Different effects of local anesthetics on extracellular signal-regulated kinase phosphorylation in rat dorsal horn neurons.

Local anesthetics, which are widely known to be neuronal voltage-gated Na(+) channel blockers, also affect a variety of other ion channels, N-methyl-d-asparate (NMDA) receptors and α-amino-3-hydroxy-5-methyl-4-izoxazolepropionic acid (AMPA) receptors. Glutamate, which is released from presynaptic fibers, activates extracellular signal-regulated kinase (ERK) through NMDA and AMPA receptors in spinal dorsal horn neurons. ERK plays a key role in central sensitization, which contributes to the chronicity of pain. We investigated the effects of four representative local anesthetics, lidocaine, tetracaine, levobupivacaine, and ropivacaine on ERK phosphorylation induced by capsaicin, which releases glutamate from presynaptic neurons, NMDA, AMPA, or ionomycin, a calcium ionophore, in dorsal neurons. We observed capsaicin-induced phosphorylation of ERK, which was suppressed by lidocaine, tetracaine, or ropivacaine, but not by levobupivacaine. NMDA-induced phosphorylation of ERK was suppressed by lidocaine, tetracaine, or levobupivacaine, but not by ropivacaine. AMPA-induced phosphorylation of ERK was suppressed by lidocaine or tetracaine, but not by levobupivacaine or ropivacaine. Finally, ionomycin-induced ERK phosphorylation was suppressed by lidocaine, tetracaine, or ropivacaine, but not by levobupivacaine. Our results suggest that local anesthetics contribute to the prevention of the incidence of persistent postsurgical pain with varying intensities and through different mechanisms of action.

Intrathecal endothelin-1 has antinociceptive effects in rat model of postoperative pain.

Endothelin-1 is known to be a potent vasoconstrictor. Administration of endothelin-1 to the central nervous system (CNS) induces antinociceptive effects. Nociceptive stimuli affect dorsal root ganglion (DRG) neurons and neurons/astrocytes/microglia in the dorsal horn of the spinal cord. Surgical incision in the plantar aspect of the rat hindpaw is a model for postoperative pain, and withdrawal thresholds reportedly decrease around the incision. We hypothesized that intrathecal endothelin-1 would have antinociceptive effects in this model, and affect DRG neurons and microglia/neurons in the dorsal horn. Intrathecal endothelin-1 partially restored the withdrawal threshold (which was decreased by plantar incision). BQ-123, and BQ-788 (specific endothelin ET(A)- and ET(B)-receptor antagonists, respectively) attenuated the increase in withdrawal threshold induced by endothelin-1. Phosphorylation of extracellular signal-regulated kinase (ERK) in DRG neurons and microglial activation/ERK phosphorylation in the dorsal horn were observed following the incision. Endothelin-1 decreased the incision-induced increase in the numbers of phosphorylated ERK-positive neurons in DRG and activated microglia in the dorsal horn, without affecting the numbers of phosphorylated ERK-positive neurons in the dorsal horn. BQ-123 or BQ-788 partially suppressed these endothelin-1-induced alterations. Our results show that the pain threshold, which is decreased by surgical stimuli, is partially restored by intrathecal endothelin-1 through both endothelin ET(A)- and ET(B)- receptors in DRG neurons and microglia in the spinal cord. Endothelin-1 administration to the CNS may be worth considering as a new candidate for the treatment of postoperative pain and to mitigate prolonged periods of pain.

The mu opioid receptor modulates neurotransmission in the rat spinal ventral horn.

