This study was designed to investigate the modulation of the Corydalis tuber on glycine-activated ion current in rat periaqueductal gray (PAG) neurons. Aqueous extract from Corydalis tuber has been widely used for pain control such as dysmenorrhea, irregular menstruation or amenorrhea with abdominal cramping, neuralgia, headache and gastrointestinal spasm. The PAG region of the brain is known to be involved heavily with nociception. Modulation of the Corydalis tuber on glycine-induced ion current in rat periaqueductal gray (PAG) neurons was studied by a nystatin-perforated patch-clamp technique. High concentrations of Corydalis tuber elicited ion current, which was suppressed by strychnine application. Low concentrations of Corydalis tuber reduced glycine-induced ion currents in the PAG neurons. Inhibitory action of Corydalis tuber on glycine-activated ion current was reduced by treatment with naltrexone, a non- selective opioid antagonist. Application of N-methylmalemide (NEM), a sulfhydryl alkylating agent, also reduced the inhibitory action of Corydalis tuber on glycine-activated ion current in the PAG neurons. These results suggest that the inhibitory effect of Corydalis tuber on glycine-activated ion current in the PAG neurons is one of the analgesic mechanisms of the Corydalis tuber, which may activate descending pain control system in PAG neurons.
Background : Amygdalin is abundant in Armeniacae semen, and it is recently reported to treat cancers and relieve pain. But modus operandi of amygdalin at the level of neuron has not been reported, yet. Objective : This study aimed to find out the effect of amygdalin on glycine- and glutamate-induced ion currents in periaqueductal gray (PAG) neurons. And it was investigated that amygdalin participates in the regulation of the descending pain control system in the level of PAG neurons. Method : We investigated that the changes of glycine- and glutamate-induced ion currents in PAG neurons through application of lipopolysaccharides (LPS) and application of amygdalin with LPS by using the nystatin-perforated patch clamp method. Result : Application of LPS on PAG neurons resulted in increased glycine-induced ion current, and in decreased glutamate-induced ion current. In contrast, application of amygdalin with LPS resulted in decreased glycine-induced ion current increased by LPS, and increased glutamate-induced ion current decreased by LPS. Conclusion : Amygdalin from Armeniacae semen controls glycine- and glutamate-induced ion current by LPS in PAG neurons, and it is suggested that amygdalin participates in the regulation of the descending pain control system in the level of PAG neurons.
Immunohistochemical technique of the c-fos primary gene protein, Fos, was used to analyze the effects of external factors on the neuronal activities in the periaqueductal gray(PAG) of the intact rats, sham-operated rats and post-operated stress control rats. In addition, the number of Fos positive neurons has been evaluated to verify the effects of cannula implantation and veratridine treatment on the neuronal activities in PAG area. The results were summerized as follow : 1. There was no significant difference in the number of Fos positive neurons observed in the caudal and middle portion of lateroventral PAG from cannula implanted rats and sham operated rats. 2. The number of Fos positive neurons in the PAG was not changed by the stress induced by connection of collecting tube to rats for 12 hours as compared to that of intact rats. 3. In the saline treated group, the Fos immunoreactivity in the PAG did not changed at 30 minutes and 1 hour after saline treatment as compared to that of intact rats. However, the number of Fos positive neurons was significantly increased at 2 hours after treatment compared to that of saline treated rats at 30 minutes after treatment. 4. The Fos immunoreactivity was dramatically increased at 30 minutes, 1 hour and 2 hours after veratridine treatment as compared to those of saline treated groups. The number of Fos immunoreative neurons showed the maximal level at 2 hours after veratridine treatment. 5. The Fos positive neurons induced by saline and veratridine treatment were mainly distributed in front of the microdialysis window. These results suggest that new microdialysis demonstrated in this study improves efficiency and accuracy to confine the neuronal activity in front of microdialysis window site. Moreover, this directional specificity allows us to locate probe tips adjacent to the brain area of the interest site rather than centering the probes within that brain area. Finally, This microdialysis method can be used to dialyse the neurotransmitters using concious and freely moving rats.
Peripheral nerve injuries are a commonly encountered clinical problem and often result in a chronic pain and severe functional deficits. The expression of c-Fos is sometimes used as a marker of increased neuronal activity. We have prepared the aqueous extract of amygdalin from Armeniacae semen for pain control. In the present study, we investigated the effects of amygdalin on the recovery rate of the locomotor function and on the expression of c-Fos in the ventrolateral periaqueductal gray (vlPAG) region following sciatic crushed nerve injury in rats. Walking track analysis for the evaluation of functional recovery and immunohistochemistry for the c-Fos expression were used in this study. In the present results, characteristic gait change with dropping of the sciatic function index (SFI) was observed and c-Fos expression in the vlPAG was suppressed following sciatic crushed nerve injury in rats. Amygdalin enhanced SFI value and restored c-Fos expression in the vlPAG to the control value. The present our study indicated that amygdalin activates neurons in the vlPAG, and it facilitates functional recovery following peripheral nerve injury.
