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Mitochondrial function is closely related to the body's oxidative stress level and lifespan. Our previous research demonstrated that the combination of blueberry extracts (BE) and apple peel extracts (APE) could synergistically promote the oxidative stress and lifespan of

In the present study, a selenium-chondroitin sulfate (SeCS) was synthesized by the sodium selenite (Na2SeO3) and ascorbic acid (Vc) redox reaction using chondroitin sulfate derived from shark cartilage as a template, and characterized by SEM, SEM-EDS, FTIR and XRD. Meanwhile, its stability was investigated at different conditions of pH and temperatures. Besides, its antioxidant activity was further determined by the DPPH and ABTS assays. The results showed the SeCS with the smallest particle size of 131.3 ± 4.4 nm and selenium content of 33.18% was obtained under the optimal condition (CS concentration of 0.1 mg/mL, mass ratio of Na2SeO3 to Vc of 1:8, the reaction time of 3 h, and the reaction temperature of 25 °C). SEM image showed the SeCS was an individual and spherical nanostructure and its structure was evidenced by FTIR and XRD. Meanwhile, SeCS remained stable at an alkaline pH and possessed good storage stability at 4 °C for 28 days. The results on scavenging free radical levels showed that SeCS exhibited significantly higher antioxidant activity than SeNPs and CS, indicating that SeCS had a potential antioxidant effect.

Increased consumption of fruit and vegetables is associated with a reduced risk of age-related functional decline and chronic diseases, which is primarily attributed to their phytochemicals. Apples and blueberries are rich in phytochemicals with a wide range of biological activities and health benefits. The objective of this study was to determine whether the combination of apple peel extracts (APE) and blueberry extracts (BE) had synergistic effects in promoting lifespan in Caenorhabditis elegans (C. elegans), and to explore the underlying mechanisms of action. The results showed that the lifespan of C. elegans treated with APE plus BE was increased by 31.4%, which is significantly more than with APE or BE alone, and extended the health span of animals, including improving motility and enhancing resistance to heat stress and UV-B radiation in C. elegans. Meanwhile, treatment with APE plus BE could regulate the expression of anti-aging related genes, and promote the migration of DAF-16 into the nucleus. In addition, administration with APE plus BE eradicated the extension of the lifespan of mutants, and inhibited the expression of the daf-16 downstream gene. In conclusion, the combination of APE and BE could synergistically promote the lifespan via the insulin signaling pathway and DAF-16 in C. elegans.

Increased consumption of fruits and vegetables is associated with a reduced risk of age-related functional decline and chronic diseases, which is primarily attributed to phytochemicals. Raspberries are rich in phytochemicals with a wide range of biological activities and health benefits. However, little is known about their effects on aging. The objective of this study was to determine whether raspberry extract (RE) could promote lifespan and stress resistance in Caenorhabditis elegans (C. elegans), and to explore the underlying mechanisms of action. The results showed that the mean lifespan of C. elegans treated with RE at 20, 40 and 80 mg mL-1 was significantly increased by 13.6%, 22.9% and 29.7%, respectively, in a dose-dependent manner. Supplementation with RE decreased the accumulation of lipofuscin and extended the healthspan of animals by improving motility and enhancing resistance to heat stress and UV-B radiation in C. elegans. Meanwhile, treatment with RE could regulate the expression of anti-aging related genes, including daf-2, age-1, akt-2, sir-2.1, daf-16, skn-1, jnk-1 and hsp-16.2, and promote the migration of DAF-16 into the nucleus. In addition, administration with RE abolished the extension of the lifespan of daf-2(e1370) mutants and RNAi (daf-16) C. elegans, and inhibited the expression of daf-16 downstream genes, including sod-3, ctl-2, dod17 and clk-1. In conclusion, RE could prolong the lifespan, improve the healthspan and enhance stress resistance in C. elegans by the insulin/IGF signaling pathway and DAF-16, providing a theoretical basis to fully exploit raspberry in the prevention of aging and healthcare.

