Sprouting phenomenon, a new model for the role of A-beta fibers in wind up

Wind up is a progressive frequency-dependent facilitation of the responses of nociceptive neurons observed on the application of repetitive (usually electrical) stimuli of constant intensity. The NMDA and NK1 receptors are essentially involved in wind up. After induction of wind up, stimulation of C-fibers show the characteristics of wind up, but stimulation of Abeta fibers for induction of wind up is controversial. In this study, we have proposed a new model for the role of Abeta fibers in wind up, through sprouting of nerve fibers in the dorsal horn of spinal cord. We named it "sprouting phenomenon". It has been reported that in some clinical hyperalgesic states induced by peripheral injury or inflammation, wind up may aggravate the pain. For example, studies have indicated the presence of wind up in post-surgical states, some neuropathic pains, fibromyalgia syndrome, and post-herpetic neuralgia. According to sprouting phenomenon, it seems that some clinical interventions can be assessed to alleviate post-inflammatory pains: (1) Immediate and complete relief of inflammation by anti-inflammatory agents to prevent repetitive excitation of C-fibers and subsequent morphological changes of dorsal horn laminae; (2) using local anesthetics in order to prevent pain signal transmission; (3) prevention of sprouting by intrathecal injection of some anti-proliferation agents; (4) using NMDA or NK1 receptor antagonists to prevent central mechanism of wind up. Some clinical trials have indicated the effectiveness of these antagonists. It is worth noting that future clinical studies are needed to validate these predictions.     

Wind-up of spinal cord neurones and pain sensation: much ado about something?

Wind-up is a frequency-dependent increase in the excitability of spinal cord neurones, evoked by electrical stimulation of afferent C-fibres. Although it has been studied over the past thirty years, there are still uncertainties about its physiological meaning. Glutamate (NMDA) and tachykinin NK1 receptors are required to generate wind-up and therefore a positive modulation between these two receptor types has been suggested by some authors. However, most drugs capable of reducing the excitability of spinal cord neurones, including opioids and NSAIDs, can also reduce or even abolish wind-up. Thus, other theories involving synaptic efficacy, potassium channels, calcium channels, etc. have also been proposed for the generation of this phenomenon. Whatever the mechanisms involved in its generation, wind-up has been interpreted as a system for the amplification in the spinal cord of the nociceptive message that arrives from peripheral nociceptors connected to C-fibres. This probably reflects the physiological system activated in the spinal cord after an intense or persistent barrage of afferent nociceptive impulses. On the other hand, wind-up, central sensitisation and hyperalgesia are not the same phenomena, although they may share common properties. Wind-up can be an important tool to study the processing of nociceptive information in the spinal cord, and the central effects of drugs that modulate the nociceptive system. This paper reviews the physiological and pharmacological data on wind-up of spinal cord neurones, and the perceptual correlates of wind-up in human subjects, in the context of its possible relation to the triggering of hyperalgesic states, and also the multiple factors which contribute to the generation of wind-up.     

Contributions of glutamate receptors to the maintenance of mustard oil-induced hyperalgesia in spinalized rats

