The role of nitric oxide in the dorsomedial periaqueductal gray(dmPAG)column in cardiovascular responses in urethane-anesthetized male rats

Background:The dorsomedial periaqueductal gray(dmPAG)is a mesencephalic area and has numerous functions including cardiovascular regulation.Because nitric oxide(NO)is present in the dmPAG,here we investigate,the probable cardiovascular effect of NO in the dmPAG.Methods:Five groups(n=6 for each group)were used as follows:(1)control;(2)L-NAME(N^(G)-nitro-L-arginine methyl ester,a NO synthase inhibitor,90 nmol);(3)Larginine(L-Arg,a precursor for NO,60 nmol);(4)Sodium nitroprusside(SNP,a NO donor,27 nmol);and(5)L-Arg+L-NAME.The cardiovascular parameters were recorded by a Power Lab device after cannulation of the femoral artery.Drugs were injected using a stereotaxic instrument.The changes(Δ)in systolic blood pressure(SBP),mean arterial pressure(MAP),and heart rate(HR)were calculated at different times and compared to the control group.Results:Microinjection of L-NAME significantly increased ΔSBP,ΔMAP,and ΔHR more than saline(from p<0.05 to p<0.001).L-Arg only significantly increased ΔHR(p<0.05).In the L-Arg+L-NAME group,the above parameters also significantly increased(from p<0.01 to p<0.05)but not as significantly as with L-NAME alone.Microinjection of SNP significantly decreased ΔSBP and ΔMAP more than in the control and L-NAME groups(from p<0.01 to p<0.001),but ΔHR did not change significantly.Conclusion:The results indicated that NO in dmPAG has an inhibitory effect on cardiovascular responses in anesthetized rats.

Different coding characteristics between flight and freezing in dorsal periaqueductal gray of mice during exposure to innate threats

Background:Flight and freezing are two vital defensive behaviors that mice display to avoid natural enemies.When they are exposed to innate threats,visual cues are processed and transmitted by the visual system into the emotional nuclei and finally transmitted to the periaqueductal gray(PAG)to induce defensive behaviors.However,how the dorsal PAG(dPAG)encodes the two defensive behaviors is unclear.Methods:Multi-array electrodes were implanted in the dPAG nuclei of C57BL/6 mice.Two kinds of visual stimuli(looming and sweeping)were used to induce defensive behaviors in mice.Neural signals under different defense behaviors were recorded,and the encoding characteristics of the two behaviors were extracted and analyzed from spike firing and frequency oscillations.Finally,synchronization of neural activity during the defense process was analyzed.Results:The neural activity between flight and freezing behaviors showed different firing patterns,and the differences in the inter-spike interval distribution were mainly reflected in the 2–10 ms period.The frequency band activities under both defensive behaviors were concentrated in the theta band;the active frequency of flight was~8to 10 Hz,whereas that of freezing behavior was~6 to 8 Hz.The network connection density under both defense behaviors was significantly higher than the period before and after defensive behavior occurred,indicating that there was a high synchronization of neural activity during the defense process.Conclusions:The dPAG nuclei of mice have different coding features between flight and freezing behaviors;during strong looming stimulation,fast neuro-i nstinctive decision making is required while encountering weak sweeping stimulation,and computable planning late behavior is predicted in the early stage.The frequency band activities under both defensive behaviors were concentrated in the theta band.There was a high synchronization of neural activity during the defense process,which may be a key factor triggering different defensive behaviors.

Effects of electrostimulation and administration of succinylcholine on the expression of Fos protein in mesencephalic periaqueductal gray matter of rats after simulated weightlessness