Increased consumption of fruits and vegetables is associated with reduced risk of age-related functional declines and chronic diseases, primarily attributed to their bioactive phytochemicals. Apples and blueberries are rich in phytochemicals with a wide range of biological activities and health benefits. Our previous research has shown the combination of apple peel extracts (APE) and blueberry extracts (BE) can synergistically promote the lifespan of Caenorhabditis elegans (C. elegans). The objectives of this study were to determine whether the extension of lifespan was involved in regulation of oxidative stress, and to explore the underlying mechanisms of action. The results showed that the combination of APE and BE could synergistically ameliorate oxidative stress by improving antioxidant enzyme activities and enhancing resistance to paraquat. Meanwhile, treatment with APE plus BE could down-regulate the overexpression of reactive oxygen species (ROS) and affect the expression of antioxidant related genes, including sod-3, cat-1, ctl-1, skn-1, mev-1 and isp-1. However, administration with APE plus BE abolished the extension of the lifespan of skn-1(zu135) mutants, and inhibited the expression of skn-1 downstream genes, including gcs-1, gst-4 and gst-7. In addition, supplementation with APE plus BE could promote the migration of SKN-1 into the nucleus, which eliminated improvement to ROS and paraquat. In conclusion, the combination of APE and BE could synergistically protect against oxidative stress in C. elegans via the SKN-1/Nrf2 pathway. This study provided the theoretical basis to explore the combination of phytochemicals in the prevention of aging regulated by oxidative stress.

Demyelinating diseases, such as multiple sclerosis, are characterized by inflammatory demyelination and neurodegeneration of the central nervous system. Therapeutic strategies that induce effective neuroprotection and enhance intrinsic repair mechanisms are central goals for future therapy of multiple sclerosis. Oestrogens and oestrogen receptor ligands are promising treatments to prevent multiple sclerosis-induced neurodegeneration. In the present study we investigated the capacity of oestrogen receptor β ligand treatment to affect callosal axon demyelination and stimulate endogenous myelination in chronic experimental autoimmune encephalomyelitis using electrophysiology, electron microscopy, immunohistochemistry and tract-tracing methods. Oestrogen receptor β ligand treatment of experimental autoimmune encephalomyelitis mice prevented both histopathological and functional abnormalities of callosal axons despite the presence of inflammation. Specifically, there were fewer demyelinated, damaged axons and more myelinated axons with intact nodes of Ranvier in oestrogen receptor β ligand-treated mice. In addition, oestrogen receptor β ligand treatment caused an increase in mature oligodendrocyte numbers, a significant increase in myelin sheath thickness and axon transport. Functional analysis of callosal axon conduction showed a significant improvement in compound action potential amplitudes, latency and in axon refractoriness. These findings show a direct neuroprotective effect of oestrogen receptor β ligand treatment on oligodendrocyte differentiation, myelination and axon conduction during experimental autoimmune encephalomyelitis.

After complete spinal cord transections that removed all supraspinal inputs in adult rats, combinations of serotonergic agonists and epidural electrical stimulation were able to acutely transform spinal networks from nonfunctional to highly functional and adaptive states as early as 1 week after injury. Using kinematics, physiological and anatomical analyses, we found that these interventions could recruit specific populations of spinal circuits, refine their control via sensory input and functionally remodel these locomotor pathways when combined with training. The emergence of these new functional states enabled full weight-bearing treadmill locomotion in paralyzed rats that was almost indistinguishable from voluntary stepping. We propose that, in the absence of supraspinal input, spinal locomotion can emerge from a combination of central pattern-generating capability and the ability of these spinal circuits to use sensory afferent input to control stepping. These findings provide a strategy by which individuals with spinal cord injuries could regain substantial levels of motor control.

Spinal cord injuries (SCIs) in humans1,2 and experimental animals3–6 are often associated with varying degrees of spontaneous functional recovery during the first months after injury. Such recovery is widely attributed to axons spared from injury that descend from the brain and bypass incomplete lesions, but its mechanisms are uncertain. To investigate the neural basis of spontaneous recovery, we used kinematic, physiological and anatomical analyses to evaluate mice with various combinations of spatially and temporally separated lateral hemisections with or without the excitotoxic ablation of intrinsic spinal cord neurons. We show that propriospinal relay connections that bypass one or more injury sites are able to mediate spontaneous functional recovery and supraspinal control of stepping, even when there has been essentially total and irreversible interruption of long descending supraspinal pathways in mice. Our findings show that pronounced functional recovery can occur after severe SCI without the maintenance or regeneration of direct projections from the brain past the lesion and can be mediated by the reorganization of descending and propriospinal connections4,7–9. Targeting interventions toward augmenting the remodeling of relay connections may provide new therapeutic strategies to bypass lesions and restore function after SCI and in other conditions such as stroke and multiple sclerosis.

