大鼠CB／CT微量注射CGRP或SP引起的对伤害性刺激反应的易化作用

探讨降钙素基因相关肽（CGRP）和P物质（SP）在SD大鼠扣带束及周围皮质（CB/CT）对伤害性刺激反应的作用。应用热板和机械压力实验法,以大鼠后抓缩抓反应潜伏期（HWL）为痛阈指标,观察CB/CT微量注射CGRP或SP后HWL的变化。CB/CT内注射0.125,0.25,0.5mol CGRP或SP均显著地降低大鼠对热板的双侧HWL,且呈量效关系;0.125nmolCGRP或0.5nmol SP能显著降低大鼠对机械压力的HWL。CB/CT微量注射CGRP或SP使大鼠对热板和机械压力刺激发生痛敏。     

PKC and PKA, but not PKG mediate LPS-induced CGRP release and [Ca(2+)](i) elevation in DRG neurons of neonatal rats.

Calcitonin gene-related peptide (CGRP), is produced in dorsal root ganglia (DRG) neurons and released from primary afferent neurons to mediate hemodynamic effects and neurogenic inflammation. In this work, we determined whether lipopolysaccharide (LPS), an inflammatory stimulator, could trigger CGRP release from cultured DRG neurons and if so, which cellular signaling pathway was involved in this response. Cytoplasmic concentration of calcium ([Ca(2+)](i)) plays a key role in neurotransmitter release, therefore [Ca(2+)](i) was also determined in cultured DRG cells using fluo-3/AM. The results showed that LPS (0.1-10 microg/ml) evoked CGRP release in a time- and concentration-dependent manner from DRG neurons. LPS also increased [Ca(2+)](i) in a concentration-dependent manner. The protein kinase C (PKC) inhibitors, calphostin C 0.5 microM or RO-31-8220 0.1 microM, and the cAMP-dependent protein kinase (PKA) specific inhibitor RP-CAMPS 30 microM or nonspecific inhibitor H8 1 microM inhibited 1 microg/ml LPS-evoked CGRP release and [Ca(2+)](i) increase from DRG neurons. The cGMP-dependent protein kinase (PKG) inhibitor Rp-8-pCPT-cGMPS 30 microM did not block the LPS response. These data suggest that LPS may stimulate CGRP release and [Ca(2+)](i) elevation through PKC and PKA, but not PKG signaling pathway in DRG neurons of neonatal rats.     

Second phase of formalin-induced excitation of spinal dorsal horn neurons in spinalized rats is reversed by sciatic nerve block

Considerable debate persists concerning peripheral vs. central mechanisms underlying the second phase of the nociceptive response in the formalin test in the rat. To gain insight into the neurophysiological basis of this pain, we investigated the effects of block of afferent nerve conduction during the second phase of formalin-evoked excitation of single nociceptive neurons recorded extracellularly from rat spinal dorsal horn segments (L(3-4)) in pentobarbital-anaesthetized, male Sprague-Dawley rats. Rats were spinally transected (T(9)) to examine exclusively peripheral and spinal nociceptive processing. In six control rats, hind paw intraplantar formalin injection (50 microL, 2.5%) induced the typical biphasic increase in the discharge rate of the six wide dynamic range neurons tested. This response consisted of a relatively brief immediate phase (approximately 5 min), followed by decreased firing. An ensuing second phase of elevated discharge began approximately 35 min after injection and persisted to at least 80 min. In this control group, 0.9% saline was applied to the exposed ipsilateral sciatic nerve after onset of the second phase (40 min after formalin injection). In a group of six test rats, application of 2% lidocaine instead of saline reversed the second phase of excitation in all six wide dynamic range neurons examined. When the firing rate was normalized to that at 40 min (100%), the time of saline or lidocaine administration, the rate at 50 min was 120 +/- 7.5% in the saline-treated group and 31 +/- 7.4% in the lidocaine-treated group; following lidocaine treatment firing rate remained markedly less than that before administration throughout the remainder of the recording. It is concluded that: (i) spinal mechanisms alone are not sufficient for induction and maintenance of second phase increased discharge of spinal nociceptive dorsal horn neurons; (ii) descending influences via supraspinal inputs are not causal in the development and maintenance of second phase increased discharge and (iii) tonic input from afferent neurons during the second phase plays a primary and essential role in generating and sustaining the second phase of elevated discharge of dorsal horn neurons and, thus, presumably the second phase of nociceptive scores in the formalin test. The data in this study reveal how much of an altered synaptically elicited response in the spinal dorsal horn can be attributed to postsynaptic plastic changes vs. how much can be simply due to increased synaptic input. The present results are important not only in the context of the formalin test but also in the context of other models related to inflammatory pain and neuropathic pain.     