N-methyl-d-aspartate (NMDA) receptors, which are widely distributed throughout the central nervous system, appear to play a critical role in several types of plasticity and long-term potentiation. In the pain system, increased sensitivity to somatosensory stimuli, known as hyperalgesia and allodynia, can arise from tissue damage or excessive C-fiber nociceptor activation. Previously, NMDA, non-NMDA ionotropic, and metabotropic glutamate receptors have been proposed to contribute to the sustained hyperalgesia following tissue injury or nociceptor activation. Although non-NMDA receptors appear to mediate both hyperalgesia and normal (nonhyperalgesic) responses and behavior, NMDA receptors have been reported to participate only in hyperalgesic responses. In contrast, other studies have implicated NMDA receptors in both hyperalgesic and normal responses. The aim of this study was to critically compare the effects of the glutamate receptor antagonists ketamine and 2-amino-5-phosphonovaleric acid (APV; NMDA receptor antagonists), 6,7-dinitroquinoxaline-2,3-dione (DNQX; non-NMDA ionotropic receptor antagonist), and 2-amino-3-phosphonopropionic acid (AP3; metabotropic receptor antagonist) on intra-articular mustard oil-induced facilitation of flexion withdrawal reflexes in spinalized rats. Our results showed that, as expected from previous studies, ketamine, APV, and DNQX dose-dependently inhibited the flexion withdrawal reflex evoked by C-fiber electrical stimulation of the sciatic nerve. Surprisingly, however, ketamine, APV, and DNQX also inhibited flexion withdrawal reflexes in normal (nonhyperalgesic) rats with similar ED50s. In contrast, AP3 had no effect in either hyperalgesic or normal rats. These results demonstrate that NMDA and non-NMDA ionotropic, but not metabotropic, glutamate receptors contribute without preference to both facilitated and normal flexion withdrawal reflexes evoked by high-intensity electrical stimulation in the spinalized rat. Thus, the apparent preference of NMDA receptors for hyperalgesic states seen in some previous studies on nociception, as well as in other model systems, may have arisen from differences in experimental paradigm, such as the intensity of sensory stimulation or excitability of the spinal cord, coupled with the voltage dependency of the NMDA conductance. Received: 15 November / Accepted: 12 June 1997     

Amyloid beta-protein1-42 increases cAMP and apolipoprotein E levels which are inhibited by beta1 and beta2-adrenergic receptor antagonists in mouse primary astrocytes

Amyloid beta-protein (Abeta) increases apolipoprotein E (apoE) levels in astrocytes which could alter lipid trafficking. The mechanism for the Abeta-induced increase in apoE levels is not well understood. It is well established that stimulation of beta-adrenergic receptors (betaARs) increases cAMP levels. Elevation of cAMP levels increases apoE abundance. The current study determined if Abeta(1-42) stimulation of cAMP and apoE levels could be inhibited by betaAR antagonists in astrocytes. We demonstrate that Abeta(1-42) but not the reverse protein Abeta(42-1) or Abeta(1-40) stimulated cAMP formation and this stimulation was inhibited by selective betaAR antagonists in mouse primary cortical astrocytes. Abeta(1-42) significantly increased apoE levels which were significantly inhibited by the betaAR selective antagonists with the greatest inhibition observed with the beta(2) antagonist. Separate lines of evidence have suggested that agonist-induced stimulation of betaARs and increases in apoE abundance may serve a neuroprotective role in astrocytes. Our results indicate a potential interaction between betaARs and apoE which may contribute to reducing Abeta(1-42) neurotoxicity.     

Acute topical application of tumor necrosis factor alpha evokes protein kinase A-dependent responses in rat sensory neurons

Local perfusion of the dorsal root ganglion (DRG) with tumor necrosis factor alpha (TNF-alpha) in rats induces cutaneous hypersensitivity to mechanical stimuli. Thus we investigated the cellular mechanisms of TNF-alpha-induced mechanical hyperalgesia. The L(4) and L(5) DRGs with the sciatic nerves attached were excised from rats for in vitro dorsal root microfilament recording. After baseline recording for 15 min, TNF-alpha (0.001, 0.01, 0.1, or 1 ng/ml) was applied to the DRG for 15 min, followed by washout for at least 30 min. Alternatively, H-89 or Rp-cAMPS, two specific cAMP-dependent protein kinase (PKA) inhibitors, was added to the perfusion solution for 15 min prior to TNF-alpha application. TNF-alpha (1 ng/ml) induced neuronal discharges in 67% (14/21) of C fibers and 27% (4/15) of Abeta fibers when applied topically to the DRG. Acute TNF-alpha application not only evoked discharges in silent fibers, but also enhanced ongoing activity of spontaneously active fibers and increased neuronal sensitivity to electrical stimulation of the peripheral nerves. H-89 (10 microM) and Rp-cAMPS (100 microM) each completely blocked the TNF-alpha-evoked response in most C and Abeta fibers tested but did not affect fiber conductivity. Our results demonstrates that exogenous inflammatory cytokines such as TNF-alpha can elicit a PKA-dependent response in sensory neurons and thus strongly suggest that endogenous TNF-alpha may contribute to the development of certain pathological pain states.     