Kim, Sung-Tae;Sung, Yun-Hee;Kim, Chang-Ju;Joo, Kwan-Joong;Han, Seung-Ho;Lee, Choong-Yeol;Kim, Youn-Sub
Journal of Physiology & Pathology in Korean Medicine
/
v.20
no.6
/
pp.1672-1677
/
2006
Sarcodon aspratus is the mushroom of Telephoracea which was been classified into Alphllophorales. The aqueous extract of Sarcodon aspratus in known to have anti-tumor activity, immune modulatory effect, and anti-oxidative action. The descending pain control system consists of three major components: the periaqueductal gray (PAG) of the midbrain, the rostroventral medulla including the nucleus raphe magnus, and the spinal dorsal horn. Glutamate is the primary excitatory neurotransmitter in the brain. Glutamate ionotropic receptors are classified as N-methyl-D-aspartate (NMDA) receptor, ${\alpha}$-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor, and kainate receptor. In the present study, the modulation of Sarcodon aspratus on the ion currents activated by glutamate, NMDA, AMPA, and kainate in the acutely dissociated PAG neurons was investigated by nystatin-perforated patch-clamp technique under boltage-clamp condition. Sarcodon aspratus increased glutamate- and NMDA-induced ion currents were not increased by Sarcodon aspratus. The present results show that Sarcodon aspratus may activate the descending pain control system in rat PAG neurons through NMDA receptor.
Background: The present study was conducted to investigate effects of microinjection of bicuculline, GABA-A receptor antagonist, into the brain stem nuclei on the dorsal horn neuron responsiveness in rats with an experimental peripheral neuropathy. Methods: An experimental neuropathy was induced by a unilateral ligation of L5~L6 spinal nerves of rats. After 2~3 weeks after the surgery, single-unit recording was made from wide dynamic range (WDR) neurons in the spinal cord dorsal horn. Results: Responses of WDR neurons to both noxious and innocuous mechanical stimuli applied to the somatic receptive fields were enhanced on the nerve injured side. These enhanced responsiveness of WDR neurons were suppressed by microinjection of bicuculline into periaqueductal gray(PAG) or nucleus reticularis gigantocellularis(Gi). A similar suppression was also observed when morphine was microinjected into PAG or Gi. Suppressive action by Gi-bicuculline was reversed by naloxonazine, ${\mu}$-opioid receptor antagonist, microinjected into PAG whereas PAG-bicuculline induced suppression was not affected by naloxonazine injection into Gi. Gi-bicuculline induced suppression were reversed by a transection of dorsolateral funiculus(DLF) of the spinal cord. Conclusions: The results suggest that endogenous opioids, via acting on GABAergic interneurons in PAG and Gi, may be involved in the control of neuropathic pain by activating the descending inhibitory pathways that project to the spinal dorsal horn through DLF to inhibit the responsiveness of WDR neurons.
Background : Acupuncture has been used as a clinical treatment in Oriental medicine for various diseases including pain relief. The descending pain control system of periaqueductal gray (PAG) is a powerful pain control system in mammalians. Expression of c-Fos is used as a marker for stimuli-induced metabolic changes of neurons. Objective : In the present study, the effects of acupuncture on analgesic effect in visceral pain were investigated through the writhing reflex and c-Fos expression in ventrolateral PAG (vlPAG) area using immunohistochemistry in mice. Method : For the writhing test, mice were divided into five groups. Immediately after finishing the behavioral test, the animals were weighed and overdosed with Zoletil. After a complete lack of response was observed, the brains of the mice were dissected into serial coronal sections, and c-Fos immunohistochemistry was performed. Statistical analysis of all data was performed using one-way ANOVA. Result : The present results showed that acupuncture affected the writhing reflex and that Choksamni (zusnali) acupoint and aspirin significantlysuppressed acetic acid treatment-induced increased writhing reflex, and the expression of c-Fos in vlPAG was significantly increased in the acupunctured group. Conclusion : The present study suggests that acupuncture has an antinociceptive effect on acetic acid-induced visceral pain by increase of c-Fos expression in mice. Aspirin also showed analgesic effect, however the mechanism is different from the acupuncture.