Involvement of ryanodine receptors in tetanic sciatic stimulation-induced long-term potentiation of spinal dorsal horn and persistent pain in rats

Tetanic stimulation of the sciatic nerve induces long‐term potentiation (LTP) of C‐fiber‐evoked field potentials in the spinal dorsal horn and persistent pain, suggesting that spinal LTP may be a substrate for central sensitization of the pain pathway. However, its cellular mechanism remains unclear. The present study provides electrophysiological and behavioral evidence for the involvement of ryanodine receptor (RyR) in the induction of spinal LTP and persistent pain in rats. The specific inhibitor of ryanodine receptor, ryanodine and dantrolene, dose dependently blocked the induction, but not maintenance, of spinal LTP and reduced persistent pain behaviors induced by tetanic sciatic stimulation. Both cyclic ADP ribose (cADPR), an endogenous agonist of RyR, and (±)‐1,4‐dihydro‐2,6‐dimethyl‐5‐nitro‐4‐[2‐(trifluromethyl)‐phenyl]‐3‐pyridine carboxylic acid methyl ester (Bay K 8644), an agonist of L‐type calcium channel, attenuated ryanodine‐induced inhibition. Immunohistochemistry and electron microscopic observation showed that RyR subtypes RyR1 and RyR3 were located in the spinal dorsal horn. The results suggest that RyRs are involved in synaptic plasticity of the spinal pain pathway and may be a novel target for treating pain. © 2012 Wiley Periodicals, Inc.     

Effects of spinal nerve ligation on immunohistochemically identified neurons in the L4 and L5 dorsal root ganglia of the rat

This study examined the effect of spinal nerve ligation on different populations of immunohistochemically identified neurons in the dorsal root ganglia (DRG) of the rat. The optical fractionator method was used to count neurons in the ipsilateral L4 and L5 DRG 1-20 weeks after ligation of the L5 and L6 spinal nerves, sham surgery, or no surgery. One week after ligation, neurons in the L5 DRG that were labeled by IB4, a marker of unmyelinated primary afferent neurons, were largely absent. The numbers of IB4-labeled neurons then progressively increased to reach control values by 20 weeks. A smaller, sustained decrease occurred in the number of small-, medium- and large-sized neurons immunoreactive for calcitonin gene-related peptide (CGRP), a marker for peptidergic primary afferents, in the L5 DRG. There was a proportionately greater decrease in the numbers of medium- to large-sized CGRP-like immunoreactive neurons. The number of myelinated afferents in the L5 DRG, identified by their staining for neurofilament protein (N52), did not change after ligation. However, closer examination revealed a significant decrease in the numbers of large-sized neurons, coupled with an increase in the numbers of small- to medium-sized neurons, and the appearance of a novel population of very small-sized neurons labeled by N52. The numbers and cell size distributions of IB4-labeled, CGRP-like immunoreactive, and N52-labeled neurons were unchanged in the adjacent L4 DRG. Unlike the L5 DRG, injury-induced changes in the expression of various receptors, neurotransmitters and neurotrophic factors in the L4 DRG are not confounded by a change in the immunohistochemical phenotype of primary afferent neurons.     