BACKGROUND： Expression of Fos in neurons of periaqueductal gray （PAG） is used to reflect the excitability. However, changes of expression of Fos in neurons of PAG are caused by injured electrostimulation after simulated weightlessness, and the relationship between pretreatment and injection of succinylcholine has not been determined yet. OBJECTIVE ： To investigate the changes of expression of Fos in PAG induced by injured electrostimulation pretreatment and injection of succinylcholine at 2 weeks after simulated weightlessness.DESIGN： Observational and controlled animal study.SETTING： Department of Physiology, Medical School, Xi＇an Jiaotong University; Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education. MATERIALS： A total of 24 adult female SD rats, of clean grade and weighing 180-220 g, were selected in this study. METHODS： The experiment was completed in the Experimental Animal Center of Xi＇an Jiaotong University.① All rats were randomly divided into 2 groups according to body mass： simulated weightlessness group and control group with 12 in each group. And then, each group was also divided into 3 subgroups： electrostimulation group, succinylcholine-pretreatment group and succinylcholine-injection group with 4 in each subgroup. ②The model of weightlessness was simulated by tail-suspended female rats, which were described and modified by Cheng Jie. Rats in normal control group were given the same interventions as simulated weightlessness group except for tail-suspended. ③ Experimental method： The rats in electrostimulation group were given nociceptive stimulus by a pair of subcutaneous electrodes inserted into 1 and 5 claw of left hindlimb. The stimulus （current： 10 mA; duration： 1 ms; interval： 1 s） lasted for 30 minutes. The rats in succinylcholine-pretreatment group received stimulus after intravenous administration of succinylcholine, rats in succinylcholine-injection group were not given stimulus, just received succinylcholine. ④ All rats were perfused and fixed after 2 hours from the end of stimulation. The brains were removed, and serial frozen sections of midbrain were stained using immunocytochemical method, observed and taken photos under light-microscope. The number and morphological characters of Fos-immunoreactive （Fos-IR） neurons in ventrolateral part of PAG were investigated. MAIN OUTCOME MEASURES： The alterations in number and morphological characters of Fos-IR neurons in ventrolateral PAG of all rats.RESULTS： A total of 24 rats were involved in the final analysis. ① The morphological changes of Fos-IR neurons： The expressions of Fos in ventrolateral part of PAG were observed in both control and simulated weightlessness groups rats after being given nociceptive stimulus. As compared with control group, Fos-IR neurons in simulated weightlessness group were dyed lightly, cellular integrity was impaired, and cellular verge was unclear. ② The numbers of Fos-IR neurons： In control group, the numbers of Fos-IR neurons in ventrolateral part of PAG in simulated weightlessness group were obviously lower than succinylcholine-pretreatment group, but obviously higher than succinylcholine-injection group （46.94±3.38, 71.06±8.96 and 35.04±4.62, respectively, P 〈 0.05）. In 14-day simulated weightlessness group, the numbers of Fos-IR neurons in electrostimulation group were also obviously lower than succinylcholine-pretreatment group, and obviously higher than succinylcholine-injection group （27.77±3.27, 32.91±2.99 and 11.75±1.00, respectively, P 〈 0.05）. The numbers of Fos-IR neurons in all subgroups in control group were obviously higher than those subgroups in simulated weightlessness group. Compared with electrostimulation group, the percentage of expression of Fos in ventrolateral part of PAG responsed to nociceptive stimulus after administration of succinylcholine （SCH） was increased to 51.83% in control group and 18.51% in simulated weightlessness group.CONCLUSION ：① The expression of Fos in neurons in ventrolateral part of PAG were increased by the pretreatment of SCH before nociceptive stimulus.② Nociceptive stimulus could increase the expression of Fos in neurons in ventrolateral part of PAG. ③ The numbers of Fos-IR neurons in ventrolateral part of PAG were decreased obviously after 2-week simulated weightlessness.

Movement Trend Alterations in the Periaqueductal Gray(PAG)-Employed Ratbot Navigation Are Correlated with Stimulation Parameters

In previous studies,Periaqueductal Gray(PAG)stimulation was used to stop ratbots from moving.Due to the homology between the PAG and the intercollicular nucleus,which has been used for forward movement in birds,we investigated the possibility of PAG application to induce forward locomotion for the first time.Using a corridor maze,the traveled distances via PAG electrical stimulation were examined in nine Wistar male rats during three sessions.A custom-designed stimulator was developed to apply the stimulation.The results showed reductions in responses to stimulation over time.Accordingly,the traveled distances had negative slopes during the consecutive trials(in 8 out of the 9 rats),and the slope mean was significantly different from zero.There was a strong correlation between the stimulation parameters(electric Charge per Phase(CPP)and the Number of Pulses(NP))and the observed slopes.The negative Movement Slopes(MS)were highly correlated with the CPP and the NP,as the Pearson's linear correlation coefficients were−0.87 and−0.79,respectively.The MS-CPP coefficients of determination(R-squared)were also between 0.76 and 0.95.In addition,the MS-NP coefficients of determination were between 0.63 and 0.87.Thus,it is concluded that the electrical stimulation parameters influence the behavioral outcomes directly.Furthermore,the PAG area may be considered a suitable candidate for forward locomotion control in the future if the area is harnessed effectively to prevent undesirable chaotic behaviors.