The aim of the present study is to examine the brainstem sites where the electrical stimulation produces a suppression of dorsal horn neuron responses of neuropathic rats. An experimental neuropathy was induced by a unilateral ligation of L5-L6 spinal nerves of rats. Ten to 15 days after surgery, the spinal cord was exposed and single-unit recording was made on wide dynamic range (WDR) neurons in the dorsal horn. Neuronal responses to mechanical stimuli applied to somatic receptive fields were examined to see if they were modulated by electrical stimulation of various brainstem sites. Electrical stimulation of periaqueductal gray (PAG), n. raphe magnus (RMg) or n. reticularis gigantocellularis (Gi) significantly suppressed responses of WDR neurons -to both noxious and non-noxious stimuli. Electrical stimulation of other brainstem areas, such as locus coeruleus. (LC) and n. reticularis paragigantocellularis lateralis (LPGi), produced little or no suppression. Microinjection of morphine into PAG, RMg, or Gi also produced a suppression as similar pattern to the case of electrical stimulation, whereas morphine injection into LC or LPGi exerted no effects. The results suggest that PAG, NRM and Gi are the principle brainstem nuclei involved in the descending inhibitory systems responsible for the control of neuropathic pain. These systems are likely activated by endogenous opioids and exert their inhibitory effect by acting on WDR neurons in the spinal cord.
Purpose : It had been suggested that pain arising from deep somatic body regions influences neural activity within periaqueductal gray(PAG) of midbrain via distinct spinal pathways. Aspirin is one of the popular non-steroidal anti-inflammatory drugs used in the management of pain. Fos expression was used as a marker for neuronal activity throughout central neurons following painful peripheral stimulation. This study was prepared to investigate changes of c-Fos immunoreactivity in midbrain by deep pain and effects of aspirin. Methods : Male Sprague-Dawley rats were injected with 0.1 mL of 5% formalin in the plantar muscle of the right hindpaw. For experimental group II, aspirin was injected intravenously before injection of formalin. An aspirin-untreated group was utilized as group I. Rats were sacrificed at 0.5, 1, 2, 6 and 24 hours after formalin injection. Rat's brains were removed and sliced in rat brain matrix. Brain slices were coronally sectioned at interaural 1.00-1.36 mm. Serial sections were immunohistochemically reacted with polyclonal c-Fos antibody. The numbers of c-Fos protein immunoreactive neurons in ventrolateral periaqueductal gray(VLPAG) and dorsomedial periaqueductal gray(DMPAG) were counted and analyzed statistically with Mann-Whitney U tests. Results : Higher numbers of c-Fos protein immunoreactive neurons were found in VLPAG. In both VLPAG and DMPAG of formalin-treated group, the numbers of c-Fos protein immunoreactive neurons were significantly higher at all time points than the formalin-untreated group, which peaked at two hours. The numbers of c-Fos immunoreactive neuron of the aspirin-treated group were less compared to the aspirin-untreated group at each time point. Conclusion : These results provide some basic knowledge in understanding the mechanism of formalin-induced deep somatic pain and the effects of aspirin.
In the present study, we designed and constructed new microdialysis probe in order to improve the efficacy and accuracy of microdialysis method. In addition, extracellular concentrations of GABA, glutamate, aspartate and glycine were monitored with new designed probe in the lateral portion of the ventrocaudal periaqueductal gray using unanesthetized and unrestrained rats. Furthermore, the effect of opiates on release of these amino acids, especially GABA, was analyzed by measuring their concentration in PAG dialysates following veratridine administration in the presence of systemic morphine. The results were summerized as follow : 1. The damaging rates of 1.0mm or 1.5mm window probe were 12.5% or 42.8%, respectively. In the group using 1.5mm window probe, the damaging area was extended into mesencephalic aqueduct because of microdialyzing pressure. 2. Because of the unique design of our probes with an opening facing one side, dialysis occurs in a hemisphere($600{\mu}m$ in mediolateral direction and $100{\mu}m$ in opposite side of the dialysis probe) around the opening rather than in a spherical shaped configuration which is typical of most commercially available probe designs. 3. Glutamate, taurine and glycine were present in the highest concentration in the dialysate sample obtained before treatment with veratridine, whereas, aspartate and GABA were present in the lowest concentration. 4. The concentration of all 5 amino acids increased significantly following $75{\mu}m$ veratridine perfusion into lateral ventrocaudal PAG. 5. There was no significant difference between basal and peak amino acid concentrations according to window sizes. 6. Morphine had no effect on baseline concentrations of amino acids in dialysates obtained from the lateral PAG as compared to saline treated controls. However, following veratridine treatment, morphine selectively affected GABA release in the lateral ventrocaudal PAG as compared to saline treated controls. These results suggest that GABAergic interneurons in the PAG are inhibited by opioids. Therefore, endogenous enkephalins or endorphins may directly inhibit intrinsic GABAergic intemeurons and block their tonic inhibition of PAG-NMR projection neurons. Moreover, new designed probes demonstrate improved efficiency and accuracy in collecting samples as compared to commercial types of microdialysis probes.
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