Modulation of sensory neuron‐specific receptors in the development of morphine tolerance and its neurochemical mechanisms

Prevention of opiate tolerance is a critical issue in pain management. The present study was designed to characterize the pharmacological properties of sensory neuron‐specific receptors (SNSR; also known as Mas‐related gene receptors, or Mrg) for their modulation in the development of morphine tolerance and to investigate the underlying mechanism(s). Daily coadministration of the SNSR agonist BAM8‐22 at a dose of 0.01 or 0.001, but not 1.0, nmol with morphine (intrathecally, or i.t., 20 μg/day) for 6 days significantly decreased the development of morphine tolerance. Coadministration of BAM8‐22 (i.t., 1.0 nmol) on days 1, 3, and 5 completely blocked tolerance to morphine‐induced analgesia. Intermittent coadministration of the structurally dissimilar SNSR agonist (Tyr⁶)‐2‐MSH‐6‐12 (MSH; 5 nmol) also produced similar modulation. Chronic administration of morphine (20 μg, i.t.) increased expression of neuronal nitric oxide synthase (nNOS) and calcitonin gene‐related peptide (CGRP) in superficial layers of the spinal cord and dorsal root ganglia. All these increases were abolished when BAM8‐22 or MSH was intermittently coadministered. Furthermore, intermittent administration of BAM8‐22 inhibited morphine‐induced increase in protein kinase Cγ (PKCγ) in both membrane and cytosol of spinal dorsal horn neurons. These results suggest that moderate activation of SNSR modulated morphine tolerance by inhibition of the PKC signaling pathway, leading to abolishment of enhancement of nNOS and CGRP. As SNSR are uniquely located ina subset of small‐sized neurons in dorsal root and trigeminal ganglia, intermittent combination of SNSR agonist could be a promising adjunct for sustained use of opiates without central nervous system side effects. © 2010 Wiley‐Liss, Inc.     

Protease‐activated receptor 4 activation increases the expression of calcitonin gene‐related peptide mRNA and protein in dorsal root ganglion neurons

Accumulating evidence demonstrates that nociceptor activation evokes a rapid change in mRNA and protein levels of calcitonin gene‐related peptide (CGRP) in dorsal root ganglion (DRG) neurons. Although the colocalization of CGRP and protease‐activated receptor‐4 (PAR4), a potent modulator of pain processing and inflammation, was detected in DRG neurons, the role of PAR4 activation in the expression of CGRP has not been investigated. In the present study, the expression of CGRP and activation (phosphorylation) of extracellular signal‐regulated kinases 1 and 2 (ERK1/2) in rat DRG neurons were measured by immunofluorescence, real‐time PCR, and Western blotting after AYPGKF‐NH2 (selective PAR4‐activating peptide; PAR4‐AP) intraplantar injection or treatment of cultured DRG neurons. The expression of CGRP in cultured DRG neurons was also assessed after treatment with AYPGKF‐NH2 with preaddition of PD98059 (an inhibitor for ERK1/2 pathway). Results showed that PAR4‐AP intraplantar injection or treatment of cultured DRG neurons evoked significant increases in DRG cells displaying CGRP immunoreactivity and cytoplasmic and nuclear staining for phospho‐ERK1/2 (p‐ERK1/2). Percentages of total DRG neurons expressing both CGRP and PAR4 or p‐ERK1/2 also increased significantly at 2 hr after PAR4‐AP treatment. Real‐time PCR and Western blotting showed that PAR4‐AP treatment significantly increased expression of CGRP mRNA and protein levels in DRG neurons. The PAR4 activation‐evoked CGRP expression both at mRNA and at protein levels was significantly inhibited after p‐ERK1/2 was inhibited by PD98059. These results provide evidence that activation of PAR4 upregulates the expression of CGRP mRNA and protein levels in DRG neurons via the p‐ERK1/2 signal pathway. © 2013 Wiley Periodicals, Inc.     

The neural pathway of galanin in the hypothalamic arcuate nucleus of rats: activation of beta-endorphinergic neurons projecting to periaqueductal gray matter

We have previously shown that microinjection of galanin into the arcuate nucleus of hypothalamus (ARC) produced antinociceptive effects in rats (Sun et al., 2003a). In this study, the neural pathway of galanin from ARC to midbrain periaqueductal gray (PAG) in nociceptive modulation was investigated. The hindpaw withdrawal latencies (HWLs) with noxious thermal and mechanical stimulation were assessed by the hotplate and the Randall Selitto tests. Intra-ARC administration of 0.1, 0.5, or 1 nmol of galanin induced significant increases in HWLs of rats. The galanin-induced increases in HWLs were inhibited by injection of 10 microg of the opioid receptor antagonist naloxone or 1 nmol of the mu-opioid receptor antagonist beta-funaltrexamine (beta-FNA) into PAG, suggesting that the antinociceptive effects induced by intra-ARC injection of galanin occur via the neural pathway from ARC to PAG. Furthermore, our results demonstrate that the galaninergic fibers directly innervated the beta-endorphinergic neurons in ARC by immunofluorescent methods. Taken together, our results suggest that galanin produces antinociceptive effects in the ARC of rats by activating the beta-endorphinergic pathway from ARC to PAG.     