Inhibition by the chromaffin cell-derived peptide serine-histogranin in the rat's dorsal horn

The heptadecapeptide histogranin, synthesized by adrenal chromaffin cells, is implicated in the analgesia produced by transplanting chromaffin cells into the spinal cord, including block of hyperalgesia mediated by NMDA-subtype glutamate receptors. To examine the neurophysiological basis for this analgesia, we applied the stable analog [Ser¹]-histogranin (SHG) by iontophoresis near extracellularly recorded wide-dynamic range (WDR) neurons in anesthetized rats. When SHG was applied during peripheral electrical stimulation of A and C fibers at 0.1 Hz, the C-fiber response was significantly inhibited but the A-fiber response was unaffected. SHG also opposed the NMDA-receptor-dependent post-tetanic facilitation (wind-up) of C-fiber responses produced by increasing the rate of peripheral afferent stimulation to 1 Hz for 20 s. To test whether block of NMDA-subtype receptors could be wholly or partially responsible for this suppression, SHG was applied during sequential pulsed iontophoresis of three agonists targeting distinct excitatory synaptic receptors: NMDA, kainate and substance P. All three excitatory effects were reversed by SHG; this reversal outlasted the 10–30 min observation period when higher SHG doses were applied (>60 nA). Histogranin therefore probably produces prolonged spinal analgesia by opposing the basal and potentiating synaptic effects of C-fibers on dorsal horn neurons. Actions besides or in addition to NMDA-receptor antagonism (e.g., agonism at inhibitory postsynaptic receptors or block of voltage-gated cation channels on C-fibers) are implied by the diversity of excitatory transmitters opposed by SHG.     

Microglial overexpression of the M-CSF receptor augments phagocytosis of opsonized Abeta

The role of microglia in Alzheimer's disease (AD) has come under intense scrutiny recently because microglia may clear amyloid beta (Abeta) by phagocytosis after immunization of transgenic mice. Increased expression of the macrophage colony-stimulating factor receptor (M-CSFR) is an important feature of microglia in AD and transgenic mouse models for AD. Increased expression of M-CSFR on mouse and human microglia accelerates phagocytosis of aggregated Abeta in part through macrophage scavenger receptors. We now show that Abeta phagocytosis by microglia overexpressing M-CSFR is further enhanced by antibody opsonization of Abeta. M-CSFR overexpression increased microglial phagocytosis of opsonized aggregated Abeta in culture medium, and accelerated ingestion of native Abeta from AD brain sections. M-CSFR overexpression also increased microglial expression of Fcgamma receptors, and blocking Fcgamma receptors attenuated the enhanced Abeta uptake observed after M-CSFR overexpression and antibody opsonization. Microglia in AD and in AD mouse models with increased expression of M-CSFR are likely to rapidly ingest opsonized Abeta after immunization, making high intracerebral antibody titers unnecessary.     

NK-1, but not NK-2, tachykinin receptors mediate plasma extravasation induced by antidromic C-fiber stimulation in rat hindpaw: demonstrated with the NK-1 antagonist CP-96,345 and the NK-2 antagonist Men 10207.