BACKGROUND: Opioids inhibit excitatory neurotransmission and produce antinociception through µ opioid receptors (MORs). Although MORs are expressed in the spinal ventral horn, their functions and effects are largely unknown. Therefore, we examined the neuromodulatory effects of µ opioids in spinal lamina IX neurons at the cellular level. METHODS: The effects of the selective µ agonist [D-Ala(2),-N-Me-Phe(4), Gly(5)-ol]enkephalin (DAMGO) on synaptic transmission were examined in spinal lamina IX neurons of neonatal rats using the whole-cell patch-clamp technique. RESULTS: DAMGO produced outward currents in 56% of the lamina IX neurons recorded, with a 50% effective concentration of 0.1 µM. Analysis of the current-voltage relationship revealed a reversal potential of approximately -86 mV. These currents were not blocked by tetrodotoxin but were inhibited by Ba(2+) or a selective µ antagonist. Moreover, the currents were suppressed by the addition of Cs(+) and tetraethylammonium or guanosine 5'-[ß-thio]diphosphate trilithium salt to the pipette solution. In addition, DAMGO decreased the frequency of spontaneous excitatory and inhibitory postsynaptic currents, and these effects were unaltered by treatment with tetrodotoxin. CONCLUSION: Our results suggest that DAMGO hyperpolarizes spinal lamina IX neurons by G protein-mediated activation of K(+) channels after activation of MORs. Furthermore, activation of MORs on presynaptic terminals reduces both excitatory and inhibitory transmitter release. Although traditionally opioids are not thought to affect motor function, the present study documents neuromodulatory effects of µ opioids in spinal lamina IX neurons, suggesting that MORs can influence motor activity.

Organization of intralaminar and translaminar neuronal connectivity in the superficial spinal dorsal horn.

The spinal dorsal horn exhibits a high degree of intrinsic connectivity that is critical to its role in the processing of nociceptive information. To examine the spatial organization of this intrinsic connectivity, we used laser-scanning photostimulation in parasagittal and transverse slices of lumbar spinal cord to stimulate presynaptic neurons by glutamate uncaging, and mapped the location of sites that provide excitatory and inhibitory synaptic input to neurons of the superficial laminae. Excitatory interneuronal connectivity within lamina II exhibited a pronounced sagittal orientation, in keeping with the somatotopic organization present in the pattern of primary afferent projections. Excitatory inputs to all classes of lamina II neurons arose from a wider rostrocaudal area than inhibitory inputs, whereas both excitatory and inhibitory input zones were restricted mediolaterally. Lamina I-II neurons exhibited cell type-specific patterns in the laminar distribution of their excitatory inputs that were related to their dorsoventral dendritic expanse. All cell types received excitatory input predominantly from positions ventral to that of their soma, but in lamina I neurons and lamina II vertical cells this ventral displacement of the excitatory input zone was greater than in the other cell types, resulting in a more pronounced translaminar input pattern. A previously unknown excitatory input to the superficial dorsal horn from lamina III-IV was identified in a subset of the vertical cell population. These results reveal a specific three-dimensional organization in the local patterns of excitatory and inhibitory connectivity that has implications for the processing of information related to both somatotopy and sensory modality.

Cytokine mechanisms of central sensitization: distinct and overlapping role of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha in regulating synaptic and neuronal activity in the superficial spinal cord.

Central sensitization, increased sensitivity in spinal cord dorsal horn neurons after injuries, plays an essential role in the induction and maintenance of chronic pain. However, synaptic mechanisms underlying central sensitization are incompletely known. Growing evidence suggests that proinflammatory cytokines (PICs), such as interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNFalpha), are induced in the spinal cord under various injury conditions and contribute to pain hypersensitivity. Using patch-clamp recordings in lamina II neurons of isolated spinal cord slices, we compared the effects of IL-1beta, IL-6, and TNFalpha on excitatory and inhibitory synaptic transmission. Whereas TNFalpha enhanced the frequency of spontaneous EPSCs (sEPSCs), IL-6 reduced the frequency of spontaneous IPSCs (sIPSCs). Notably, IL-1beta both enhanced the frequency and amplitude of sEPSCs and reduced the frequency and amplitude of sIPSCs. Consistently, TNFalpha and IL-1beta enhanced AMPA- or NMDA-induced currents, and IL-1beta and IL-6 suppressed GABA- and glycine-induced currents. Furthermore, all the PICs increased cAMP response element-binding protein (CREB) phosphorylation in superficial dorsal horn neurons and produced heat hyperalgesia after spinal injection. Surprisingly, soluble IL-6 receptor (sIL-6R) produced initial decrease of sEPSCs, followed by increase of sEPSCs and CREB phosphorylation. Spinal injection of sIL-6R also induced heat hyperalgesia that was potentiated by coadministration with IL-6. Together, our data have demonstrated that PICs induce central sensitization and hyperalgesia via distinct and overlapping synaptic mechanisms in superficial dorsal horn neurons either by increasing excitatory synaptic transmission or by decreasing inhibitory synaptic transmission. PICs may further induce long-term synaptic plasticity through CREB-mediated gene transcription. Blockade of PIC signaling could be an effective way to suppress central sensitization and alleviate chronic pain.