Localization of calcitonin receptor-like receptor and receptor activity modifying protein 1 in enteric neurons, dorsal root ganglia, and the spinal cord of the rat

Calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) comprise a receptor for calcitonin gene related peptide (CGRP) and intermedin. Although CGRP is widely expressed in the nervous system, less is known about the localization of CLR and RAMP1. To localize these proteins, we raised antibodies to CLR and RAMP1. Antibodies specifically interacted with CLR and RAMP1 in HEK cells coexpressing rat CLR and RAMP1, determined by Western blotting and immunofluorescence. Fluorescent CGRP specifically bound to the surface of these cells and CGRP, CLR, and RAMP1 internalized into the same endosomes. CLR was prominently localized in nerve fibers of the myenteric and submucosal plexuses, muscularis externa and lamina propria of the gastrointestinal tract, and in the dorsal horn of the spinal cord of rats. CLR was detected at low levels in the soma of enteric, dorsal root ganglia (DRG), and spinal neurons. RAMP1 was also localized to enteric and DRG neurons and the dorsal horn. CLR and RAMP1 were detected in perivascular nerves and arterial smooth muscle. Nerve fibers containing CGRP and intermedin were closely associated with CLR fibers in the gastrointestinal tract and dorsal horn, and CGRP and CLR colocalized in DRG neurons. Thus, CLR and RAMP1 may mediate the effects of CGRP and intermedin in the nervous system. However, mRNA encoding RAMP2 and RAMP3 was also detected in the gastrointestinal tract, DRG, and dorsal horn, suggesting that CLR may associate with other RAMPs in these tissues to form a receptor for additional peptides such as adrenomedullin.     

Differential expression patterns of K+/Cl− cotransporter 2 in neurons within the superficial spinal dorsal horn of rats

γ‐Aminobutyric acid (GABA)‐ and glycine‐mediated hyperpolarizing inhibition is associated with a chloride influx that depends on the inwardly directed chloride electrochemical gradient. In neurons, the extrusion of chloride from the cytosol primarily depends on the expression of an isoform of potassium–chloride cotransporters (KCC2s). KCC2 is crucial in the regulation of the inhibitory tone of neural circuits, including pain processing neural assemblies. Thus we investigated the cellular distribution of KCC2 in neurons underlying pain processing in the superficial spinal dorsal horn of rats by using high‐resolution immunocytochemical methods. We demonstrated that perikarya and dendrites widely expressed KCC2, but axon terminals proved to be negative for KCC2. In single ultrathin sections, silver deposits labeling KCC2 molecules showed different densities on the surface of dendritic profiles, some of which were negative for KCC2. In freeze fracture replicas and tissue sections double stained for the β3‐subunit of GABA_A receptors and KCC2, GABA_A receptors were revealed on dendritic segments with high and also with low KCC2 densities. By measuring the distances between spots immunoreactive for gephyrin (a scaffolding protein of GABA_A and glycine receptors) and KCC2 on the surface of neurokinin 1 (NK1) receptor‐immunoreactive dendrites, we found that gephyrin‐immunoreactive spots were located at various distances from KCC2 cotransporters; 5.7 % of them were recovered in the middle of 4–10‐µm‐long dendritic segments that were free of KCC2 immunostaining. The variable local densities of KCC2 may result in variable postsynaptic potentials evoked by the activation of GABA_A and glycine receptors along the dendrites of spinal neurons. J. Comp. Neurol. 523:1967–1983, 2015 © 2015 Wiley Periodicals, Inc.     