Calcitonin gene-related peptide (CGRP) is abundant in the central terminals of primary afferents. However, the function of CGRP receptors in the spinal cord remains unclear. CGRP receptors are heterodimers of calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 1 (RAMP1). We studied the localization of CRLR and RAMP1 in the rat dorsal horn using well-characterized antibodies against them, which labeled numerous puncta in laminae I-II. In addition, RAMP1 was found in cell bodies, forming patches at the cell surface. The CRLR- and RAMP1-immunoreactive puncta were further characterized using double and triple labeling. Colocalization was quantified in confocal stacks using Imaris software. CRLR did not colocalize with primary afferent markers, indicating that these puncta were not primary afferent terminals. CRLR- and RAMP1-immunoreactive puncta contained synaptophysin and vesicular glutamate transporter-2 (VGLUT2), showing that they were glutamatergic presynaptic terminals. Electron microscopic immunohistochemistry confirmed that CRLR immunoreactivity was present in axonal boutons that were not in synaptic glomeruli. Using tyramide signal amplification for double labeling with the CRLR and RAMP1 antibodies, we found some clear instances of colocalization of CRLR with RAMP1 in puncta, but their overall colocalization was low. In particular, CRLR was absent from RAMP1-containing cells. Many of the puncta stained for CRLR and RAMP1 were labeled by anti-opioid and anti-enkephalin antibodies. CRLR and, to a lesser extent, RAMP1 also colocalized with adrenergic alpha(2C) receptors. Triple label studies demonstrated three-way colocalization of CRLR-VGLUT2-synaptophysin, CRLR-VGLUT2-opioids, and CRLR-opioids-alpha(2C) receptors. In conclusion, CRLR is located in glutamatergic presynaptic terminals in the dorsal horn that contain alpha(2C) adrenergic receptors and opioids. Some of these terminals contain RAMP1, which may form CGRP receptors with CRLR, but in others CRLR may form other receptors, possibly by dimerizing with RAMP2 or RAMP3. These findings suggest that CGRP or adrenomedullin receptors modulate opioid release in the dorsal horn.

Neurotransmitter receptors that inhibit the release of opioid peptides in the spinal cord may play an important role in modulating pain. Serotonin is an important neurotransmitter in bulbospinal descending pathways, and 5-HT(1) receptors have been shown to inhibit synaptic transmission. Our goal was to determine whether 5-HT(1A) receptors inhibit opioid release in the spinal cord. Opioid release was evoked from rat spinal cord slices by electrically stimulating one dorsal horn, and measured in situ through the internalization of micro-opioid receptors in dorsal horn neurons. Stimulation with 1000 square pulses at 500 Hz produced internalization in 60% of the mu-opioid receptor neurons in the stimulated dorsal horn, but not in the contralateral one. The selective 5-HT(1A) receptor agonist 8-hydroxy-2-dipropylaminotetralin (8-OH-DPAT) inhibited the evoked mu-opioid receptor internalization by about 50%, with an approximate IC(50) of 50 nM. The effect of 8-OH-DPAT was attributed to inhibition of opioid release and not of the receptor internalization process, because 8-OH-DPAT did not inhibit the internalization induced by incubating the slices with a micro-opioid receptor agonist (endomorphin-2, 100 nM). The selective 5-HT(1A) receptor antagonist WAY100135 (10 microM) blocked the inhibition produced by 1 microM 8-OH-DPAT. These results show that 5-HT(1A) receptors inhibit opioid release in the spinal dorsal horn, probably from a subpopulation of enkephalin-containing presynaptic terminals. Therefore, 5-HT(1A) receptors likely decrease the analgesia produced by endogenously released opioids.