Switching from morphine to fentanyl attenuates the decline of μ-opioid receptor expression in periaqueductal gray of rats with morphine tolerance

Background Opioid switching is a therapeutic maneuver to improve analgesic response and/or reduce adverse side effects although the underlying mechanisms remain unknown.The μ-opioid receptor （MOR） has an important role in mediating the actions of morphine and other analgesic agents.This study is aimed at exploring the changes of MOR in the periaqueductal gray （PAG） in rats when morphine is substituted for equianalgesic fentanyl.Methods Forty rats were randomly assigned to five treatment groups：7 days normal saline group （N group）,7 days fentanyl group （F group）,7 days morphine group （M group）,7 days morphine and 7 days fentanyl-switching group （MF group）,and 14 days morphine group （MM group）.Rats repeatedly received subcutaneous injections of morphine sulfate （10 mg/kg） or equianalgesic fentanyl sulfate （0.1 mg/kg） twice daily.Rats＇ antinociceptive response to thermal pain was evaluated by the tail flick latency assay.MOR mRNA and protein expression in the PAG were measured using RT-PCR and Western blotting analyses respectively.Results This study showed that after morphine was substituted with fentanyl on day 8,the tail flick latency （TFL） increased from （3.9±0.4） seconds to （11.4±0.4） seconds.The results also demonstrated that both MOR mRNA and protein expression in the PAG of rats in the MF group were less than that in the M group （P〈0.05） but more than that in MM group （P〈0.05）.Conclusions Equianalgesic fentanyl was still antinociceptive effective in rats with morphine tolerance,which may be due to the switching from morphine to fentanyl attenuating the decline of MOR expression in the PAG of rats.

THE INTERACTION BETWEEN PERIAQUEDUCTAL GRAY AND NUCLEUS ACCUMBENS IN ACCELER-ATING THE RELEASE OF ENKEPHALINS AND β-ENDORPHIN

There is a growing body of evidence showing that periaqueductal gray (PAG) and nucleus accumbens (N. accumbens) play important roles in the modulation of nociception. In our previous study of the mechanisms of morphine analgesia we found that opioid antagonist naloxone injected into either of the two nuclei, PAG or N. accumbens, could

The Periaqueductal Gray and Its Extended Participation in Drug Addiction Phenomena

The periaqueductal gray(PAG)is a complex mesencephalic structure involved in the integration and execution of active and passive self-protective behaviors against imminent threats,such as immobility or flight from a predator.PAG activity is also associated with the integration of responses against physical discomfort(e.g.,anxiety,fear,pain,and disgust)which occurs prior an imminent attack,but also during withdrawal from drugs such as morphine and cocaine.The PAG sends and receives projections to and from other well-documented nuclei linked to the phenomenon of drug addiction including:(i)the ventral tegmental area;(ii)extended amygdala;(iii)medial prefrontal cortex;(iv)pontine nucleus;(v)bed nucleus of the stria terminalis;and(vi)hypothalamus.Preclinical models have suggested that the PAG contributes to the modulation of anxiety,fear,and nociception(all of which may produce physical discomfort)linked with chronic exposure to drugs of abuse.Withdrawal produced by the major pharmacological classes of drugs of abuse is mediated through actions that include participation of the PAG.In support of this,there is evidence of functional,pharmacological,molecular.And/or genetic alterations in the PAG during the impulsive/compulsive intake or withdrawal from a drug.Due to its small size,it is difficult to assess the anatomical participation of the PAG when using classical neuroimaging techniques,so its phys-iopathology in drug addiction has been underestimated and poorly documented.In this theoretical review,we discuss the involvement of the PAG in drug addiction mainly via its role as an integrator of responses to the physical discomfort associated with drug withdrawal.

Analgesic target:NMDA NR2B receptors in the rodent midbrain periaqueductal grey

Hyperoside (Hyp) is one of the flavonoids compound isolated from Balbisia calycina (Gris.). It exhibits anti-inflammation and analgesic activities in the mice. The

A short-chain α-neurotoxin from Naja naja atra produces potent cholinergic-dependent analgesia