Protein kinase C gamma interneurons in the rat medullary dorsal horn: Distribution and synaptic inputs to these neurons,and subcellular localization of the enzyme

The γ isoform of protein kinase C (PKCγ), which is concentrated in interneurons in the inner part of lamina II (II_i) of the dorsal horn, has been implicated in the expression of tactile allodynia. Lamina II_i PKCγ interneurons were shown to be activated by tactile inputs and to participate in local circuits through which these inputs can reach lamina I, nociceptive output neurons. That such local circuits are gated by glycinergic inhibition and that A‐ and C‐fibers low threshold mechanoreceptors (LTMRs) terminate in lamina II_i raise the general issue of synaptic inputs to lamina II_i PKCγ interneurons. Combining light and electron microscopic immunochemistry in the rat spinal trigeminal nucleus, we show that PKCγ‐immunoreactivity is mostly restricted to interneurons in lamina II_i of the medullary dorsal horn, where they constitute 1/3 of total neurons. The majority of synapses on PKCγ‐immunoreactive interneurons are asymmetric (likely excitatory). PKCγ‐immunoreactive interneurons appear to receive exclusively myelinated primary afferents in type II synaptic glomeruli. Neither large dense core vesicle terminals nor type I synaptic glomeruli, assumed to be the endings of unmyelinated nociceptive terminals, were found on these interneurons. Moreover, there is no vesicular glutamate transporter 3‐immunoreactive bouton, specific to C‐LTMRs, on PKCγ‐immunoreactive interneurons. PKCγ‐immunoreactive interneurons contain GABA_Aergic and glycinergic receptors. At the subcellular level, PKCγ‐immunoreactivity is mostly concentrated on plasma membranes, close to, but not within, postsynaptic densities. That only myelinated primary afferents were found to contact PKCγ‐immunoreactive interneurons suggests that myelinated, but not unmyelinated, LTMRs play a critical role in the expression of mechanical allodynia. J. Comp. Neurol. 522:393–413, 2014. © 2013 Wiley Periodicals, Inc.     

ATP induces long-term potentiation of C-fiber-evoked field potentials in spinal dorsal horn: the roles of P2X4 receptors and p38 MAPK in microglia

Many studies have shown that adenosine triphosphate (ATP), as a neurotransmitter, is involved in plastic changes of synaptic transmission in central nervous system. In the present study, we tested whether extracellular ATP can induce long-term potentiation (LTP) of C-fiber-evoked field potentials in spinal dorsal horn. The results showed the following: (1) ATP at a concentration of 0.3 mM induced spinal LTP of C-fiber-evoked field potentials, lasting for at least 5 h; (2) spinal application of 2',3'-O-(2,4,6-trinitrophenyl)adenosine-5-triphosphate (TNP-ATP; an antagonist of P2X(1-4) receptors), but not pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; an antagonist of P2X(1,2,3,5,7) receptors), 30 min before ATP blocked ATP-induced LTP, indicating that ATP may induce spinal LTP by activation of P2X(4) receptors; (3) at 60 min after LTP induction the level of phospho-p38 mitogen-activated protein kinase (p-p38 MAPK) was significantly elevated and at 180 min after LTP the number of P2X(4) receptors increased significantly; both p-p38 and P2X(4) receptors were exclusively co-located with the microglia marker, but not with neuronal or astrocyte marker; (4) spinal application of TNP-ATP but not PPADS prevented p38 activation; (5) spinal application of SB203580, a p38 MAPK inhibitor, prevented both spinal LTP and the upregulation of P2X(4) receptors. The results suggested that ATP may activate p38 MAPK by binding to intrinsic P2X(4) receptors in microglia, and subsequently enhance the expression of P2X(4) receptors, contributing to spinal LTP.     

The acute and chronic administration of (+/-)-8-hydroxy-2-(Di-n-propylamino)tetralin significantly alters the activity of spontaneously active midbrain dopamine neurons in rats: an in vivo electrophysiological study