The effects of intradermal injection of CP-96,345 and Men 10207, selective antagonists for NK-1 and NK-2 tachykinin receptors, respectively, on the extravasation of plasma protein induced by antidromic stimulation of unmyelinated sensory fibers in the sciatic nerve was studied in rat hindpaw. Activation of unmyelinated fibers by antidromic sciatic nerve stimulation (1 Hz, 5 min) consistently evoked a localized plasma extravasation of Evans blue on the skin area of the hindpaw innervated by the sciatic nerve, which was not inhibited by intradermal injection of saline or Men 10207 (9 and 35 nmol). In contrast, CP-96,345 (3 and 9 nmol, but not 1 nmol), injected intradermally 15 min prior to nerve stimulation dose-dependently inhibited this response. Plasma extravasation induced by intravenously injected substance P was also inhibited by CP-96,345. Since CP-96,345 is a highly selective antagonist for NK-1 tachykinin receptors, it is suggested that the plasma extravasation induced by antidromic C-fiber stimulation and by systemically applied tachykinins is mediated by NK-1 tachykinin receptors.     

Differential regulation of Abeta42-induced neuronal C1q synthesis and microglial activation

Expression of C1q, an early component of the classical complement pathway, has been shown to be induced in neurons in hippocampal slices, following accumulation of exogenous Abeta42. Microglial activation was also detected by surface marker expression and cytokine production. To determine whether C1q induction was correlated with intraneuronal Abeta and/or microglial activation, D-(-)-2-amino-5-phosphonovaleric acid (APV, an NMDA receptor antagonist) and glycine-arginine-glycine-aspartic acid-serine-proline peptide (RGD, an integrin receptor antagonist), which blocks and enhances Abeta42 uptake, respectively, were assessed for their effect on neuronal C1q synthesis and microglial activation. APV inhibited, and RGD enhanced, microglial activation and neuronal C1q expression. However, addition of Abeta10-20 to slice cultures significantly reduced Abeta42 uptake and microglial activation, but did not alter the Abeta42-induced neuronal C1q expression. Furthermore, Abeta10-20 alone triggered C1q production in neurons, demonstrating that neither neuronal Abeta42 accumulation, nor microglial activation is required for neuronal C1q upregulation. These data are compatible with the hypothesis that multiple receptors are involved in Abeta injury and signaling in neurons. Some lead to neuronal C1q induction, whereas other(s) lead to intraneuronal accumulation of Abeta and/or stimulation of microglia.     

Amyloid beta prevents activation of calcium/calmodulin-dependent protein kinase II and AMPA receptor phosphorylation during hippocampal long-term potentiation

Accumulation of amyloid beta-peptides (Abeta) in the brain has been linked with memory loss in Alzheimer's disease and its animal models. However, the synaptic mechanism by which Abeta causes memory deficits remains unclear. We previously showed that acute application of Abeta inhibited long-term potentiation (LTP) in the hippocampal perforant path via activation of calcineurin, a Ca2+ -dependent protein phosphatase. This study examined whether Abeta could also inhibit Ca2+/calmodulin dependent protein kinase II (CaMKII), further disrupting the dynamic balance between protein kinase and phosphatase during synaptic plasticity. Immunoblot analysis was conducted to measure autophosphorylation of CaMKII at Thr286 and phosphorylation of the GluR1 subunit of AMPA receptors in single rat hippocampal slices. A high-frequency tetanus applied to the perforant path significantly increased CaMKII autophosphorylation and subsequent phosphorylation of GluR1 at Ser831, a CaMKII-dependent site, in the dentate area. Acute application of Abeta1-42 inhibited dentate LTP and associated phosphorylation processes, but was without effect on phosphorylation of GluR1 at Ser845, a protein kinase A-dependent site. These results suggest that activity-dependent CaMKII autophosphorylation and AMPA receptor phosphorylation are essential for dentate LTP. Disruption of such mechanisms could directly contribute to Abeta-induced deficits in hippocampal synaptic plasticity and memory.     