Distinct roles of matrix metalloproteases in the early- and late-phase development of neuropathic pain.

Treatment of neuropathic pain, triggered by multiple insults to the nervous system, is a clinical challenge because the underlying mechanisms of neuropathic pain development remain poorly understood. Most treatments do not differentiate between different phases of neuropathic pain pathophysiology and simply focus on blocking neurotransmission, producing transient pain relief. Here, we report that early- and late-phase neuropathic pain development in rats and mice after nerve injury require different matrix metalloproteinases (MMPs). After spinal nerve ligation, MMP-9 shows a rapid and transient upregulation in injured dorsal root ganglion (DRG) primary sensory neurons consistent with an early phase of neuropathic pain, whereas MMP-2 shows a delayed response in DRG satellite cells and spinal astrocytes consistent with a late phase of neuropathic pain. Local inhibition of MMP-9 by an intrathecal route inhibits the early phase of neuropathic pain, whereas inhibition of MMP-2 suppresses the late phase of neuropathic pain. Further, intrathecal administration of MMP-9 or MMP-2 is sufficient to produce neuropathic pain symptoms. After nerve injury, MMP-9 induces neuropathic pain through interleukin-1beta cleavage and microglial activation at early times, whereas MMP-2 maintains neuropathic pain through interleukin-1beta cleavage and astrocyte activation at later times. Inhibition of MMP may provide a novel therapeutic approach for the treatment of neuropathic pain at different phases.

Neurokinin-1 receptor enhances TRPV1 activity in primary sensory neurons via PKCepsilon: a novel pathway for heat hyperalgesia.

The neuropeptide substance P (SP) is expressed in unmyelinated primary sensory neurons and represents the best known "pain" neurotransmitter. It is generally believed that SP regulates pain transmission and sensitization by acting on neurokinin-1 receptor (NK-1), which is expressed in postsynaptic dorsal horn neurons. However, the expression and role of NK-1 in primary sensory neurons are not clearly characterized. Our data showed that NK-1 was expressed in both intact and dissociated dorsal root ganglion (DRG) neurons. In particular, NK-1 was mainly coexpressed with the capsaicin receptor TRPV1 (transient receptor potential vanilloid subtype 1), a critical receptor for the generation of heat hyperalgesia. NK-1 agonist [Sar(9), Met(O2)(11)]-substance P (Sar-SP) significantly potentiated capsaicin-induced currents and increase of [Ca2+]i in dissociated DRG neurons. NK-1 antagonist blocked not only the potentiation of TRPV1 currents but also heat hyperalgesia induced by intraplantar Sar-SP. NK-1 antagonist also inhibited capsaicin-induced spontaneous pain, and this inhibition was enhanced after inflammation. To analyze intracellular cross talking of NK-1 and TRPV1, we examined downstream signal pathways of G-protein-coupled NK-1 activation. Sar-SP-induced potentiation of TRPV1 was blocked by inhibition of G-protein, PLCbeta (phospholipase C-beta), or PKC but not by inhibition of PKA (protein kinase A). In particular, PKCepsilon inhibitor completely blocked both Sar-SP-induced TRPV1 potentiation and heat hyperalgesia. Sar-SP also induced membrane translocation of PKCepsilon in a portion of small DRG neurons. These results reveal a novel mechanism of NK-1 in primary sensory neurons via a possible autocrine and paracrine action of SP. Activation of NK-1 in these neurons induces heat hyperalgesia via PKCepsilon-mediated potentiation of TRPV1.

Nerve conduction blockade in the sciatic nerve prevents but does not reverse the activation of p38 mitogen-activated protein kinase in spinal microglia in the rat spared nerve injury model.

