Tetanus-induced asynchronous GABA release in cultured hippocampal neurons

Asynchronous GABA release was studied in cultured hippocampal neurons using paired whole-cell recordings. Tetanization of the presynaptic GABAergic neuron was accompanied by a train of IPSCs which showed tetanic depression. Asynchronous IPSCs (asIPSCs) also developed during the train and continued for 1.85±0.3 s after the stimulation. The threshold frequency for evoking asIPSCs was 10 Hz, while maximal asynchronous activity was achieved at 40 Hz. Perfusion with EGTA-AM blocked asIPSCs. The elevation of [Ca²⁺]_i that accompanies presynaptic action potential firing triggers asynchronous release of GABA vesicles, thereby counteracting tetanic depression of synchronous IPSCs.     

Suppression of morphine withdrawal by electroacupuncture in rats: dynorphin and kappa-opioid receptor implicated

Our previous work has demonstrated that 100-Hz electroacupuncture (EA) or 100-Hz transcutaneous electrical nerve stimulation (TENS) was very effective in ameliorating the morphine withdrawal syndrome in rats and humans. The mechanism was obscure. (1) Rats were made dependent on morphine by repeated morphine injections (5-140 mg/kg, s.c., twice a day) for eight days. They were then given 100-Hz EA for 30 min 24 h after the last injection of morphine. A marked increase in tail flick latency (TFL) was observed. This effect of 100-Hz EA could be blocked by naloxone (NX) at 20 mg/kg, but not at 1 mg/kg, suggesting that 100-Hz EA-induced analgesia observed in morphine-dependent rats is mediated by kappa-opioid receptors. (2) A significant decrease of the concentration of dynorphin A (1-17) immunoreactivity (-ir) was observed in the spinal perfusate in morphine-dependent rats, that could be brought back to normal level by 100-Hz EA. (3) 100-Hz EA was very effective in suppressing NX-precipitated morphine withdrawal syndrome. This effect of EA could be prevented by intrathecal administration of nor-BNI (2.5 micrograms/20 microliters), a kappa-opioid receptor antagonist, or dynorphin A (1-13) antibodies (25 micrograms/20 microliters) administered 10 min prior to EA. In conclusion, while the steady-state spinal dynorphin release is low in morphine-dependent rats, it can be activated by 100-Hz EA stimulation, which may be responsible for eliciting an analgesic effect and ameliorating morphine withdrawal syndrome, most probably via interacting with kappa-opioid receptor at spinal level.     

Effects of antibodies to dynorphin A and beta-endorphin on lateral hypothalamic self-stimulation in ad libitum fed and food-deprived rats

Many laboratories have reported that systemically administered naloxone has little or no effect on lateral hypothalamic self-stimulation (LH ICSS). In the present study, lateral ventricular infusion of beta-endorphin antiserum and a high dose of naloxone (100 micrograms) produced small but significant increases in stimulation frequency threshold for LH ICSS. beta-Endorphin activity, mediated by a non-mu (e.g. delta or epsilon) receptor, may therefore be involved in the reinforcement of self-stimulation behavior. When rats are deprived of food for 24 h, LH ICSS thresholds decline. Under this condition, systemic naloxone elevates the LH ICSS threshold, often returning it to the pre-deprivation level. In the present study, lateral ventricular infusion of dynorphin A(1-13) antiserum similarly reversed the threshold-lowering effect of food deprivation. The effects of systemic naloxone and intraventricular dynorphin A antiserum on LH ICSS, which are specific to food-deprived animals, may be related to previous findings that these two treatments elevate LH stimulation threshold for eliciting feeding behavior. Results of the ICSS and stimulation-induced feeding studies suggest a model for the mediation of incentive stimuli by dynorphin A activity that is afferent to LH 'reward' neurons and positively gated by 'hunger'. An hypothesized role for 'hunger'-gated dynorphin A release in potentiating the hedonic response to alimentary stimuli and drugs of abuse is discussed.     