Amyloid-beta peptides induce several chemokine mRNA expressions in the primary microglia and Ra2 cell line via the PI3K/Akt and/or ERK pathway

Alzheimer's disease (AD) is characterized by the presence of senile plaques composed primarily of amyloid-beta peptide (Abeta) in the brain. Microglia have been reported to surround these Abeta plaques, which have opposite roles, provoking a microglia-mediated inflammatory response that contributes to neuronal cell loss or the removal of Abeta and damaged neurons. To perform these tasks microglia migrate to the sites of Abeta secretion. We herein analyzed the process of chemokine expression induced by Abeta stimulation in primary murine microglia and Ra2 microglial cell line. We found that Abeta1-42 induced the expressions of CCL7, CCL2, CCL3, CCL4 and CXCL2 in the microglia. The signal transduction pathway for the expression of CCL2 and CCL7 mRNA induced by Abeta1-42 was found to depend on phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK), whereas the pathway for CCL4 depended only on PI3K/Akt. These inflammatory chemokine expressions by Abeta stimulation emphasize the contribution of neuroinflammatory mechanisms to the pathogenesis of AD.     

GABA(A) receptor facilitation of neurokinin release from primary afferent terminals in the rat spinal cord

Our goal was to test the following hypotheses: 1) GABA_A receptors facilitate neurokinin release from primary afferent terminals; 2) they do this by suppressing an inhibitory effect of GABA_B receptors; 3) the activation of these two receptors is controlled by the firing frequency of primary afferents. We evoked neurokinin release by stimulating the dorsal root attached to spinal cord slices, and measured it using neurokinin 1 receptor (NK1R) internalization. Internalization evoked by root stimulation at 1 Hz (but not at 100 Hz) was increased by the GABA_A receptor agonists muscimol (effective concentration of drug for 50% of the increase [EC₅₀] 3 μM) and isoguvacine (EC₅₀ 4.5 μM). Internalization evoked by root stimulation at 100 Hz was inhibited by the GABA_A receptor antagonists bicuculline (effective concentration of drug for 50% of the inhibition [IC₅₀] 2 μM) and picrotoxin (IC₅₀ 243 nM). Internalization evoked by incubating the root with capsaicin (to selectively recruit nociceptive fibers) was increased by isoguvacine and abolished by picrotoxin. Therefore, GABA_A receptors facilitate neurokinin release. Isoguvacine-facilitated neurokinin release was inhibited by picrotoxin, low Cl⁻, low Ca²⁺, Ca²⁺ channel blockers and N-methyl-d-aspartate receptor antagonists. Bumetanide, an inhibitor of the Na⁺-K⁺-2Cl⁻ cotransporter, inhibited isoguvacine-facilitated neurokinin release, but this could be attributed to a direct inhibition of GABA_A receptors. The GABA_B agonist baclofen inhibited NK1R internalization evoked by 100 Hz root stimulation (IC₅₀ 1.5 μM), whereas the GABA_B receptor antagonist (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid (CGP-55845) increased NK1R internalization evoked by 1 Hz root stimulation (EC₅₀ 21 nM). Importantly, baclofen inhibited isoguvacine-facilitated neurokinin release, and CGP-55845 reversed the inhibition of neurokinin release by bicuculline. In conclusion, 1) GABA_B receptors located presynaptically in primary afferent terminals inhibit neurokinin release; 2) GABA_A receptors located in GABAergic interneurons facilitate neurokinin release by suppressing GABA release onto these GABA_B receptors; 3) high frequency firing of C-fibers stimulates neurokinin release by activating GABA_A receptors and inhibiting GABA_B receptors, whereas low frequency firing inhibits neurokinin release by the converse mechanisms.     

Neuroprotective effects of estrogen against beta-amyloid toxicity are mediated by estrogen receptors in cultured neuronal cells

Although estrogen is known to exert beneficial effects on Alzheimer's disease, its underlying cellular mechanisms have not been clear. In this study we investigated whether or not neuroprotective effects of estrogen are mediated by estrogen receptors (ERs). Treatment of estrogen (1.8 nM) reduced beta-amyloid (Abeta)-induced death of ER-expressing W4 cells. This effect of estrogen was blocked by a specific ER blocker ICI 182,780. When estrogen was treated to HT22 cells, which lack functional ERs, Abeta-induced cell death was not affected. Transfection of HT22 cells with human ERalpha, but not ERbeta, restored protective action of estrogen against Abeta. Hoechst staining revealed that estrogen protected ERalpha-expressing cells by blocking Abeta-induced apoptosis. These results indicate that estrogen blocks Abeta-induced cell death via ERalpha-dependent pathways.     