BACKGROUND: Current evidence indicates that p38 mitogen-activated protein kinase activation in spinal microglia contributes to the development of neuropathic pain. However, how nerve injury activates p38 in spinal microglia is incompletely unknown. Nerve injury-induced ectopic spontaneous activity is essential for the generation of neuropathic pain. The authors examined whether peripheral neural activity is necessary for p38 activation in spinal microglia. METHODS: To examine whether spinal microglia activation depends on peripheral activity in the rat spared nerve injury (SNI) model, the authors blocked conduction in the sciatic nerve before or 2 days after SNI. The block was produced by applying bupivacaine-loaded microspheres above the nerve injury site. The p38 activation was examined by p38 phosphorylation using a phosphorylated p38 antibody, and neuropathic pain-related behavior was evaluated before and after intrathecal infusion of a p38 inhibitor. RESULTS: Three days after SNI, there was a marked p38 activation in the medial two thirds of the dorsal horn, where the injured tibial and peroneal nerves terminated and where isolectin B4 staining was lost. Phosphorylated p38 was only colocalized with the microglial surface marker OX-42, indicating a microglial localization of phosphorylated p38 in the SNI model. Bupivacaine microspheres produced persistent block (loss of sensory and motor function) of the sciatic nerve for the whole period of the study (3 days). This blockade prevented but did not reverse p38 activation in spinal microglia. Intrathecal infusion of the p38 inhibitor FR167653 prevented and reversed mechanical allodynia on post-SNI day 3. CONCLUSIONS: After nerve injury, activity in the peripheral nerve is required for the induction but not the maintenance of p38 activation in spinal microglia.

Differential wiring of local excitatory and inhibitory synaptic inputs to islet cells in rat spinal lamina II demonstrated by laser scanning photostimulation.

The substantia gelatinosa (lamina II) of the spinal dorsal horn contains inhibitory and excitatory interneurons that are thought to play a critical role in the modulation of nociception. However, the organization of the intrinsic circuitry within lamina II remains poorly understood. We used glutamate uncaging by laser scanning photostimulation to map the location of neurons that give rise to local synaptic inputs to islet cells, a major class of inhibitory interneuron in lamina II. We also mapped the distribution of sites on the islet cells that exhibited direct (non-synaptic) responses to uncaging of excitatory and inhibitory transmitters. Local synaptic inputs to islet cells arose almost entirely from within lamina II, and these local inputs included both excitatory and inhibitory components. Furthermore, there was a striking segregation in the location of sites that evoked excitatory versus inhibitory synaptic inputs, such that inhibitory presynaptic neurons were distributed more proximal to the islet cell soma. This was paralleled in part by a differential distribution of transmitter receptor sites on the islet cell, in that inhibitory sites were confined to the peri-somatic region while excitatory sites were more widespread. This differential organization of excitatory and inhibitory inputs suggests a principle for the wiring of local circuitry within the substantia gelatinosa.

Role of the CX3CR1/p38 MAPK pathway in spinal microglia for the development of neuropathic pain following nerve injury-induced cleavage of fractalkine.

Accumulating evidence suggests that microglial cells in the spinal cord play an important role in the development of neuropathic pain. However, it remains largely unknown how glia interact with neurons in the spinal cord after peripheral nerve injury. Recent studies suggest that the chemokine fractalkine may mediate neural/microglial interaction via its sole receptor CX3CR1. We have examined how fractalkine activates microglia in a neuropathic pain condition produced by spinal nerve ligation (SNL). SNL induced an upregulation of CX3CR1 in spinal microglia that began on day 1, peaked on day 3, and maintained on day 10. Intrathecal injection of a neutralizing antibody against CX3CR1 suppressed not only mechanical allodynia but also the activation of p38 MAPK in spinal microglia following SNL. Conversely, intrathecal infusion of fractalkine produced a marked p38 activation and mechanical allodynia. SNL also induced a dramatic reduction of the membrane-bound fractalkine in the dorsal root ganglion, suggesting a cleavage and release of this chemokine after nerve injury. Finally, application of fractalkine to spinal slices did not produce acute facilitation of excitatory synaptic transmission in lamina II dorsal horn neurons, arguing against a direct action of fractalkine on spinal neurons. Collectively, our data suggest that (a) fractalkine cleavage (release) after nerve injury may play an important role in neural-glial interaction, and (b) microglial CX3CR1/p38 MAPK pathway is critical for the development of neuropathic pain.

Induction of CB1 cannabinoid receptor by inflammation in primary afferent neurons facilitates antihyperalgesic effect of peripheral CB1 agonist.