Opioid μ- and δ-receptors are present on the central terminals of primary afferents, where they are thought to inhibit neurotransmitter release. This mechanism may mediate analgesia produced by spinal opiates; however, when they used neurokinin 1 receptor (NK1R) internalization as an indicator of substance P release, Trafton et al. (1999) noted that this evoked internalization was altered only modestly by morphine delivered intrathecally at spinal cord segment S1-S2. We reexamined this issue by studying the effect of opiates on NK1R internalization in spinal cord slices andin vivo. In slices, NK1R internalization evoked by dorsal root stimulation at C-fiber intensity was abolished by the μ agonist [d-Ala2,N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO) (1 μm) and decreased by the δ agonist [d-Phe2,5]-enkephalin (DPDPE) (1 μm).In vivo, hindpaw compression induced NK1R internalization in ipsilateral laminas I-II. This evoked internalization was significantly reduced by morphine (60 nmol), DAMGO (1 nmol), and DPDPE (100 nmol), but not by the κ agonisttrans-(1S,2S)-3,4-dichloro-N-mathyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide hydrochloride (200 nmol), delivered at spinal cord segment L2 using intrathecal catheters. These doses of the μ and δ agonists were equi-analgesic as measured by a thermal escape test. Lower doses neither produced analgesia nor inhibited NK1R internalization. In contrast, morphine delivered by percutaneous injections at S1-S2 had only a modest effect on thermal escape, even at higher doses. Morphine decreased NK1R internalization after systemic delivery, but at a dose greater than that necessary to produce equivalent analgesia. All effects were reversed by naloxone. These results indicate that lumbar opiates inhibit noxious stimuli-induced neurotransmitter release from primary afferents at doses that are confirmed behaviorally as analgesic.

To determine what neural pathways trigger opioid release in the dorsal horn, we stimulated the dorsal root, the dorsal horn, or the dorsolateral funiculus (DLF) in spinal cord slices while superfusing them with peptidase inhibitors to prevent opioid degradation. Internalization of μ-opioid receptors (MOR) and neurokinin 1 receptors (NK1R) was measured to assess opioid and neurokinin release, respectively. Dorsal root stimulation at low, high, or mixed frequencies produced abundant NK1R internalization but no MOR internalization, indicating that primary afferents do not release opioids. Moreover, capsaicin and NMDA also failed to produce MOR internalization. In contrast, dorsal horn stimulation elicited MOR internalization that increased with the frequency, being negligible at d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2(CTAP), in the presence of low Ca2+and by the Na+channel blocker lidocaine, confirming that it was caused by opioid release and neuronal firing. DLF stimulation in “oblique” slices (encompassing the DLF and the dorsal horn of T11-L4) produced MOR internalization, but only in areas near the stimulation site. Moreover, cutting oblique slices across the dorsal horn (but not across the DLF) eliminated MOR internalization in areas distal to the cut, indicating that it was produced by signals traveling in the dorsal horn and not via the DLF. These findings demonstrate that some dorsal horn neurons release opioids when they fire at high frequencies, perhaps by integrating signals from the rostral ventromedial medulla, primary afferents, and other areas of the spinal cord.

Capsaicin stimulates neurokinin release in the spinal cord when applied both centrally and peripherally. To determine whether these two actions have different mechanisms, we measured neurokinin 1 receptor (NK1R) internalization in rat spinal cord slices elicited by incubating the whole slice or just the dorsal root with capsaicin. NK1R internalization produced by incubating the slices with capsaicin was abolished by the NK1R antagonist RP-67580, by the vanilloid receptor 1 (VR1) antagonist capsazepine, and by eliminating Ca(2+) from the medium, but was not affected by the Na(+) channel blocker lidocaine. Therefore, the internalization was due to neurokinin release mediated by Ca(2+) entry through VR1 receptors, but did not require the firing of action potentials. Incubating the root with capsaicin produced NK1R internalization in the ipsilateral dorsal horn that was abolished when capsazepine or lidocaine was included in, or when Ca(2+) was omitted from, the medium surrounding the root. Therefore, the internalization was mediated by Ca(2+) entry in the axons through VR1, and required firing of action potentials. The efficacy of capsaicin when applied to the root (36+/-3%) was lower than when applied to the slice (91+/-3%), but its potency was the same (0.49 microM and 0.37 microM, respectively). We also investigated whether presynaptic N-methyl-D-aspartate (NMDA) and GABA(B) receptors modulate these two actions of capsaicin. Neither the NMDA receptor blocker MK-801 nor the GABA(B) agonist baclofen decreased NK1R internalization produced by 1 microM capsaicin applied to the slices, but they inhibited the internalization produced by 0.3 microM capsaicin applied to the slices or 1 microM capsaicin applied to the root. Therefore, capsaicin can produce neurokinin release from primary afferents 1) by a direct action on their central terminals and 2) by increasing the firing of action potentials on their axons. The first effect largely bypasses other modulatory mechanism, but the second does not.