Objective To investigate the analgesia induced by cobrotoxin （CT） from venom of Naja naja atra, and the effects of atropine and naloxone on the antinociceptive activity of CT in rodent pain models. Methods CT was administered intraperitoneally （33.3, 50, 75 μg/kg）, intra-cerebral venticularly （2.4 μg/kg） or microinjected into periaqueductal gray （PAG, 1.2 μg/kg）. The antinociceptive action was tested using the hot-plate test and the acetic acid writhing test in mice and rats. The involvement of cholinergic system and the opioid system in CT-induced analgesia was examined by pretreatment of animals with atropine （0.5 mg/kg, im or 10 mg/kg, ip） or naloxone （3 mg/kg, ip）. The effect of CT on motor activity was tested using the Animex test. Results CT （33.3, 50 and 75 μg/kg, ip） exhibited a dosedependent analgesic action in mice as determined with hot-plate test and acetic acid writhing test. In the mouse acetic acid writhing test, the intra-cerebral ventricle administration of CT 2.4 μg/kg （1/23th of a systemic dose） produced marked analgesic effects. Microinjection of CT 1.2 μg/kg （1/46th of systemic dose） into the PAG also elicited a robust analgesic action in the hot-plate test in rats. Atropine at 0.5 mg/kg （ira） or naloxone at 3 mg/kg （ip） failed to block the analgesic effects of CT, but atropine at 10 mg/kg （ip） did antagonize the analgesia mediated by CT in the mouse acetic acid writhing test. At the highest effective dose of antinociception （75 μg/kg）, CT did not change the spontaneous mobility of mice. Conclusion These results suggest that CT from Naja naja atra venom has analgesic effects. Central nervous system may be involved in CT＇ analgesic effects and the PAG may be the primary central site where CT exerts its effects. The central cholinergic system but not opioid system appears to be involved in the antinociceptive action of CT.

Role of 5-hydroxytryptamine type 3 receptors in the regulation of anxiety reactions

5-Hydroxytryptamine(5-HT)type 3 receptor(5-HT_(3)R)is the only type of ligand-gated ion channel in the 5-HT receptor family.Through the high permeability of Na+,K+,and Ca2+and activation of subsequent voltage-gated calcium channels(VGCCs),5-HT_(3)R induces a rapid increase of neuronal excitability or the release of neurotransmitters from axon terminals in the central nervous system(CNS).5-HT_(3)Rs are widely expressed in the medial prefrontal cortex(mPFC),amygdala(AMYG),hippocampus(HIP),periaqueductal gray(PAG),and other brain regions closely associated with anxiety reactions.They have a bidirectional regulatory effect on anxiety reactions by acting on different types of cells in different brain regions.5-HT_(3)Rs mediate the activation of the cholecystokinin(CCK)system in the AMYG,and theγ-aminobutyric acid(GABA)“disinhibition”mechanism in the prelimbic area of the mPFC promotes anxiety by the activation of GABAergic intermediate inhibitory neurons(IINs).In contrast,a 5-HT_(3)R-induced GABA“disinhibition”mechanism in the infralimbic area of the mPFC and the ventral HIP produces anxiolytic effects.5-HT_(2)R-mediated regulation of anxiety reactions are also activated by 5-HT_(3)R-activated 5-HT release in the HIP and PAG.This provides a theoretical basis for the treatment of anxiety disorders or the production of anxiolytic drugs by targeting 5-HT_(3)Rs.However,given the circuit specific modulation of 5-HT_(3)Rs on emotion,systemic use of 5-HT_(3)R agonism or antagonism alone seems unlikely to remedy anxiety,which deeply hinders the current clinical application of 5-HT_(3)R drugs.Therefore,the exploitation of circuit targeting methods or a combined drug strategy might be a useful developmental approach in the future.

Preoperative Acute Sleep Deprivation Causes Postoperative Pain Hypersensitivity and Abnormal Cerebral Function

Preoperative sleep loss can amplify post-operative mechanical hyperalgesia.However,the underlying mechanisms are still largely unknown.In the current study,rats were randomly allocated to a control group and an acute sleep deprivation(ASD)group which experienced 6 h ASD before surgery.Then the variations in cerebral function and activity were investigated with multi-modal techniques,such as nuclear magnetic resonance,functional magnetic resonance imaging,c-Fos immunofluorescence,and electrophysiology.The results indicated that ASD induced hyperalgesia,and the metabolic kinetics were remarkably decreased in the striatum and midbrain.The functional connectivity(FC)between the nucleus accumbens(NAc,a subregion of the ventral striatum)and the ventrolateral periaqueductal gray(vLPAG)was significantly reduced,and the c-Fos expression in the NAc and the vLPAG was suppressed.Furthermore,the electrophysiological recordings demonstrated that both the neuronal activity in the NAc and the vLPAG,and the coherence of the NAc-vLPAG were suppressed in both resting and task states.This study showed that neuronal activity in the NAc and the vLPAG were weakened and the FC between the NAc and the vLPAG was also suppressed in rats with ASD-induced hyperalgesia.This study highlights the importance of preoperative sleep management for surgical patients.