Cannabinoids act on various regions in the nervous system to modulate neuronal activity including nociception. Here, we investigated CB1 receptor expression in primary afferent neurons in the dorsal root ganglion (DRG) and the efficacy of a local (intraplantar) application of the selective CB1 agonist, 2-arachidonyl-2-chloroethylamide (ACEA), on inflammatory thermal hyperalgesia. In situ hybridization showed normal CB1 mRNA expression in 28% of DRG neurons. Peripheral inflammation by CFA (complete Freund's adjuvant) significantly increased the ratio of CB1 mRNA-positive neurons to 43%, primarily with increase in NF200-negative C-fiber nociceptors. Furthermore, CB1 and TRPV1 (transient potential receptor vanilloid subtype-1) co-localization was increased from 41% before inflammation to 67% two days after inflammation. Inflammation also increased CB1 immunoreactivity in DRG neurons and in nerve fibers of the hindpaw dermis, indicating increased CB1 transport from the cell body to the peripheral nerve. The intraplantar application of ACEA attenuated CFA-induced thermal hyperalgesia. The antinociceptive properties of ACEA became more prominent at 2 days after inflammation, compared with those in non-inflamed and inflamed animals at 8 h. These results suggest that CB1 expression in primary afferent neurons is increased by inflammation and that the subsequent increase in CB1 transport to peripheral axons contributes to the increased antihyperalgesic efficacy of locally administered CB1 agonist.

Different effects of opioid and cannabinoid receptor agonists on C-fiber-induced extracellular signal-regulated kinase activation in dorsal horn neurons in normal and spinal nerve-ligated rats.

Nerve injury results in neuropathic pain, a debilitating pain condition. Whereas cannabinoids are consistently shown to attenuate neuropathic pain, the efficacy of opioids is highly controversial. Molecular mechanisms underlying analgesic effects of opioids and cannabinoids are not fully understood. We have shown that the signaling molecule ERK (extracellular signal-regulated kinase) is activated by C-fiber stimulation in dorsal horn neurons and contributes to pain sensitization. In this study, we examined whether opioids and cannabinoids can affect C-fiber-induced ERK phosphorylation (pERK) in dorsal horn neurons in spinal cord slices from normal and spinal nerve-ligated rats. In normal control spinal slices, capsaicin induced a drastic pERK expression in superficial dorsal horn neurons, which was suppressed by morphine (10 microM), the selective mu-opioid receptor agonist DAMGO [[d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (1 microM)], and the selective CB1 receptor ACEA agonist [arachidonyl-2'-chloroethylamide (5 microM)]. One week after spinal nerve ligation when neuropathic pain is fully developed, capsaicin induced less pERK expression in the injured L(5)-spinal segment. This pERK induction was not suppressed by morphine (10 microM) and DAMGO (1 microM) but was enhanced by high concentration of DAMGO (5 microM). In contrast, ACEA (10 microM) was still very effective in inhibiting capsaicin-induced pERK expression. In the adjacent L(4) spinal segment, both DAMGO and ACEA significantly suppressed pERK induction by capsaicin. These results indicate that, after nerve injury, opioids lose their capability to suppress C-fiber-induced spinal neuron activation in the injured L(5) but not in the intact L(4) spinal segment, whereas cannabinoids still maintain their efficacy.

Possible role of spinal astrocytes in maintaining chronic pain sensitization: review of current evidence with focus on bFGF/JNK pathway.

Although pain is regarded traditionally as neuronally mediated, recent progress shows an important role of spinal glial cells in persistent pain sensitization. Mounting evidence has implicated spinal microglia in the development of chronic pain (e.g. neuropathic pain after peripheral nerve injury). Less is known about the role of astrocytes in pain regulation. However, astrocytes have very close contact with synapses and maintain homeostasis in the extracellular environment. In this review, we provide evidence to support a role of spinal astrocytes in maintaining chronic pain. In particular, c-Jun N-terminal kinase (JNK) is activated persistently in spinal astrocytes in a neuropathic pain condition produced by spinal nerve ligation. This activation is required for the maintenance of neuropathic pain because spinal infusion of JNK inhibitors can reverse mechanical allodynia, a major symptom of neuropathic pain. Further study reveals that JNK is activated strongly in astrocytes by basic fibroblast growth factor (bFGF), an astroglial activator. Intrathecal infusion of bFGF also produces persistent mechanical allodynia. After peripheral nerve injury, bFGF might be produced by primary sensory neurons and spinal astrocytes because nerve injury produces robust bFGF upregulation in both cell types. Therefore, the bFGF/JNK pathway is an important signalling pathway in spinal astrocytes for chronic pain sensitization. Investigation of signaling mechanisms in spinal astrocytes will identify new molecular targets for the management of chronic pain.

