In contrast, TRPC3/6 DKO mice demonstrated a less marked deviation from baseline behaviour upon CFA-induced inflammation. Furthermore, the relative drop in response thresholds to von Frey hairs in the inflamed state over basal(pre-CFA) state occurred to a significantly lesser extent in TRPC3/6 DKO mice as compared to wildtype mice. In addition, TRPC3/6 DKO mice showed a significantly lower magnitude of thermal hyperalgesia than wildtype mice. We infer from the above that the development of primary mechanical and thermalhyperalgesia as well as mechanical allodynia following somatic inflammation is impaired by a loss of TRPC3 and TRPC6 channels. These results strongly suggest TRPC3 and TRPC6 channels play a crucial role in the development of hyperalgesia under pathological conditions.A growing body of evidence accumulated that upon injury or inflammation, the injured site releases a variety of inflammatory mediators, including bradykinin, histamine,prostaglandins, substance P, CGRP, etc. These inflammatory mediators can interact with their receptors located in nociceptors, leading to the reduction of threshold, enhancement of responsiveness of nociceptor. This is called peripheral sensitization. Amongst which,bradykinin is the most potent mediators which could induce pronociceptive effect. It has been shown that peripheral administration of bradykinin induces both acute spontaneous pain response and long-lasting mechanical and thermal hyperalgesia. We are further interested to know whether TRPC3/TRPC6 channels are involved in the bradykinin-induced pain hypersensitivity. To address this question, bradykinin was administered into the intraplantar surface of mice hindpaw. Upon bradykinin delivery,mice displayed a spontaneous nociceptive reponses, manifesting as lifting, licking and biting of the injected paw. When compared to wildtype mice, TRPC3/TRPC6 DKO mice demonstrated a much lowered spontaneous pain response.We further assess the effect of TRPC3/TRPC6 channels in the motor function of mice by using rota-rod test. In striking contrast to dramatic effect on pain sensation, TRPC3 and TRPC6 channels did not exert obvious effects on motor coordination.Part 2: To assess the effect of TRPC3 and TRPC6 channels on the excitability of nociceptive DRG neurons.Objective: To assess the effect of TRPC3/6 channels on the excitability of nociceptive DRG neuronsMethods: By using whole-cell patch-clamp recordings in intact DRG preparation,passive and active membrane properties was assessed in small nociceptive DRG neurons derived from wildtype and TRPC3/6 DKO mice under basal states and 24 hours after CFAinjection.Results:1) Passive and active membrane properties were assessed in small nociceptive DRG neurons derived from wildtype and TRPC3/6 DKO mice under basal states. Passive membrane properties including resting membrane potential(RMP) and membrane resistance(Rm) as well as membrane capacitance(Cm) of small DRG neurons tested were not significantly different between wildtype and TRPC3/6 DKO mice. However, the active membrane properties of small DRG neurons from TRPC3/6 DKO mice represented significant differences compared with wildtype controls. Small DRG neurons from TRPC3/6 DKO displayed reduced excitability, manifesting as increased rheobase to evoke actional potential(AP) and reduced AP frequency induced by a depolarizing step current as compared to that from wildtype mice. This result suggests that TRPC3 and TRPC6 channels are involved in determining the excitability of DRG neurons.2) Passive and active membrane properties were assessed in small nociceptive DRG neurons derived from wildtype and TRPC3/6 DKO mice under inflammatory pathological states. Following CFA inflammation, small DRG neurons from wildtype mice become hyperexcitable, manifesting as decreased rheobase to evoke AP and enhanced AP frequency in response to depolarizing current injection. In contrast, TRPC3/6 DKO mice demonstrated a significantly lower excitability than wildtype mice after CFA inflammation.It can be inferred from the above that TRPC3 and TRPC6 channels are key determinants in the hyperexcitability of nociceptive DRG neurons under pathological states.Part 3: To evaluate the influence of TRPC3 and TRPC6 channels on the up-regulated expression of c-Fos and p-ERK in the spinal cord induced by irritantsObjective:In order to get further evidence of TRPC3/6 channel restrain the pain reaction.Methods : The formalin-induced inflammatory pain models were set up with the above methods. Two hours later, the lumbar enlargement was removed afterparaformaldehyde perfusion and fixation, then post-fixed, dehydrated, frozen sectioned and dyed by immunohistochemical method.Result:To further address whether TRPC3 and TRPC6 channels affect excitability of spinal dorsal horn neurons, we studied induction of c-Fos and phosphorylated ERK1/2(p ERK1/2), markers of neuronal activity, following formalin injection in wildtype and TRPC3/6 DKO mice. In the spinal cord, strong Fos-immunoreactivity was seen in the superficial and deep lamina of the dorsal horn where the nociceptive primary afferents mainly terminates following formalin injection in wildtype mice. In striking contrast,TRPC3/6 DKO mice showed a much less