This study examined the effect of the acute and chronic systemic administration of (+/-)-8-Hydroxy-2-(Di-n-propylamino)Tetralin(8-OH-DPAT) on the number and firing pattern of spontaneously active dopamine (DA) neurons in the ventral tegmental area (VTA or A10) and substantia nigra pars compacta (SNC or A9) in anesthetized male rats. These parameters were measured using extracellular in vivo electrophysiology. A single s.c. injection of 0.01, 0.1, or 1 mg/kg of 8-OH-DPAT did not significantly alter the number of spontaneously active SNC DA neurons compared to vehicle-treated animals (controls). The acute administration of 0.01 or 0.1 mg/kg of 8-OH-DPAT did not significantly alter, whereas the 1 mg/kg dose significantly decreased the number of spontaneously active VTA DA neurons compared to controls. The acute administration of 8-OH-DPAT significantly increased the percentage of VTA DA neurons firing in a bursting pattern. In contrast, there was a significant decrease in the percentage of SNC DA neurons firing in a bursting pattern following the acute administration of 8-OH-DPAT. The number of spontaneously active SNC DA neurons was not significantly altered by the chronic s.c. administration of 8-OH-DPAT (0.01, 0.1, or 1 mg/kg s.c.) as compared to controls. However, the chronic s.c. administration of all doses of 8-OH-DPAT significantly decreased the number of spontaneously active VTA DA neurons compared to controls. The i.v. administration of (+)-apomorphine (50 microg/kg) did not reverse the 8-OH-DPAT-induced decrease in the number of spontaneously active VTA DA neurons, suggesting that this effect is unlikely due to depolarization blockade. The percentage of VTA DA neurons exhibiting burst firing was significantly increased by 0.01 and 0.1 mg/kg, but significantly decreased by 1 mg/kg of 8-OH-DPAT. Overall, the systemic administration of 8-OH-DPAT preferentially affects the activity of spontaneously active A10 DA neurons in rats.     

Role of opioid receptors (μ, δ1, δ2) in modulating responses of nociceptive neurons in the superficial and deeper dorsal horn of the medulla (trigeminal nucleus caudalis) in the rat

This report describes the effects of intravenously administered agonists and antagonists at μ-, δ₁- and δ₂-opioid receptors on the Aδ- and C-fiber-evoked responses of trigeminal nociceptive neurons in anesthetized rats. Extracellular single unit recordings were made from 61 nociceptive neurons (23 NS, 38 WDR) in the superficial and 37 nociceptive neurons (3 NS, 34 WDR) in the deeper dorsal horn of the medulla (trigeminal nucleus caudalis). Administration of either the δ₁-receptor agonist [

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-Pen^2,5]enkephalin (DPDPE; 0.05–2 mg/kg), the δ₂-receptor agonist [

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-Ala²,Glu⁴]deltorphin (DELT; 1–2 mg/kg) or the μ-receptor agonist [

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-Ala²,N-MePhe⁴,Gly⁵-ol]enkephalin (DAMGO; 0.05–1 mg/kg) inhibited the Aδ- and C-fiber-evoked responses of nociceptive neurons in the superficial and deeper dorsal horn. The inhibitory effect was more pronounced on the C-fiber-evoked responses than on the Aδ-fiber-evoked responses. In other neurons, DPDPE also produced facilitation, or inhibition followed by facilitation, or differential effects (inhibition of the C-fiber-evoked responses and facilitation of the Aδ-fiber-evoked responses) on the Aδ- and C-fiber-evoked responses. The effects of DPDPE were antagonized by 7-benzylidenenaltrexone (BNTX, 0.4–1 mg/kg), a δ₁-receptor antagonist, in 88% (7/8) of neurons. Naltriben (NTB, 0.7–1 mg/kg), a δ₂-receptor antagonist, antagonized the effects of both DELT and DPDPE. A smaller dose of NTB (0.3 mg/kg), which failed to reverse the effects of DPDPE in 100% (4/4) of neurons, effectively antagonized the effects of DELT in 100% (6/6) of neurons. The inhibitory action of DAMGO was completely antagonized by naloxone (0.2 mg/kg) in 100% (6/6) of neurons. The results of the present investigation suggest that: (1) μ-, δ₁- and δ₂-opioid receptors play an important role in the inhibitory modulation of the Aδ- and C-fiber-evoked responses of nociceptive neurons in the superficial and deeper dorsal horn of the medulla; (2) selective inhibition of the C-fiber-evoked responses by activation of opioid receptors may account for the opioid-mediated selective suppression of second or persistent pain as compared to first pain; and (3) NTB, in a limited dose range, can discriminate between δ₁- and δ₂-opioid receptor subtypes.