Astroglial scars surround damaged tissue after trauma, stroke, infection, or autoimmune inflammation in the CNS. They are essential for wound repair, but also interfere with axonal regrowth. A better understanding of the cellular mechanisms, regulation, and functions of astroglial scar formation is fundamental to developing safe interventions for many CNS disorders. We used wild-type and transgenic mice to quantify and dissect these parameters. Adjacent to crush spinal cord injury (SCI), reactive astrocytes exhibited heterogeneous phenotypes as regards proliferation, morphology, and chemistry, which all varied with distance from lesions. Mature scar borders at 14 d after SCI consisted primarily of newly proliferated astroglia with elongated cell processes that surrounded large and small clusters of inflammatory, fibrotic, and other cells. During scar formation from 5 to 14 d after SCI, cell processes deriving from different astroglia associated into overlapping bundles that quantifiably reoriented and organized into dense mesh-like arrangements. Selective deletion of STAT3 from astroglia quantifiably disrupted the organization of elongated astroglia into scar borders, and caused a failure of astroglia to surround inflammatory cells, resulting in increased spread of these cells and neuronal loss. In cocultures, wild-type astroglia spontaneously corralled inflammatory or fibromeningeal cells into segregated clusters, whereas STAT3-deficient astroglia failed to do so. These findings demonstrate heterogeneity of reactive astroglia and show that scar borders are formed by newly proliferated, elongated astroglia, which organize via STAT3-dependent mechanisms to corral inflammatory and fibrotic cells into discrete areas separated from adjacent tissue that contains viable neurons.

Biomaterial vehicles that can provide sustained, site-specific molecular delivery in the central nervous system (CNS) have potential for therapeutic and investigative applications. Here, we present in vitro and in vivo proof of principle tests of diblock copolypeptide hydrogels (DCH) to serve as depots for sustained local release of protein effector molecules. We tested two DCH, K(180)L(20) and E(180)L(20), previously shown to self-assemble into biocompatible, biodegradable deposits that persist four to eight weeks after injection into mouse forebrain. In vitro tests demonstrated sustained release from dialysis cassettes of the representative protein, lysozyme, dissolved in K(180)L(20) or E(180)L(20) hydrogels. Release time in vitro varied in relation to DCH charge and mechanical properties, and ionic strength of the media. To evaluate bioactive protein delivery in vivo, we used nerve growth factor (NGF) and measured the size of mouse forebrain cholinergic neurons, which respond to NGF with cellular hypertrophy. For in vivo tests, the storage modulus of DCH depots was tuned to just below that of CNS tissue. In comparison with NGF injected in buffer, depots of NGF dissolved in either K(180)L(20) or E(180)L(20) provided significantly longer delivery of NGF bioactivity, maintaining hypertrophy of local forebrain cholinergic neurons for at least 4 weeks and inducing hypertrophy a further distance away (up to 5 mm) from injection sites. These findings show that depots of DCH injected into CNS can provide sustained delivery within the blood-brain barrier of a bioactive protein growth factor that exerts a predicted, quantifiable effect on local cells over a prolonged subacute time.

