Plastic Changes And Their Underlying Mechanisms Of The Insular Cortex-basolateral Amygdala Pathway Induced By Neuropathic Pain Stimulation

Chronic pain is a prevalent clinical disease, among which neuropathic pain is the most complex and intractable. Neuropathic pain is mainly induced by tissure or nerve injuries. It is often accompanied with negative emotions such as anxiety and depression. Due to the interaction between persistent pain symptom and negative emotions, neuropathic pain exert huge physical and mental burden on patients. Although certain progresses was made in the peripheral nervous system and spinal cord levels, the mechanisms of higher brain centers that modulate neuropathic pain remains to be investigated.Recently, the insular cortex(IC) has attracted many attentions. It is reported that bilateral IC lesions significantly alleviate inflammatory and neuropathic pain(NP) behaviors in rats. Moreover, patients who undergone IC resection suffered from glioma or cerebral infarction showed reduction in nociception. These data suggested a key role of IC in nociceptive sensation and modulation. The induction and maintenance of NP-related negative emotions involves limbic system component amygdala, which can be divided into central amygdaloid nucleus(CeA) medially and lateral amygdaloid nucleus(LA)/ basolateral amygdaloid nucleus(BLA) laterally. The link between cortex and amygdala is mainly mediated via BLA. Modulation of medial prefrontol cortex(mPFC)-BLA pathway leads to changes of anxiety and fear memory in rodents. However, dose similar pathway between IC and BLA exist? If yes, what kinds of changes are undergoing in chronic pain, especially NP? What’ more? Is such pathway involved in NP and its related negative emotions?Given these problems, we conducted current study by using morphological, molecular neurobiological, electrophysiological and behavioral methods to investigate the morphological and functional plasticity alterations of IC-BLA pathway under NP conditions. Furthermore, we modulated this pathway in vivo by optogenetics and chemogenetics in order to detect its role in NP. The contents of the present study are as follows: 1. Efferents and afferents connections of the IC in rats.1) The afferents connections of the IC: We injected the retrograde tracer Fluoro-gold(FG) into anterior IC(AIC) or posterior IC(PIC), respectively. The slices were observed directely or followed by immumohistochemical staining after cutting. The results showed that FG retrogradely-labelled neurons were mainly located in the following: contralateral IC, ipsilateral primary motor cortex(M1), ventral orbital cortex(VO), medial thalamic nucleus, BLA, substantia nigra compacta part(SNC), ventral tegmental area(VTA), dorsal raphe nucleus(DR), parabrachial nuclei(PB),and locus coeruleus(LC).2) The efferents connections of the IC: We next iontophoretically injected an anterograde tracer Phaseolusvulgaris-leukoagglutinin(PHA-L) into the AIC or PIC, respectively. Two weeks after injection, the rats were perfused and brains were cut into slices followed by immumohistochemical staining. Brain areas and nucleus containing PHA-L were observed under microscope. As a result, we detected that PHA-L labeled fibers and terminals were mainly found in the following: contralateral IC, ipsilateral dorsolateral orbital cortex(DLO), prelimbic cortex(PrL), accumnens nucleus core(AcbC), BLA, mediodorsal thalamic nucleus(MD), central medial thalamic nucleus(CM), ventral posterior thalamic nucleus, parvicellular part(VPPC), lateral hypothalamic area(LH), periaqueductal gray(PAG), and contralateral raphe magnus nucleus(RMg), nucleus of the solitary tract(Sol), and trigeminal caudal subnucleus(Vc).3) The neurochemical properties of IC-projecting neurons: double-immunofluorescent staining was used to identify the neurochemical properties of FG retrogradely labeled neurons in the DR, LC, and SN/VTA. Specifically, the majority of FG labeled neurons contained 5-HT(89.8%) in the DR. All the FG labeled neurons contained norepinephrine(NE) in the LC. And 91.3% FG labeled neurons contained dopamine(DA) in the SN/VTA.These results indicate that the IC could receive sensory information from medial thalamic nucleus, and thus send projections to several nociceptive-related brain regions. Moreover, many types of typical neurotransmitters may be involved in the neurotransmission modulation within the IC. 2. The morphological alterations of the IC-BLA pathway in NP rats.1) NP induced upregulation of Fos in the IC contralateral to surgery: We use spared nerve injury(SNI) to establish NP model, and thus observed that Fos expression in bilateral IC showed obviously upregulation, with a predominance contralaterally after SNI surgery, indicating that IC neurons were widely activated by NP.2) The IC-BLA pathway was activated by NP stimulation in rats: FG retraogradely labeled neurons were detected throughout the IC after FG injection into the BLA. These neurons were mainly located in the layer III or V of IC. It showed increasing trends from AIC to PIC as well as from deep to superficial laminae. To further investigate the neurotransmitters of such projection neurons, we used fluorescent in situ hybridization combined with double-immunofluorescent staining and observed that all FG labeled neurons colocalized with vesicular glutamate transporter 1(VGluT1), and showed nucleus Fos staining, indicating that NP could activate the glutamatergic IC-BLA projecting neurons.3) The synaptic plasticity changes of the IC-BLA projecting neurons in NP rats: For the purpose of figuring out the changes of IC-BLA projecting neurons in NP, we used pre-bedding immunoelectron microscopy(EM) to