Actions of brain-derived neurotrophic factor on spinal nociceptive transmission during inflammation in the rat.

The aim of the current study was to investigate whether, and if so how, brain-derived neurotrophic factor (BDNF) acts to develop the spinal sensitization underlying inflammation-induced hyperalgesia. In spinal cord slice preparations from rats with inflammation induced by complete Freund's adjuvant (CFA), BDNF, but not nerve growth factor (NGF) or neurotrophin-3 (NT-3), acted presynaptically to increase the frequency of excitatory miniature EPSCs in substantia gelatinosa (SG) neurones of the CFA-treated, but not untreated rats, through activation of lidocaine (lignocaine)-sensitive, TTX-resistant Na+ channels. This effect was observed in the spinal cord slices of the CFA-treated rat only 2-4 days after the CFA injection. On the other hand, the number of monosynaptic Abeta afferent inputs to the SG significantly increased 1 week after the onset of the inflammation, and this increase was significantly suppressed by treatment with anti-BDNF antiserum administered 1 day before and just after the CFA injection. In addition, the treatment with anti-BDNF antiserum significantly attenuated the CFA-induced hyperalgesia and/or allodynia. These findings, taken together, suggest that BDNF, which is considered to be released from the sensitized primary afferents, increases the excitability of SG neurones through its action on the presynaptic terminals. BDNF may thereafter induce monosynaptic Abeta afferents to the SG, thereby developing hyperalgesia and/or allodynia during inflammation.

Ionotropic and metabotropic receptors, protein kinase A, protein kinase C, and Src contribute to C-fiber-induced ERK activation and cAMP response element-binding protein phosphorylation in dorsal horn neurons, leading to central sensitization.

Molecular mechanisms underlying C-fiber stimulation-induced ERK (extracellular signal-regulated kinase) activation in dorsal horn neurons and its contribution to central sensitization have been investigated. In adult rat spinal slice preparations, activation of C-fiber primary afferents by a brief exposure of capsaicin produces an eightfold to 10-fold increase in ERK phosphorylation (pERK) in superficial dorsal horn neurons. The pERK induction is reduced by blockade of NMDA, AMPA/kainate, group I metabotropic glutamate receptor, neurokinin-1, and tyrosine receptor kinase receptors. The ERK activation produced by capsaicin is totally suppressed by inhibition of either protein kinase A (PKA) or PKC. PKA or PKC activators either alone or more effectively together induce pERK in superficial dorsal horn neurons. Inhibition of calcium calmodulin-dependent kinase (CaMK) has no effect, but pERK is reduced by inhibition of the tyrosine kinase Src. The induction of cAMP response element binding protein phosphorylation (pCREB) in spinal cord slices in response to C-fiber stimulation is suppressed by preventing ERK activation with the MAP kinase kinase inhibitor 2-(2-diamino-3-methoxyphenyl-4H-1-benzopyran-4-one (PD98059) and by PKA, PKC, and CaMK inhibitors. Similar signaling contributes to pERK induction after electrical stimulation of dorsal root C-fibers. Intraplantar injection of capsaicin in an intact animal increases expression of pCREB, c-Fos, and prodynorphin in the superficial dorsal horn, changes that are prevented by intrathecal injection of PD98059. Intrathecal PD98059 also attenuates capsaicin-induced secondary mechanical allodynia, a pain behavior reflecting hypersensitivity of dorsal horn neurons (central sensitization). We postulate that activation of ionotropic and metabotropic receptors by C-fiber nociceptor afferents activates ERK via both PKA and PKC, and that this contributes to central sensitization through post-translational and CREB-mediated transcriptional regulation in dorsal horn neurons.

AM404 enhances the spontaneous release of L-glutamate in a manner sensitive to capsazepine in adult rat substantia gelatinosa neurones.

In 84% of substantia gelatinosa (SG) neurones examined in adult rat spinal cord slices, an anandamide transport inhibitor, AM404, increased the frequency of spontaneous excitatory postsynaptic currents in a manner similar to that of capsaicin. AM404 was without actions in the presence of a vanilloid TRPV1 receptor antagonist, capsazepine. We conclude that AM404 enhances the spontaneous release of L-glutamate by activating TRPV1 receptors in the SG.

Regulation by equilibrative nucleoside transporter of adenosine outward currents in adult rat spinal dorsal horn neurons.