Amphiphilic diblock copolypeptide hydrogels (DCHs) are synthetic materials whose properties can be varied readily and predictably by altering copolymer chain length or composition and which are of potential interest for biomaterial applications. We tested the biocompatibility in the central nervous system (CNS) of DCH composed of lysine, homoarginine or glutamate in combination with leucine. A range of DCH formulations with rheological properties similar to brain tissue were injected into mouse forebrain and examined after 1-8 weeks using light microscopy, immunohistochemistry and electron microscopy. DCH deposits elicited no more gliosis, inflammation, or toxicity to neurons, myelin or axons than did injections of physiological saline. The size, rigidity, and density of DCH deposits could be varied subtly by altering DCH composition and concentration. For any given DCH formulation, increased concentration correlated with increased gel strength in vitro and increased deposit size in vivo. DCHs of lysine and leucine (K(m)L(n)) were selected for detailed analyses because these formed deposits with desirable physical properties and since lysine is routinely used as a substrate for neural cell cultures. Deposits of unmodified K(180)L(20) exhibited time-dependent in-growth of blood vessels and of certain glial cells, and limited in-growth of nerve fibers. These findings show that DCHs are injectable, re-assemble in vivo to form 3-dimensional deposits, exhibit little or no detectable toxicity in the CNS, integrate well with brain tissue and represent a new class of synthetic biomaterials with potential for applications as depots or scaffolds in the CNS.

Signaling mechanisms that regulate astrocyte reactivity and scar formation after spinal cord injury (SCI) are not well defined. We used the Cre recombinase (Cre)-loxPsystem under regulation of the mouse glial fibrillary acidic protein (GFAP) promoter to conditionally delete the cytokine and growth factor signal transducer, signal transducer and activator of transcription 3 (STAT3), from astrocytes. After SCI in GFAP-Cre reporter mice, >99% of spinal cord cells that exhibited Cre activity as detected by reporter protein expression were GFAP-expressing astrocytes. Conditional deletion (or knock-out) of STAT3 (STAT3-CKO) from astrocytes in GFAP-Cre-loxPmice was confirmedin vivoandin vitro. In uninjured adult STAT3-CKO mice, astrocytes appeared morphologically similar to those in STAT3+/+ mice except for a partially reduced expression of GFAP. In STAT3+/+ mice, phosphorylated STAT3 (pSTAT3) was not detectable in astrocytes in uninjured spinal cord, and pSTAT3 was markedly upregulated after SCI in astrocytes and other cell types near the injury. Mice with STAT3-CKO from astrocytes exhibited attenuated upregulation of GFAP, failure of astrocyte hypertrophy, and pronounced disruption of astroglial scar formation after SCI. These changes were associated with increased spread of inflammation, increased lesion volume and partially attenuated motor recovery over the first 28 d after SCI. These findings indicate that STAT3 signaling is a critical regulator of certain aspects of reactive astrogliosis and provide additional evidence that scar-forming astrocytes restrict the spread of inflammatory cells after SCI.

Neurotransmitter receptors that control the release of opioid peptides in the spinal cord may play an important role in pain modulation. Norepinephrine, released by a descending pathway originating in the brainstem, is a powerful inducer of analgesia in the spinal cord. Adrenergic alpha2C receptors are present in opioid-containing terminals in the dorsal horn, where they could modulate opioid release. The goal of this study was to investigate this possibility. Opioid release was evoked from rat spinal cord slices by incubating them with the sodium channel opener veratridine in the presence of peptidase inhibitors (actinonin, captopril and thiorphan), and was measured in situ through the internalization of mu-opioid receptors in dorsal horn neurons. Veratridine produced internalization in 70% of these neurons. The alpha2 receptor agonists clonidine, guanfacine, medetomidine and UK-14304 inhibited the evoked mu-opioid receptor internalization with IC50s of 1.7 microM, 248 nM, 0.3 nM and 22 nM, respectively. However, inhibition by medetomidine was only partial, and inhibition by UK-14304 reversed itself at concentrations higher than 50 nM. None of these agonists inhibited mu-opioid receptor internalization produced by endomorphin-2, showing that they inhibited opioid release and not the internalization itself. The inhibitions produced by clonidine, guanfacine or UK-14304 were completely reversed by the selective alpha2C antagonist JP-1203. In contrast, inhibition by guanfacine was not prevented by the alpha2A antagonist BRL-44408. These results show that alpha2C receptors inhibit the release of opioids in the dorsal horn. This action may serve to shut down the opioid system when the adrenergic system is active.