The role of non-N-methyl-D-aspartate ionotropic glutamate receptors in the spinal transmission of nociception in normal animals and animals with carrageenan inflammation.

The role of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate receptors in spinal nociceptive transmission in both normal animals and animals with carrageenan inflammation was investigated using the AMPA/kainate receptor antagonist 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione (NBQX) and the selective GluR5 kainate receptor antagonist LY382884 [3S,4aR,6S,8aR-6-(4-carboxyphenyl)methyl-1,2,3,4,4a,5,6,7,8, 8a-deca-hydroisoquinoline-3-carboxylic acid]. In normal animals, spinal administration of 100 microg of LY382884 produced a significant inhibition of both the C-fibre-evoked response and post-discharge of dorsal horn neurons, with the wind-up of the neurons being reduced by both 50 and 100 microg of LY382884. The spinal actions of LY382884 were enhanced following 3 h of carrageenan inflammation, such that doses of 20 microg and above were able to produce significant inhibitions of the noxious-evoked response of the neurons. Spinal administration of NBQX in normal animals (5-50 microg) inhibited the C-fibre-evoked response of the dorsal horn neurons, but only 50 microg of NBQX was able to inhibit the wind-up and post-discharge of the neurons. Following 3 h of carrageenan inflammation, the ability of NBQX to inhibit the wind-up and post-discharge of the neurons was markedly enhanced. These data suggest that both AMPA and kainate GluR5 receptors play an enhanced role in spinal nociceptive processing following the development of peripheral inflammation, as antagonists at both receptors are more effective against nociceptive responses, including wind-up under these inflammatory conditions.     

N-methyl-D-aspartate receptors and large conductance calcium-sensitive potassium channels inhibit the release of opioid peptides that induce mu-opioid receptor internalization in the rat spinal cord

Endogenous opioids in the spinal cord play an important role in nociception, but the mechanisms that control their release are poorly understood. To simultaneously detect all opioids able to activate the mu-opioid receptor, we measured mu-opioid receptor internalization in rat spinal cord slices stimulated electrically or chemically to evoke opioid release. Electrical stimulation of the dorsal horn in the presence of peptidase inhibitors produced mu-opioid receptor internalization in half of the mu-opioid receptor neurons. This internalization was rapidly abolished by N-methyl-D-aspartate (IC50=2 microM), and N-methyl-D-aspartate antagonists prevented this effect. mu-Opioid receptor internalization evoked by high K+ or veratridine was also inhibited by N-methyl-D-aspartate receptor activation. N-methyl-D-aspartate did not affect mu-opioid receptor internalization induced by exogenous endomorphins, confirming that the effect of N-methyl-D-aspartate was on opioid release. We hypothesized that this inhibition was mediated by large conductance Ca2+-sensitive K+ channels BK(Ca2+). Indeed, inhibition by N-methyl-D-aspartate was prevented by tetraethylammonium and by the selective BK(Ca2+) blockers paxilline, penitrem A and verruculogen. Paxilline did not increase mu-opioid receptor internalization in the absence of N-methyl-D-aspartate, indicating that it does not produce an increase in opioid release unrelated to the inhibition by N-methyl-d-aspartate. The BK(Ca2+) involved appears to be a subtype with slow association kinetics for iberiotoxin, which was effective only with long incubations. The BK(Ca2+) opener NS-1619 also inhibited the evoked mu-opioid receptor internalization, and iberiotoxin prevented this effect. We concluded that Ca2+ influx through N-methyl-D-aspartate receptors causes the opening of BK(Ca2+) and hyperpolarization in opioid-containing dorsal horn neurons, resulting in the inhibition of opioid release. Since mu-opioid receptors in the dorsal horn mediate analgesia, inhibition of spinal opioid release could contribute to the hyperalgesic actions of spinal N-methyl-D-aspartate receptors.     