Fos-positive cells in both the superficial and deep layers of spinal dorsal horn. Similarly, upon formalin injection, intense p ERK1/2expression was observed in the lamina I of spinal dorsal horn of wildtype mice, whereas a dramatic reduction of p ERK1/2 expression was seen after deletion of TRPC3/TRPC6 channels. These results indicate that TRPC3 and TRPC6 channels are involved in the increased neuronal activity in spinal dorsal horn under pathological states.Part 4: To reveal the role of TRPC3 and TRPC6 channels on spinal synaptic transmission and plasiticity in spinal lamina I neurons.Objective : Further confirmed the cellular and molecular mechanism of TRP3/6channes induced pain sensitivityMethods:Adopt the mice after born 15-21 days, under the normal state and after chronic inflammatory pain models induced by the 24 hours after CFA injection, producing spinal cord slice sample, transverse spinal slices were obtained and whole cell patch clamp recordings of spinal dorsal horn lamina I neurons were performed. Observe the difference of excitability of spinal dorsal horn I layer neurons between knockout mice and wild-type mice. Observe the influence of TRPC3/6 channels to the primary afferent synaptic transmission efficiency and the change of plasticity. Record the passivity and active membrane properties of noxious DRG neurons and spinal cord dorsal horn I layer neurons,and record the evoked excitatory postsynaptic currents(e EPSCs) of spinal cord dorsal horn I layer neurons induced by stimulate the noxious primary afferent fibers. In thecondition of chronic inflammatory pain, record the spontaneous excitatory postsynaptic currents(s EPSCs) of spinal cord dorsal horn I layer neurons.Results:1) Under basal states, excitatory synaptic currents(e EPSCs) in spinal lamina I neurons were evoked by electrical stimulation of dorsal root entry zone. Input-output curve(I-O curve) demonstrated that the amplitude of e EPSC increases with the increase of stimulation intensity. As compared to wildtype mice, TRPC3/6 DKO mice showed the characteristic right-ward and downward shift in the I-O curve of e EPSCs, reflecting the reduced basal synaptic transmission.2) Following CFA inflammation, I-O curve of e EPSCs from wildtype mice demonstrated a dramatic left-ward and upward shift over basal curves, reflecting synaptic potentiation. In strking contrast, TRPC3/6 DKO mice exhibited a significantly less potentiation of synaptic transmission than wildtype mice after CFA inflammation. We can infer from the above that TRPC3 and TRPC6 channels are involved in the synaptic potentation under inflammatory pathological states.3) To further elucidate whether the effect of TRPC3/TRPC6 channels on synaptic potentiation is presynaptic or postsynaptic in origin, we performed two analysis, s EPSCs and PPR analysis. Analysis of s EPSCs revealed that deletion of TRPC3 and TRPC6 channels was able to reduce the frequency of s EPSCs, but without obvious effect on the amplitude of s EPSCs in CFA-inflammed mice. This result suggests that TRPC3 and TRPC6 channels may facilitate spinal synaptic transmission and plasticity by presynaptic mechanisms.To further consolidate the pre- or post-synaptic mechanisms, we focused on the analysis of e EPSCs elicited by paired-pulse stimulation, which represents a short-lasting changes in the second evoked EPSC when it follows shortly after the first and is well accepted as an indication of presynaptic mechanisms. In spinal slices derived from CFA-inflamed mice, we found evidence for paired-pulse facilitation(PPF) as well as paired-pulse depression(PPD) of e EPSCs in wildtype mice. Deletion of TRPC3 and TRPC6 channels led to a significant change of PPR. In conclusion, s EPSCs and PPRanalysis strongly support the inference that the potentiation of synaptic transmission by TRPC3 and TRPC6 channels under inflammatory states comes about via presynaptic mechanisms involving a decrease in release probability.Conclusions:1. By utilizing TRPC3 and TRPC6 DKO mouse model, we demonstrated for the firsttime that TRPC3 and TRPC6 channels play an important role in the basal mechanicaland thermal nociception. Furthermore, TRPC3 and TRPC6 channels exertpronociceptive effect under inflammatory pathological states.2. We demonstrated for the first time that TRPC3 and TRPC6 channels can modulate theexcitability of nociceptive DRG neurons under basal states. Upon injury or inflammation, TRPC3 and TRPC6 channels mediates peripheral sensitization of nociceptive DRG neurons, which might underly their pronociceptive effects under patholgocial states.3. We demonstrated for the first time that TRPC3 and TRPC6 channels make a greatcontribution in spinal synaptic transmission and synaptic plasticity in spinal dorsal horn. Upon injury or inflammation, peripheral sensitization and spinal synaptic plasticity has been assumed as the cellular basis of chronic pain. TRPC3 and TRPC6 channels exert marked facilitating effects on spinal synaptic transmission and plasticity.s EPSCs and PPR analysis strongly support the inference that the potentiation of synaptic transmission by TRPC3 and TRPC6 channels under inflammatory states comes about via presynaptic mechanisms involving a decrease in release probability.