Veratridine and high K+-induced micro-opioid receptor (MOR) internalization in rat spinal cord slices by evoking opioid release. Veratridine induced up to 75% MOR internalization but showed an atypical concentration-response: its effect increased steeply from 5 microM to 10 microM, and declined thereafter to disappear at 100 microM. At 100 microM, veratridine also inhibited of MOR internalization induced by exogenous endomorphin-2. This inhibition was caused by Na+ entry, since the Na+ ionophore monensin (50 microM) also inhibited endomorphin-induced MOR internalization. In contrast, veratridine induced neurokinin 1 receptor internalization (by evoking substance P release) without any inhibition at high concentrations. KCl evoked up to 80% MOR internalization, which disappeared in the presence of lidocaine or in the absence of peptidase inhibitors, indicating that it involved neuronal firing and peptide release. Unlike veratridine, KCl did not inhibit MOR internalization at high concentrations. However, both KCl and veratridine evoked more MOR internalization when applied for 2 min than for 20 min because of a direct inhibition of MOR internalization with the longer incubation times. These results show that short incubations with 20 microM veratridine or 30 mM KCl are optimal stimuli to evoke opioid release and MOR internalization in the spinal cord.

The internalization of mu-opioid receptors (MORs) provides an ideal way to locate areas of opioid peptide release. We used this method to study opioid release in the spinal cord evoked by noxious stimuli in anesthetized rats. Previous studies have shown that opioids released in the spinal cord produce MOR internalization only when they are protected from peptidase degradation. Accordingly, rats were implanted with chronic intrathecal catheters that were used to inject a mixture of peptidase inhibitors (amastatin, captopril and phosphoramidon) onto the lumbar spinal cord. Five minutes later, a noxious stimulus was delivered to the paw. Lumbar spinal segments were double-stained with antibodies against MORs and neurokinin 1 receptors (NK1Rs) using immunofluorescence. Mechanical stimulation of the hindpaw consisted of repeated 10 s clamps with a hemostat for 10 min. In the ipsilateral dorsal horn, the stimulus produced abundant NK1R internalization in segments L3-L6, and a more modest but significant MOR internalization in segments L5 and L6. In the contralateral dorsal horn, NK1R was substantially lower and MOR internalization was negligible. The same mechanical stimulus applied to a forepaw did not produce NK1R or MOR internalization in the lumbar spinal cord. Thermal stimulation consisted of immersing a hindpaw in water at 52 degrees C for 2 min. It produced substantial NK1R internalization ipsilaterally in segment L6, but no MOR internalization. These results show that mechanical stimulation induces segmental opioid release, i.e., in the dorsal horn receiving the noxious signals and not in other spinal segments.

Opioid receptors in the spinal cord produce strong analgesia, but the mechanisms controlling their activation by endogenous opioids remain unclear. We have previously shown in spinal cord slices that peptidases preclude mu-opioid receptor (MOR) internalization by opioids. Our present goals were to investigate whether enkephalin-induced analgesia is also precluded by peptidases, and whether it is mediated by MORs or delta-opioid receptors (DORs). Tail-flick analgesia and MOR internalization were measured in rats injected intrathecally with Leu-enkephalin and peptidase inhibitors. Without peptidase inhibitors, Leu-enkephalin produced neither analgesia nor MOR internalization at doses up to 100 nmol, whereas with peptidase inhibitors it produced analgesia at 0.3 nmol and MOR internalization at 1 nmol. Leu-enkephalin was 10 times more potent to produce analgesia than to produce MOR internalization, suggesting that DORs were involved. Selective MOR or DOR antagonists completely blocked the analgesia elicited by 0.3 nmol Leu-enkephalin (a dose that produced little MOR internalization), indicating that it involved these two receptors, possibly by an additive or synergistic interaction. The selective MOR agonist endomorphin-2 produced analgesia even in the presence of a DOR antagonist, but at doses substantially higher than Leu-enkephalin. Unlike Leu-enkephalin, endomorphin-2 had the same potencies to induce analgesia and MOR internalization. We concluded that low doses of enkephalins produce analgesia by activating both MORs and DORs. Analgesia can also be produced exclusively by MORs at higher agonist doses. Since peptidases prevent the activation of spinal opioid receptors by enkephalins, the coincident release of opioids and endogenous peptidase inhibitors may be required for analgesia.