Capsaicin inhibits the in vitro binding of peptides selective for mu- and kappa-opioid, and nociceptin-receptors

Capsaicin inhibited the equilibrium specific binding of endogenous opioid-like peptide ligands such as endomorphin-1, nociceptin, and dynorphin((1-17)) in rat brain membrane preparations. We studied the in vitro effect of capsaicin (1-10 microM) on homologous and heterologous competitive binding of opioid ligands, using unlabeled synthetic peptides and the following tritiated compounds: [(3)H]endomorphin-1, [(3)H]endomorphin-2, [(3)H]nociceptin((1-17)) and [(3)H]dynorphin((1-17)). Capsaicin-dependent inhibition was also observed in [(35)S]GTPgammaS stimulation assays in the presence of certain opioid peptides. The inhibition of opioid binding was further investigated using other synthetic and natural mu-opioid ligands such as [D-Ala(2),(NMe)Phe(4),Gly(5)-ol]enkephalin (DAMGO), morphine and naloxone. The decrease in opioid ligand affinity upon capsaicin treatments was most apparent with endomorphin-1, followed by nociceptin and dynorphin. The binding of other investigated opioids were not affected in the presence of capsaicin. In [(3)H]endomorphin-1 binding assays, capsazepine antagonized the inhibitory effect of capsaicin in rat brain membranes suggesting the involvement of TRPV1 receptors. In Chinese hamster ovary (CHO) cells stably expressing mu-opioid receptors, but lacking vanilloid receptors, the inhibition by capsaicin on the binding of [(3)H]endomorphin-1 was not present. It is concluded that the inhibitory effect of capsaicin on the receptor binding affinity of endogenous opioid peptides in brain membrane preparations seems not to be a direct effect, it is rather a negative feedback interaction with opioid receptors.     

Suppression of morphine withdrawal by electroacupuncture in rats: dynorphin and κ-opioid receptor implicated

Our previous work has demonstrated that 100-Hz electroacupuncture (EA) or 100-Hz transcutaneous electrical nerve stimulation (TENS) was very effective in ameliorating the morphine withdrawal syndrome in rats and humans. The mechanism was obscure. (1) Rats were made dependent on morphine by repeated morphine injections (5–140 mg/kg, s.c., twice a day) for eight days. They were then given 100-Hz EA for 30 min 24 h after the last injection of morphine. A marked increase in tail flick latency (TFL) was observed. This effect of 100-Hz EA could be blocked by naloxone (NX) at 20 mg/kg, but not at 1 mg/kg, suggesting that 100-Hz EA-induced analgesia observed in morphine-dependent rats is mediated by κ-opioid receptors. (2) A significant decrease of the concentration of dynorphin A (1–17) immunoreactivity (-ir) was observed in the spinal perfusate in morphine-dependent rats, that could be brought back to normal level by 100-Hz EA. (3) 100-Hz EA was very effective in suppressing NX-precipitated morphine withdrawal syndrome. This effect of EA could be prevented by intrathecal administration of nor-BNI (2.5 μg/20 μl), a κ-opioid receptor antagonist, or dynorphin A (1–13) antibodies (25 μg/20 μl) administered 10 min prior to EA. In conclusion, while the steady-state spinal dynorphin release is low in morphine-dependent rats, it can be activated by 100-Hz EA stimulation, which may be responsible for eliciting an analgesic effect and ameliorating morphine withdrawal syndrome, most probably via interacting with κ-opioid receptor at spinal level.     

Calcium currents in rat motor nerve terminals.

Ca2+ currents in response to an action potential were recorded extracellularly under non-voltage clamped conditions from rat motor nerve terminals. The Ca2+ current was blocked by Cd2+, Co2+, and Ni2+. A residual component that could not be blocked by inorganic cations was inhibited completely by tetrodotoxin (TTX). The Ca2+ current was also moderately sensitive to the N- and L-type Ca2+ channel-blocker omega-conotoxin but was insensitive to the L-type channel-specific dihydropyridines. When a fraction of the terminal K+ currents was blocked by 10 mM tetraethylammonium (TEA), the Ca2+ current duration decreased only slightly as stimulation frequency increased from 0.5 to 20 Hz. When K+ currents were blocked by TEA plus 3,4-diaminopyridine (250 microM) though, the Ca2+ current duration decreased from greater than 70 ms to 8-10 ms as stimulation frequency increased from 0.5 to 20 Hz. Recovery of the duration following 20-Hz stimulation occurred faster during subsequent stimulation at 0.5 Hz than at 2 Hz. ATP and ACh inhibit Ca2+ currents at stimulation frequencies ranging from 0.5 to 20 Hz; however, when the purinergic and cholinergic autoreceptors are blocked by theophylline (100 microM) and pirenzepine (3 microM), respectively, the frequency-induced decrease in current duration persisted. Thus, motor nerve terminal Ca2+ current duration is determined by stimulus repetition frequency; this appears to involve intracellular Ca2+ accumulation, although effects secondary to variability in the time course of changes in terminal membrane potentials cannot be ruled out.     