investigate the distance between AMPA receptors and postsynaptic active zone. Moreover, Western blot(WB) assay was used to measure the expression of GluR1 and phosphorylation of NR2B(pNR2B) within postsynaptic density district(PSD). The result showed that NP induced AMPA GluR1 receptors moving closely to the postsynaptic membranes, namely AMPA insertion. WB data suggested that GluR1 and pNR2 B expression were elevated in the PSD. These data indicated that projection neurons in the IC undergone morphological plasticity changes after NP.Consequently, NP resulted in activation of glutamatergic IC-BLA projecting neurons, GluR1 insertions into the postsynaptic membranes, and upregulations of GluR1 and pNR2 B in the PSD. Taken together, glutamatergic IC-BLA projecting neurons were activated and undergone morphological plasticity changes at molecular level after NP. 3. The electrophysiological study on the IC-BLA pathway in NP rats.We used tetramethylrhodamine-dextran(TMR) to retrogradely label the IC-BLA projecting neurons and thus patch these TMR-positive neurons in the IC of brain slices in the following electrophysiological study.1) NP significantly increased the frequency and amplitude of spontaneous excitatory postsynaptic currents(sEPSCs): in the voltage clamp, both the frequency and amplitude of sEPSCs in NP were increased compared with the Sham, indicating presynaptic release of glutamate and postsynaptic receptor responses were both elevated in IC-BLA projecting neurons after NP.2) NP significantly increased the excitability of IC-BLA projecting neurons: we determine the number of action potentials(AP) elicited by intracellular injection of 10 to 90 pA depolarizing currents for 400 ms in current clamp mode and found out that the spike numbers of the recorded neurons were significantly increased compared to sham, suggesting intrinsic excitability of IC-BLA projecting neurons were increased in NP.3) SNI significantly increased the excitatory synaptic transmissions of IC-BLA projecting neurons: AMPA input-output curve was steeper after SNI than that in the sham control, indicating that AMPA-mediated excitatory synaptic transmissions were elevated after NP. Similarly, we incubated CNQX into the perfusion fluid and observed that NMDA input-output curve was also steeper after SNI, which suggested that NMDA-mediated excitatory synaptic transmissions were also increased after NP.These data demonstrated that spontaneous discharge, intrinsic excitability, and synaptic transmissions of IC-BLA projecting neurons were elevated during NP conditions.4. The roles of IC-BLA pathway on NP and its related negative emotions.Finally, we utilized the optogenetics and chemogenetics to specifically manipulate the IC-BLA pathway in vivo in order to detect the roles of IC-BLA pathway on NP and its related negative emotions. AAV-CaMKII-ChR2-eYFP/AAV-CaMKII-eYFP viruses were injected into bilateral IC in mice, and optical fibers were implanted just above the bilateral BLA. Mechanical paw withdrawl threshold(PWT), open field test(OF), and elevated plus maze test(EPM) were performed 6 w after virus injection. In vitro brain slices, blue light stimulation could induce AP of infected neurons in the IC as well as the EPSCs in the BLA neurons. For the chemogenetics, AAV-hSyn-DIO-HM4Di-mCherry /AAV-hSyn-DIO-mCherry virus were selected and injected into the bilateral IC of CaMKII-Cre mice. CNO was microinjected into bilateral BLA via cannula implanted into the BLA previously 1 h before behavioral tests.1) Blue light stimulation induced APs and EPSC in brain slices in vitro: 5 Hz and 10 Hz blue light stimulation could induce APs of virus infected neurons in the IC by whole-cell patch-clamp recording. Furthermore, similar blue light stimulation protocol could induce EPSC in the BLA neurons. AMPA receptor antagonist CNQX effectively blocked this EPSC, which indicating EPSC induced by light stimulation were mediated by AMPA receptor in the BLA.2) Optogenetics activation of IC-BLA pathway resulted in nociception and also anxiety/depression-like negative emotions: bilateral activation IC-BLA pathway obviously reduced the PWT in AAV-CaMKII-ChR2-eYFP injection mice. Moreover, Optogenetics activation of IC-BLA pathway in AAV-CaMKII-ChR2-eYFP injection mice showed increased anxiety/depression-like behaviours revealed by decreased center time% in OF as well as reduced OA entries % and OA times % in the EPM.3) Chemogenetics inhibition of IC-BLA pathway led to antinociception and anti-anxiety/depression effects in NP mice. One hour after CNO administration, AAV-hSyn-DIO-HM4Di-mCherry(HM4Di) injection mice showed increased PWT compared with AAV-hSyn-DIO-mCherry(mCherry) control mice with NP. Moreover, chemogenetics inhibition of IC-BLA pathway in HM4 Di injection mice showed decreased anxiety/depression-like behaviours revealed by increased center time% in the OF as well as elevated OA entries % and OA times % in the EPM under NP conditions.These results suggested that activation of IC-BLA pathway could reduce PWT and produce anxiety/depression-like behaviours; on the contrary, inhibition of such pathway could alleviate NP and its related negative emotions.In conclusion, the present study demonstrated that 1) the IC is widely connceted with other regions throughout the central nervous system; 2) the IC sent direct projections to the BLA, namely IC-BLA pathway; 3) maladaptive activations were observed in the IC-BLA pathway during NP, resulted in morphological and functional plasiticity alterations of IC-BLA projecting neurons and exaggerating NP; 4) specifically inhibition of glutamatergic IC-BLA projecting neurons in vivo could alleviate NP and its related negative emotions.