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 μ-opioid receptor, we measured μ-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 μ-opioid receptor internalization in half of the μ-opioid receptor neurons. This internalization was rapidly abolished by N -methyl- d -aspartate (IC 50 =2μM), and N -methyl- d -aspartate antagonists prevented this effect. μ-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 μ-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 Ca 2+ -sensitive K + channels BK(Ca 2+ ). Indeed, inhibition by N -methyl- d -aspartate was prevented by tetraethylammonium and by the selective BK(Ca 2+ ) blockers paxilline, penitrem A and verruculogen. Paxilline did not increase μ-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(Ca 2+ ) involved appears to be a subtype with slow association kinetics for iberiotoxin, which was effective only with long incubations. The BK(Ca 2+ ) opener NS-1619 also inhibited the evoked μ-opioid receptor internalization, and iberiotoxin prevented this effect. We concluded that Ca 2+ influx through N -methyl- d -aspartate receptors causes the opening of BK(Ca 2+ ) and hyperpolarization in opioid-containing dorsal horn neurons, resulting in the inhibition of opioid release. Since μ-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.

The ability of peptidases to restrict neurokinin 1 receptor (NK1R) activation by exogenously applied or endogenously released neurokinins was investigated by measuring NK1R internalization in rat spinal cord slices. Concentration-response curves for substance P and neurokinin A were obtained in the presence and absence of 10 microm thiorphan, an inhibitor of neutral endopeptidase (EC 3.4.24.11), plus 10 microm captopril, an inhibitor of dipeptidyl carboxypeptidase (EC 3.4.15.1). These inhibitors significantly decreased the EC50 of substance P to produce NK1R internalization from 32 to 9 nm, and the EC50 of neurokinin A from 170 to 60 nm. Substance P was significantly more potent than neurokinin A, both with and without these peptidase inhibitors. In the presence of peptidase inhibitors, neurokinin B was 10 times less potent than neurokinin A and 64 times less potent than substance P (EC50=573 nm). Several aminopeptidase inhibitors (actinonin, amastatin, bacitracin, bestatin and puromycin) failed to further increase the effect of thiorphan plus captopril on the NK1R internalization produced by 10 nm substance P. Electrical stimulation of the dorsal root produced NK1R internalization by releasing endogenous neurokinins. Thiorphan plus captopril increased NK1R internalization produced by 1 Hz stimulation, but not by 30 Hz stimulation. Therefore, NEN and DCP restrict NK1R activation by endogenous neurokinins when they are gradually released by low-frequency firing of primary afferents, but become saturated or inhibited when primary afferents fire at a high frequency.

N-methyl-D-aspartate (NMDA) receptors in sensory afferents participate in chronic pain by mediating peripheral and central sensitization. We studied the presence of NMDA receptor subunits in different types of primary afferents. Western blots indicated that rat dorsal root ganglia (DRG) contain NR1, NR2B, NR2C, and NR2D but not NR2A. Real-time RT-PCR showed that NR2B and NR2D were expressed at higher levels than NR2A and NR2C in DRG. Immunofluorescence with an antibody that recognized NR1 and another that recognized NR2A and NR2B showed that NR1 and NR2B colocalized in 90% of DRG neurons, including most A-fibers (identified by the presence of neurofilament 200 kDa). In contrast, an antibody recognizing NR2C and NR2D labeled only neurofilament-negative DRG profiles. This antibody stained practically all DRG cells that contained calcitonin gene-related peptide and neurokinins and those that bound isolectin B4. The percentage of cells immunoreactive for NR1, NR2A/NR2B, and NR2C/NR2D were the same in the T9, T12, L4, and L6 DRG. The intracellular distribution of the NR2 subunits was strikingly different: Whereas NR2A/NR2B immunoreactivity was found in the Golgi apparatus and occasionally at the plasma membrane, NR2C/NR2D immunoreactivity was found in the cytoplasm but not in the Golgi. The NR1 subunit was present throughout the cytoplasm and was more intense in the Golgi. These findings indicate that DRG neurons have two different NMDA receptors, one containing the NR1, NR2D, and possibly the NR2C subunits, found only in C-fibers, and the diheteromer NR1/NR2B, present in the Golgi apparatus of both A- and C-fibers.