Slow-decaying presynaptic calcium dynamics gate long-lasting asynchronous release at the hippocampal mossy fiber to CA3 pyramidal cell synapse

Action potentials trigger two modes of neurotransmitter release, with a fast synchronous component and a temporally delayed asynchronous release. Asynchronous release contributes to information transfer at synapses, including at the hippocampal mossy fiber (MF) to CA3 pyramidal cell synapse where it controls the timing of postsynaptic CA3 pyramidal neuron firing. Here, we identified and characterized the main determinants of asynchronous release at the MF–CA3 synapse. We found that asynchronous release at MF–CA3 synapses can last on the order of seconds following repetitive MF stimulation. Elevating the stimulation frequency or the external Ca²⁺ concentration increased the rate of asynchronous release, thus, arguing that presynaptic Ca²⁺ dynamics is the major determinant of asynchronous release rate. Direct MF bouton Ca²⁺ imaging revealed slow Ca²⁺ decay kinetics of action potential (AP) burst-evoked Ca²⁺ transients. Finally, we observed that asynchronous release was preferentially mediated by Ca²⁺ influx through P/Q-type voltage-gated Ca²⁺ channels, while the contribution of N-type VGCCs was limited. Overall, our results uncover the determinants of long-lasting asynchronous release from MF terminals and suggest that asynchronous release could influence CA3 pyramidal cell firing up to seconds following termination of granule cell bursting.     

Effect of morphine on dynorphin B and GABA release in the basal ganglia of rats

In vivo microdialysis was used to study the effects of systemic, as well as intracerebral administration of morphine and naloxone on dynorphin B release in neostriatum and substantia nigra of rats. The release of dopamine (DA), γ-aminobutyric acid (GABA), glutamate (Glu) and aspartate (Asp) was also investigated. Systemic injection of morphine (1 mg/kg s.c.) induced long-lasting increases in extracellular dynorphin B and GABA levels in the substantia nigra, whereas DA, Glu and Asp levels, measured in the same region, were not significantly affected. No effect on striatal neurotransmitter levels was observed following systemic morphine administration. Local perfusion of the substantia nigra with morphine (100 μM) through the microdialysis probe also increased nigral dynorphin B and GABA levels. Perfusion of the neostriatum with morphine (100 μM) significantly increased GABA and dynorphin B levels in the ipsilateral substantia nigra, but no effect was observed locally. Naloxone blocked the effect of systemic morphine administration on nigral dynorphin B and GABA release, already at a dose of 0.2 mg/kg s.c. Naloxone alone, given either systemically (0.2–4 mg/kg s.c.) or intracerebrally (1–100 μM), did not affect dynorphin B or amino acid levels, either in neostriatum or in substantia nigra. However, naloxone produced a concentration-dependent increase in DA levels. The present results indicate that systemic morphine administration stimulates the release of dynorphin B in the substantia nigra, probably by activating the μ-subtype of opioid receptor, since the effect of morphine on nigral dynorphin B and GABA was antagonized by a low dose of naloxone. The increase in extracellular DA levels produced by high concentrations of naloxone, both in neostriatum and substantia nigra, indicates a disinhibitory effect of this drug on DA release, probably via a non-μ subtype of opioid receptors located on nigro-striatal DA neurones.     

Anti-nociceptive effect of neuropeptide Y in periaqueductal grey in rats with inflammation

Experimental inflammation was induced by subcutaneous injection of carrageenan into the left hindpaw of rats. Intra-periaqueductal grey (PAG) injection of 0.02 or 0.1 nmol of neuropeptide Y (NPY), but not 0.004 nmol, induced significant increases in hindpaw withdrawal latency (HWL) to thermal and mechanical stimulation in rats with inflammation. Furthermore, the anti-nociceptive effect of NPY was blocked partly by following intra-PAG injection of the Y1 receptor antagonist NPY28-36. The results demonstrated that NPY plays an anti-nociceptive role in PAG in rats with inflammation, in which Y1 receptor is involved.     