A current response induced by superfusing adenosine was examined in substantia gelatinosa (SG) neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. In 78% of the neurons examined, adenosine induced an outward current at -70 mV [18.8 +/- 1.1 pA (n = 98) at 1mM] in a dose-dependent manner (EC(50) = 177 microM). A similar current was induced by A(1) agonist N(6)-cyclopentyladenosine (1 microM), whereas A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (1 microM) reversed the adenosine action. The adenosine current reversed its polarity at a potential being close to the equilibrium potential for K(+), and was attenuated by Ba(2+) (100 microM) and 4-aminopyridine (5mM) but not tetraethylammonium (5mM). The adenosine current was enhanced in duration by equilibrative nucleoside-transport (rENT1) inhibitor S-(4-nitrobenzyl)-6-thioinosine (1 microM) and adenosine deaminase (ADA) inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (1 microM), and slowed in falling phase by adenosine kinase (AK) inhibitor iodotubercidine (1 microM). We conclude that a Ba(2+)- and 4-aminopyridine-sensitive K(+) channel in SG neurons is opened via the activation of A(1) receptors by adenosine whose level is possibly regulated by rENT1, adenosine deaminase and adenosine kinase. Considering that intrathecally-administered adenosine analogues produce antinociception, the regulatory systems of adenosine may serve as targets for antinociceptive drugs.

Enhancement of the releases of GABA and glycine during ischemia in rat spinal dorsal horn.

The present study examined a change in spontaneous inhibitory postsynaptic currents (sIPSCs) following ischemia in substantia gelatinosa (SG) neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. At about 10 min after superfusion of an oxygen- and glucose-free medium, sIPSCs were remarkably increased in amplitude and frequency when compared with those in the control. In a phase of the increase in sIPSC activities, GABAergic and glycinergic sIPSCs, which were observed in the presence of strychnine and bicuculline, respectively, with TTX, were increased greatly in frequency with a minimal change in their amplitudes. It is concluded that the in vitro ischemia increases the spontaneous quantal releases of GABA and glycine to SG neurons from nerve terminals; a part of this enhancement is possibly due to an increase in spontaneous activities of inhibitory interneurons. GABA released thus might serve to inhibit the release of l-glutamate from nerve terminals.

Alpha 2 adrenoceptor-mediated presynaptic inhibition of primary afferent glutamatergic transmission in rat substantia gelatinosa neurons.

BACKGROUND: Although intrathecal administration of norepinephrine is known to produce analgesia, cellular mechanisms for this action have not yet been fully understood. METHODS: The actions of norepinephrine (50 microm) on glutamatergic transmission were examined by using the whole cell patch clamp technique in substantia gelatinosa neurons of an adult rat spinal cord slice with an attached dorsal root. RESULTS: Norepinephrine inhibited the amplitude of monosynaptically evoked A delta-fiber and C-fiber excitatory postsynaptic currents in a reversible manner. When compared in magnitude between the A delta-fiber and C-fiber excitatory postsynaptic currents, the former inhibition (50 +/- 4%, n = 20) was significantly larger than the latter one (28 +/- 4%, n = 8). Both actions of norepinephrine were mimicked by an alpha2 adrenoceptor agonist, clonidine (10 microm), and an alpha 2A agonist, oxymetazoline (10 microm), but not by an alpha1 agonist, phenylephrine (10 microm), and a beta agonist, isoproterenol (40 microm). The inhibitory actions were antagonized by an alpha 2 antagonist, yohimbine (1 microm), all of the results of which indicate an involvement of alpha 2 adrenoceptors. Norepinephrine did not affect the amplitude of miniature excitatory postsynaptic current and of a response of substantia gelatinosa neurons to AMPA, indicating that its action on evoked excitatory postsynaptic currents is presynaptic in origin. CONCLUSIONS: Norepinephrine inhibits A delta-fiber- and C-fiber-mediated sensory transmission to substantia gelatinosa neurons through the activation of the alpha 2 adrenoceptor (possibly alpha2A type, based on the current, published behavioral and anatomical data) existing in primary afferent terminals; this action of norepinephrine is more effective in A delta-fiber than C-fiber transmission. This could contribute to at least a part of inhibitory modulation of pain sensation in the substantia gelatinosa by intrathecally administered norepinephrine.