Stimulation of the nucleus basalis of Meynert increases cerebral cortical blood flow in rats

Focal electrical stimulation of the magnocellular nucleus of the basal forebrain (nucleus basalis of Meynert; NBM) or a microinjection of L-glutamate (50 nmol) into the NBM increased cerebral cortical blood flow in the parietal lobe in urethane-anesthetized rats. The vasodilative responses were elicited only ipsilateral to the site of stimulation. Most of the vasodilative responses were abolished by intravenous administrations of muscarinic and nicotinic cholinergic blocking agents (atropine 0.5 mg/kg and mecamylamine 2 mg/kg). This suggests that the cholinergic projecting system sending fibers from the NBM to the parietal lobe contributes to the vasodilation of the cortex by activating muscarinic and nicotinic cholinergic receptors.     

Primary afferent fibers that contribute to increased substance P receptor internalization in the spinal cord after injury

Upon noxious stimulation, substance P (SP) is released from primary afferent fibers into the spinal cord where it interacts with the SP receptor (SPR). The SPR is located throughout the dorsal horn and undergoes endocytosis after agonist binding, which provides a spatial image of SPR-containing neurons that undergo agonist interaction. Under normal conditions, SPR internalization occurs only in SPR+ cell bodies and dendrites in the superficial dorsal horn after noxious stimulation. After nerve transection and inflammation, SPR immunoreactivity increases, and both noxious as well as nonnoxious stimulation produces SPR internalization in the superficial and deep dorsal horn. We investigated the primary afferent fibers that contribute to enhanced SPR internalization in the spinal cord after nerve transection and inflammation. Internalization evoked by electrical stimulation of the sciatic nerve was examined in untreated animals, at 14 days after sciatic nerve transection or sham surgery and at 3 days after hindpaw inflammation. Electrical stimulation was delivered at intensities to excite Abeta fibers only, Abeta and Adelta fibers or A and C fibers as determined by the compound action potential recorded from the tibial nerve. Electrical stimuli were delivered at a constant rate of 10 Hz for a duration of 5 min. Transection of the sciatic nerve and inflammation produced a 33.7 and 32.5% increase in SPR and immunoreactivity in lamina I, respectively. Under normal conditions, stimulation of Adelta or C fibers evoked internalization that was confined to the superficial dorsal horn. After transection or inflammation, there was a 20-24% increase in the proportion of SPR+ lamina I neurons that exhibited internalization evoked by stimulation of Adelta fibers. The proportion of lamina I SPR+ neurons that exhibited internalization after stimulation of C-fibers was not altered by transection or inflammation because this was nearly maximal under normal conditions. Moreover, electrical stimulation sufficient to excite C fibers evoked SPR internalization in 22% of SPR+ lamina III neurons after nerve transection and in 32-36% of SPR+ neurons in lamina III and IV after inflammation. Stimulation of Abeta fibers alone never evoked internalization in the superficial or deep dorsal horn. These results indicate that activation of small-caliber afferent fibers contributes to the enhanced SPR internalization in the spinal cord after nerve transection and inflammation and suggest that recruitment of neurons that possess the SPR contributes to hyperalgesia.     

Selective inhibition of cholera toxin- and catecholamine-stimulated lipolysis by blocking agents.