L-type calcium channels are involved in fast endocytosis at the mouse neuromuscular junction

We used fluorescence microscopy of FM dyes-labeled synaptic vesicles and electrophysiological recordings to examine the functional characteristics of vesicle recycling and study how different types of voltage-dependent Ca²⁺ channels (VDCCs) regulate the coupling of exocytosis and endocytosis at mouse neuromuscular junction.
Our results demonstrate the presence of at least two different pools of recycling vesicles: a high-probability release pool (i.e. a fast destaining vesicle pool), which is preferentially loaded during the first 5 s (250 action potentials) at 50 Hz; and a low-probability release pool (i.e. a slow destaining vesicle pool), which is loaded during prolonged stimulation and keeps on refilling after end of stimulation.
Our results suggest that a fast recycling pool mediates neurotransmitter release when vesicle use is minimal (i.e. during brief high-frequency stimulation), while vesicle mobilization from a reserve pool is the prevailing mechanism when the level of synaptic activity increases.
We observed that specific N- and L -type VDCC blockers had no effect on evoked transmitter release upon low-frequency stimulation (5 Hz). However, at high-frequency stimulation (50 Hz), L -type Ca²⁺ channel blocker increased FM2-10 destaining and at the same time diminished quantal release. Furthermore, when L -type channels were blocked, FM2-10 loading during stimulation was diminished, while the amount of endocytosis after stimulation was increased.
Our experiments suggest that L -type VDCCs promote endocytosis of synaptic vesicles, directing the newly formed vesicles to a high-probability release pool where they compete against unused vesicles.     

Differential antagonism of endomorphin-1 and endomorphin-2 spinal antinociception by naloxonazine and 3-methoxynaltrexone

To determine the role of spinal mu-opioid receptor subtypes in antinociception induced by intrathecal (i.t.) injection of endomorphin-1 and -2, we assessed the effects of β-funaltrexamine (a selective mu-opioid receptor antagonist) naloxonazine (a selective antagonist at the mu₁-opioid receptor) and a novel receptor antagonist (3-methoxynaltrexone) using the paw-withdrawal test. Antinociception of i.t. endomorphins and [ -Ala², MePhe⁴, Gly(ol)⁵]enkephalin (DAMGO) was completely reversed by pretreatment with β-funaltrexamine (40 mg/kg s.c.). Pretreatment with a variety of doses of i.t. or s.c. naloxonazine 24 h before testing antagonized the antinociception of endomorphin-1, -2 and DAMGO. Judging from the ID₅₀ values of naloxonazine, the antinociceptive effect of endomorphin-2 was more sensitive to naloxonazine than that of endomorphin-1 or DAMGO. The selective morphine-6β-glucuronide antagonist, 3-methoxynaltrexone, which blocked endomorphin-2-induced antinociception at each dose (0.25 mg/kg s.c. or 2.5 ng i.t.) that was inactive against DAMGO, did not affect endomorphin-1-induced antinociception but shifted the dose–response curve of endomorphin-2 3-fold to the right. These findings may be interpreted as indicative of the existence of a novel mu-opioid receptor subtype in spinal sites, where antinociception of morphine-6β-glucuronide and endomorphin-2 are antagonized by 3-methoxynaltrexone. The present results suggest that endomorphin-1 and endomorphin-2 may produce antinociception through different subtypes of mu-opioid receptor.     

中枢P物质参与电针镇痛的证据

本文采用放射免疫分析和甩尾测试法观察不同频率电针对大鼠脊髓Ｐ物质（ＳＰ）释放的影响。发现在电针有效组，２Ｈｚ（低频）电针时大鼠脊髓灌流液中ＳＰ—ｉｒ明显减少，而１５Ｈｚ（中频）、１００Ｈｚ（高频）和２／１５Ｈｚ（变频）刺激时，ＳＰ—ｉｒ明显增加。在电针无效组，各种频率电针时的ＳＰ—ｉｒ测定均无明显变化。脊髓蛛网膜下腔（ｉ．ｔ．）注射高选择性非肽类ＳＰ受体拮抗剂ＲＰ６７５８０可明显阻断１５Ｈｚ、１００Ｈｚ和２／１５Ｈｚ电针镇痛，而对ＺＨＺ电针镇痛无影响。注射ＲＰ６７５８０的同分异构体ＲＰ６８６５１则不能阻断上述各频率的电针镇痛作用。鉴于中、高频和变频电针促进脊髓ＳＰ的释放，而阻断ＳＰ受体可阻断上述电针镇痛，提示电针引起大鼠脊髓中释放的ＳＰ不是参与伤害感受，而是发挥了镇痛作用．关键词     