Pharmacologically inhibiting GluR2 internalization alleviates neuropathic pain

Neuropathic pain is of serious clinical concern and only about half of patients achieve partial relief with currently-available treatments,so it is critical to find new drugs for this condition.Recently,the cellsurface trafficking of pain-related receptors has been suggested as an important mechanism underlying persistent neuropathic pain.Here,we used the short peptide GluA_（2-3y）,which specifically inhibits the GluA2-dependent endocytosis of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors,and tested its anti-nociceptive effect in the periaqueductal grey（PAG） of intact rats and rats with neuropathic pain.Intra-PAG injection of 0.15,1.5,7.5,and 15 pmol of GluA_（2-3y） induced dose-dependent increases in hindpaw withdrawal latencies to noxious thermal and mechanical stimuli in intact rats,suggesting that GluA2 cell-surface trafficking in the PAG is involved in pain modulation.Furthermore,GluA_（2-3y） had much stronger anti-nociceptive effects in rats with neuropathic pain induced by sciatic nerve ligation.Interestingly,the intra-PAG injection of 15 pmol GluA_（2-3y） had an analgesic effect similar to 10 ug（35nmol） morphine in rats with neuropathic pain.Taken together,our results suggested that GluA2 trafficking in the PAG plays a critical role in pain modulation,and inhibiting GluA2 endocytosis with GluA_（2-3y） has potent analgesic effects in rats with neuropathic pain.These findings strongly support the recent hypothesis that targeting receptor trafficking could be a new strategy for the treatment of neuropathic pain.

PET imaging on neurofunctional changes after optogenetic stimulation in a rat model of panic disorder

Panic disorder (PD) is an acute paroxysmal anxiety disorder with poorly understood pathophysiology. The dorsal periaqueductal gray (dPAG) is involved in the genesis of PD. However, the downstream neurofunctional changes of the dPAG during panic attacks have yet to be evaluated in vivo. In this study, optogenetic stimulation to the dPAG was performed to induce panic-like behaviors, and in vivo positron emission tomography (PET) imaging with ,8F-flurodeoxyglucose (18F-FDG) was conducted to evaluate neurofunctional changes before and after the optogenetic stimulation. Compared with the baseline, post-optogenetic stimulation PET imaging demonstrated that the glucose metabolism significantly increased (P < 0.001) in dPAG, the cuneiform nucleus, the cerebellar lobule, the cingulate cortex, the alveus of the hippocampus, the primary visual cortex, the septohypothalamic nucleus, and the retrosplenial granular cortex but significantly decreased (P < 0.001) in the basal ganglia, the frontal cortex, the forceps minor corpus callosum, the primary somatosensory cortex, the primary motor cortex, the secondary visual cortex, and the dorsal lateral geniculate nucleus. Taken together, these data indicated that in vivo PET imaging can successfully detect downstream neurofunctional changes involved in the panic attacks after optogenetic stimulation to the dPAG.

Different genes involved in the amelioration of chronic neuropathic pain and comorbid anxiety by electroacupuncture with different intensities

Background:With its complex and prevalent symptoms,chronic neuropathic pain(CNP)is frequently combined with negative emotions.Electroacupuncture(EA)is a widely-used and effective treatment for CNP and pain-induced comorbid anxiety-like behaviors(PABs).Although different effects may result from EA with different intensities,the underlying mechanisms remain unclear.Objective:The study aimed to investigate the effects of EA with different intensities on CNP and PABs and the underlying mechanisms,by exploring the different genes through RNASeq in the spinal cord dorsal horn(SCDH)and periaqueductal gray(PAG).Methods:A spared nerve injury(SNI)model was established using male C57BL/6 J mice,and treated with EA of different intensities(0.1 mA or 0.3 mA,both at 100 Hz).The von-Frey tests,open field tests and elevated plus maze tests were used to measure the mechanical paw withdrawal threshold(PWT)and PABs of the mice.The tissues of SCDH and PAG regions were extracted to identify the changes in gene expression,and biofunctional analysis of differentially expressed genes(DEGs)was further performed by RNA-seq analysis.The expression of key DEGs and corresponding proteins was assessed using quantitative real-time PCR(qPCR)and protein immunofluorescence detection,respectively.Results:Both 0.1 mA and 0.3 mA EA at 100 Hz alleviated PABs compared with the SNI group.Furthermore,0.3 mA EA was effective in increasing PWT in SNI mice,whereas 0.1 mA EA was not.A total of 108 DEGs was identified in the SCDH,and 254 DEGs were identified in the PAG.Specifically,the expression of Tnr in SNI mice decreased after the treatment with 0.3 mA EA at 100 Hz,and the expression of Ptprb,Net1,Abcb1a and Adgrf5 in SNI mice decreased in the PAG after the treatment with 0.1 mA EA at 100 Hz.Conclusion:The effect of 0.3 mA EA on chronic pain may be related to the decrease of Tnr expression in SCDH,and the effect of 0.1 mA EA on PABs may be achieved by decreased expression of Ptprb and Net1 in PAG.