Vibrio cholerae enterotoxin stimulates lipolysis in rat epididymal fat cell suspensions. Like hormones this toxin increases adenylate cyclase activity, raising levels of cyclic adenosine 3',5'-monophosphate (cAMP), which activates a cellular lipase. Using specific blocking agents, we studied the responses to the adrenergic lipolytic hormones epinephrine, norepinephrine, and isoproterenol, and to cholera toxin. All stimulators were used at 100 x threshold dose. Propranolol (34 muM), a beta blocking agent, inhibited epinephrine stimulation (P less than 0.001) but not that of toxin (P greater than 0.2). Choleragenoid (25 mug/ml), a natural toxoid of cholera toxin, blocked stimulation by toxin (P less than 0.001) but not that of the adrenergic agents (P greater than 0.2). A beta blocker, practolol (3 mM), inhibited stimulation by the catecholamines tested (P less than 0.005) but not that of toxin (P greater than 0.05). Higher concentrations of propranolol (340 muM) and the alpha blocking agents phenoxybenzamine (3 mM) and phentolamine (1.6 mM) inhibited all agonists (P less than 0.001). The response to theophylline was inhibited by all blockers (P less than 0.05) except propranolol at the lower concentration (34 muM). A combined beta and alpha blockade using propranolol and epinephrine together did not inhibit toxin-mediated lipolysis. It appears that stimulation by cholera toxin is independent of beta adrenergic receptors. A major inhibition of theophylline-mediated lipolysis by alpha blocking drugs indicated a nonspecific effect of these agents at the concentrations used. The uninhibited response to toxin in the presence of propranolol and epinephrine suggests a lack of relationship of the toxin receptor to either alpha or beta receptors.     

NOS inhibitor antagonism of PGE2-induced mechanical sensitization of cutaneous C-fiber nociceptors in the rat

Prostaglandins, metabolites of arachidonic acid, released during tissue injury and inflammation sensitize primary afferent nociceptors. While it has been suggested that this effect on nociceptors is mediated mainly via the cAMP second messenger system, recent evidence suggests that nitric oxide (NO) is also involved in peripheral pain mechanisms. To test the hypothesis that NO contributes to the sensitization of nociceptors to mechanical stimuli induced by hyperalgesic prostaglandins, we compared von Frey hair mechanical threshold as well as the response evoked by 10-s sustained threshold mechanical stimulation before and after injection of prostaglandin E2 (PGE2) alone, and NOS inhibitor NG-methyl-L-arginine (L-NMA) or its inactive stereoisomer NG-methyl-D-arginine (D-NMA) plus PGE2, adjacent to the receptive field of C-fiber nociceptors. The reduction of mechanical threshold and increase in number of action potentials to sustained mechanical stimulation induced by intradermal application of PGE2 was blocked by L-NMA, but not D-NMA. It is suggested that NO contributes to nociceptor sensitization induced by hyperalgesic prostaglandins.     

Cerebral responses following stimulation of unmyelinated C-fibers in humans: electro- and magneto-encephalographic study

There are two kinds of pain, a sharp pain ascending through Adelta fibers (first pain) and a second burning pain ascending though C fibers (second pain). By using a novel method, the application of a low intensity CO(2) laser beam to a tiny area of skin using a very thin aluminum plate with numerous tiny holes as a spatial filter, we succeeded in selectively stimulating unmyelinated C fibers of the skin in humans, and could record consistent and clear brain responses using electroencephalography (EEG) and magnetoencephalography (MEG). The conduction velocity (CV) of the C fibers of the peripheral nerve and spinal cord, probably spinothalamic tract (STT), is approximately 1-4 m/s, which is significantly slower than that of Adelta (approximately 10-15 m/s) and Abeta fibers (approximately 50-70 m/s). This method should be very useful for clinical application. Following C fiber stimulation, primary and secondary somatosensory cortices (SI and SII) are simultaneously activated in the cerebral hemisphere contralateral to the stimulation, and then, SII in the hemisphere ipsilateral to the stimulation is activated. These early responses are easily detected by MEG. Then, probably limbic systems such as insula and cingulate cortex are activated, and those activities reflected in EEG components. Investigations of the cortical processing in pain perception including both first and second pain should provide a better understanding of pain perception and, therefore, contribute to pain relief in clinical medicine.