Glutamate Depresses Release by Activating Non-conventional Glutamate Receptors at Crayfish Nerve Terminals

The present study shows that release of glutamate from crayfish nerve terminals is inhibited at low depolarizing current pulses by glutamate, N -methyl-D-aspartate (NMDA) and quisqualate. These agonists elicit inhibitory effects at concentrations as low as 10^-8 M (quisqualate) and 10^-7 M (glutamate and NMDA). The NMDA-mediated inhibition is blocked by (±)-2-amino-5-phosphonovaleric acid (APV). The quisqualate-mediated inhibition is blocked by 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX). Both CNQX and APV are needed to block glutamate-mediated inhibition. The inhibition of release is not accompanied by a detectable change in presynaptic membrane conductance at the secondary branch. Using fura-2, Ca²⁺ accumulation during repetitive stimulation (100 Hz) was monitored in single release boutons. Inhibition of release, elicited by 10^-4 M glutamate, was not associated with a reduction in the accumulation of Ca²⁺. We show that the glutamate released from a single or a few release boutons during normal activity acts similarly to glutamate added externally, i.e. it inhibits its own release.     

Dynorphin-mediated antinociceptive effects of L-arginine and SIN-1 (an NO donor) in mice

The antinociceptive response of mice to the amino acid L-arginine (L-ARG) has been attributed to either an opioid mechanism or a non-opioid but nitric oxide (NO)-dependent mechanism. Earlier it was reported that the mechanism of nitrous oxide-induced antinociception involved opioid components and was also dependent on brain NO. This study was designed to determine whether the antinociceptive effects of L-ARG and the NO donor 3-morpholinosydnoimine (SIN-1) might be mediated by brain mechanisms similar to those that are responsible for nitrous oxide (N(2)O) antinociception. L-ARG and SIN-1 were administered to mice intracerebroventricularly (i.c.v.), and antinociception was assessed using the acetic acid abdominal constriction test. Both L-ARG and SIN-1 caused dose-related antinociceptive effects that were blocked by naloxone and norbinaltorphimine. The antinociceptive effects of both SIN-1 and L-ARG were also blocked to a greater extent by i.c.v. administration of a rabbit antiserum against rat dynorphin 1-13 than an antiserum against methionine-enkephalin, suggesting that the SIN-1 and L-ARG effects may be related to stimulated release of dynorphin. The antinociceptive effect of L-ARG was antagonized by an inhibitor of neuoronal NO synthase enzyme, indicating that L-ARG had to be converted to NO for its antinociceptive action. These findings indicate that the mechanisms of antinociceptive action of L-ARG and SIN-1 are both mediated by dynorphin and dependent on NO.     

Repeated peripheral electrical stimulations suppress both morphine-induced CPP and reinstatement of extinguished CPP in rats: accelerated expression of PPE and PPD mRNA in NAc implicated

Previous studies have shown that peripheral electrical stimulation (PES) can suppress morphine-induced conditioned place preference (CPP) and the reinstatement of extinguished CPP in the rat. The present study was performed to elucidate if preproenkephalin (PPE) and preprodynorphin (PPD) mRNAs in the nucleus accumbens (NAc) play a role in this event. Rats were trained with morphine for 4 days to establish CPP paradigm. They were then given 15-min test once a day for eight consecutive days for extinction trial. Twenty-four hours after the 8th session of extinction trials, rats were given peripheral electrical stimulation (PES) at 2 or 100 Hz once a day for 3 days, then a morphine-priming injection at a dose of 1, 2, or 4 mg/kg to reinstate the extinguished CPP. At the end of the experiment, PPE and PPD mRNA levels in the nucleus acccumbens (NAc) were determined by the semiquantitative RT-PCR technique. The results showed that PES at 2- and 100-Hz administered 30 min a day for 3 days suppressed both the expression of morphine-induced CPP and the reinstatement of extinguished CPP. PES at 2 Hz increased preproenkephalin (PPE) mRNA levels, whereas PES of 100 Hz that of preprodynorphin (PPD) mRNA levels in the NAc. These findings suggest that enkephalin and dynorphin in NAc may play important roles in the mechanisms underlying the inhibitory effect of PES on the expression and reinstatement of morphine-induced CPP in rats.     

Involvement of CGRP and CGRP1 receptor in nociception in the nucleus accumbens of rats

The present study was performed to investigate the role of calcitonin gene-related peptide (CGRP) and its antagonist CGRP8-37 on nociception in the nucleus accumbens of rats. Hindpaw withdrawal latencies (HWLs) to noxious stimulation induced by hot plate and Randall Selitto tests were measured. The HWL to both thermal and mechanical stimulation increased significantly after intra-nucleus accumbens administration of 0.5 or 1 nmol of CGRP, but not 0.1 nmol, indicating that CGRP plays an anti-nociceptive effect in the nucleus accumbens of rats. The anti-nociceptive effect induced by intra-nucleus accumbens administration of 1 nmol of CGRP was blocked significantly by following intra-nucleus accumbens administration of 1 nmol of CGRP8-37, a selective antagonist of CGRP1 receptor. Furthermore, the HWLs to both thermal and mechanical stimulation decreased significantly after intra-nucleus accumbens administration of 0.02, 0.1 and 0.5 nmol of CGRP8-37 alone. The hyperalgesic effect of intra-nucleus accumbens administration of CGRP8-37 lasted for more than 60 min after the injection, suggesting that CGRP1 receptor is involved in anti-nociception in the nucleus accumbens of rats. The results indicate that CGRP and CGRP1 receptor have important roles in nociceptive modulation in the nucleus accumbens of rats.     

Relieving effects of electroacupuncture on mechanical allodynia in neuropathic pain model of inferior caudal trunk injury in rat: mediation by spinal opioid receptors

The relieving effects of electroacupuncture (EA) on mechanical allodynia and its mechanism related to the spinal opioid system were investigated in a rat model of neuropathic pain. To produce neuropathic pain in the tail, the right superior caudal trunk was resected between the S1 and S2 spinal nerves. Two weeks after the surgery, EA stimulation (2 or 100 Hz, 0.3 ms, 0.2-0.3 mA) was delivered to Zusanli (ST36) for 30 min. The degree of mechanical allodynia was evaluated quantitatively by touching the tail with von Frey hair (2.0 g) at 10 min intervals. These rats were then subjected to an i.t. injection with one of the three specific opioid agonists in successive ways: the mu agonist (DAMGO 25, 50 and 100 pmol), the delta agonist (DADELT II 0.5, 1 and 2 nmol), and the kappa agonist (U50488H 5, 10 and 20 nmol) separated by 10 min in cumulative doses. During 30 min of EA stimulation, specific opioid antagonists were subjected to i.t. injection: the mu antagonist (beta-FNA 5, 10 and 20 nmol), the delta antagonist (naltrindole 5, 10 and 20 nmol), and the kappa antagonist (nor-BNI 3, 6 and 12 nmol) separated by 10 min in cumulative doses. As a result, EA reduced the behavioral signs of mechanical allodynia. Two Hz EA induced a robust and longer lasting effect than 100 Hz. All three opioid agonists also showed relieving effects on mechanical allodynia. However, nor-BNI could not block the EA effects on mechanical allodynia, whereas beta-FNA or naltrindole significantly blocked EA effects. These results suggest that the mu and delta, but not kappa, opioid receptors in the spinal cord of the rat, play important roles in mediating relieving effects on mechanical allodynia induced by 2 Hz EA.     

Involvement of opioid receptors in electroacupuncture-produced anti-hyperalgesia in rats with peripheral inflammation

Our previous study showed that electroacupuncture (EA) significantly attenuated inflammatory hyperalgesia. It has also been reported that EA analgesia in uninjured animals is mediated by mu and delta opioid receptors at 2-15 Hz and by kappa opioid receptor at 100 Hz. Because persistent pain changes neural response to external stimulation, we hypothesized that (1) the mechanisms of EA anti-hyperalgesia may be different under conditions of persistent pain and that (2) combining EA with a sub-effective dose of morphine could enhance EA anti-hyperalgesia. Hyperalgesia, decreased paw withdrawal latency (PWL) to a noxious thermal stimulus, was induced by subcutaneously injecting complete Freund's adjuvant (CFA) into the hind paws of rats. Selective antagonists against mu (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-ThrNH2, CTOP), delta (naltrinodole, NTI) and kappa (nor-binaltorphimine, BNI) opioid receptors were administered intrathecally 10 min before each of two EA treatments at acupoint Huantiao (GB30), one immediately post and the other 2 h post-CFA. Morphine was given (i.p.) 40 min before the second EA treatment. PWL was measured before and 2.5 and 5 h post-CFA. Both 10 and 100 Hz EA-produced anti-hyperalgesia were blocked spinally by mu- and delta- but not kappa-receptor antagonists. EA combined with a sub-threshold dose of morphine (2.5 mg/kg) enhanced anti-hyperalgesia additively (10 Hz EA) or synergistically (100 Hz EA) compared to that produced by each component alone. These results suggest selective involvement of mu and delta, but not kappa, receptors in EA-produced anti-hyperalgesia in rats. A combined EA and opioid drug protocol may provide an improved treatment strategy for inflammatory pain.     

Chemical stimulation of the ST36 acupoint reduces both formalin-induced nociceptive behaviors and spinal astrocyte activation via spinal alpha-2 adrenoceptors

Spinal astrocytes have emerged as important mechanistic contributors to pathological and chronic pain. Recently, we have demonstrated that injection of diluted bee venom (DBV) into the Zusanli (ST36) acupoint produces a potent anti-nociceptive effect via the activation of spinal alpha-2 adrenoceptors. However, it is unclear if this anti-nociceptive effect is associated with alterations in spinal astrocytes. Thus, the present study was designed to determine: (1) whether DBV's anti-nociceptive effect in the formalin test involves suppression of spinal astrocyte activation; (2) whether DBV-induced astrocyte inhibition is mediated by spinal alpha-2 adrenoceptors; and (3) whether this glial modulation is potentiated by intrathecal administration of the glial metabolic inhibitor, fluorocitrate (FC) in combination with DBV injection. DBV was injected directly into the ST36 acupoint, and spinal expression of the astrocytic marker, glial fibrillary acidic protein (GFAP), was assessed together with effects on formalin-induced nociception. DBV treatment reduced pain responses in the late phase of the formalin test and significantly blocked the formalin-evoked increase in spinal GFAP expression. These effects of DBV were prevented by intrathecal pretreatment with selective alpha-2A and alpha-2C adrenoceptor antagonists. Moreover, low dose intrathecal injection of FC in conjunction with low dose DBV injection into the ST36 acupoint synergistically suppressed pain responses and GFAP expression. These results demonstrate that DBV stimulation of the ST36 acupoint inhibits the formalin-induced activation of spinal astrocytes and nociceptive behaviors in this inflammatory pain model and this inhibition is associated with the activation of spinal alpha-2 adrenoceptors.     

Involvement of nociceptin/orphanin FQ and its receptor in electroacupuncture-produced anti-hyperalgesia in rats with peripheral inflammation

The neuropeptide nociceptin/orphanin FQ (N/OFQ), the endogenous agonist of the N/OFQ peptide receptor (NOP receptor), has been demonstrated to be involved in many physiological and pathological functions including pain regulation. In the present study, the involvement of N/OFQ-NOP receptor system in electroacupuncture (EA)-produced anti-hyperalgesia was investigated in rats with peripheral inflammation. Intrathecal (i.t.) administration of N/OFQ (15 nmol) or EA at acupoints GB30 and GB34 could significantly attenuate hyperalgesia which was induced by subcutaneously injecting complete Freund's adjuvant (CFA) into one hindpaw of rats, manifesting as decreased paw withdrawal latency (PWL) to the noxious thermal stimulus. The anti-nociceptive effect of N/OFQ or EA was significantly blocked by intrathecal injection of [Nphe(1)]nociceptin(1-13)NH(2) (20 nmol), a selective antagonist of the NOP receptor, indicating the NOP-receptor-mediated mechanism. Additionally, the combination of N/OFQ injection with EA treatment could enhance anti-hyperalgesia compared to that produced by each component alone. These findings suggested that the spinal N/OFQ-NOP system might be involved in EA analgesia, which may be one of the mechanisms underlying the anti-nociceptive effect of EA in rat's peripheral inflammatory pain.