The Pivotal Role of Neuropeptide Crosstalk from Ventromedial-PACAP to Dorsomedial-Galanin in the Appetite Regulation in the Mouse Hypothalamus.

We have previously shown that pituitary adenylate cyclase-activating polypeptide (PACAP) in the ventromedial hypothalamus (VMH) enhances feeding during the dark cycle and after fasting, and inhibits feeding during the light cycle. On the other hand, galanin is highly expressed in the hypothalamus and has been reported to be involved in feeding regulation. In this study, we investigated the involvement of the VMH-PACAP to the dorsomedial hypothalamus (DMH)-galanin signaling in the regulation of feeding. Galanin expression in the hypothalamus was significantly increased with fasting, but this increment was canceled in PACAP-knockout (KO) mice. Furthermore, overexpression of PACAP in the VMH increased the expression of galanin, while knockdown (KD) of PACAP in the VMH decreased the expression of galanin, indicating that the expression of galanin in the hypothalamus might be regulated by PACAP in the VMH. Therefore, we expressed the synaptophysin-EGFP fusion protein (SypEGFP) in PACAP neurons in the VMH and visualized the neural projection to the hypothalamic region where galanin was highly expressed. A strong synaptophysin-EGFP signal was observed in the DMH, indicating that PACAP-expressing cells of the VMH projected to the DMH. Furthermore, galanin immunostaining in the DMH showed that galanin expression was weak in PACAP-KO mice. When galanin in the DMH was knocked down, food intake during the dark cycle and after fasting was decreased, and food intake during the light cycle was increased, as in PACAP-KO mice. These results indicated that galanin in the DMH may regulate the feeding downstream of PACAP in the VMH.

A subset of spinal dorsal horn interneurons crucial for gating touch-evoked pain-like behavior.

A cardinal, intractable symptom of neuropathic pain is mechanical allodynia, pain caused by innocuous stimuli via low-threshold mechanoreceptors such as Aß fibers. However, the mechanism by which Aß fiber-derived signals are converted to pain remains incompletely understood. Here we identify a subset of inhibitory interneurons in the spinal dorsal horn (SDH) operated by adeno-associated viral vectors incorporating a neuropeptide Y promoter (AAV-NpyP+) and show that specific ablation or silencing of AAV-NpyP+ SDH interneurons converted touch-sensing Aß fiber-derived signals to morphine-resistant pain-like behavioral responses. AAV-NpyP+ neurons received excitatory inputs from Aß fibers and transmitted inhibitory GABA signals to lamina I neurons projecting to the brain. In a model of neuropathic pain developed by peripheral nerve injury, AAV-NpyP+ neurons exhibited deeper resting membrane potentials, and their excitation by Aß fibers was impaired. Conversely, chemogenetic activation of AAV-NpyP+ neurons in nerve-injured rats reversed Aß fiber-derived neuropathic pain-like behavior that was shown to be morphine-resistant and reduced pathological neuronal activation of superficial SDH including lamina I. These findings suggest that identified inhibitory SDH interneurons that act as a critical brake on conversion of touch-sensing Aß fiber signals into pain-like behavioral responses. Thus, enhancing activity of these neurons may offer a novel strategy for treating neuropathic allodynia.

Expression of the pacemaker channel HCN4 in excitatory interneurons in the dorsal horn of the murine spinal cord.

In the central nervous system, hyperpolarization-activated, cyclic nucleotide-gated (HCN1-4) channels have been implicated in neuronal excitability and synaptic transmission. It has been reported that HCN channels are expressed in the spinal cord, but knowledge about their physiological roles, as well as their distribution profiles, appear to be limited. We generated a transgenic mouse in which the expression of HCN4 can be reversibly knocked down using a genetic tetracycline-dependent switch and conducted genetically validated immunohistochemistry for HCN4. We found that the somata of HCN4-immunoreactive (IR) cells were largely restricted to the ventral part of the inner lamina II and lamina III. Many of these cells were either parvalbumin- or protein kinase Cγ (PKCγ)-IR. By using two different mouse strains in which reporters are expressed only in inhibitory neurons, we determined that the vast majority of HCN4-IR cells were excitatory neurons. Mechanical and thermal noxious stimulation did not induce c-Fos expression in HCN4-IR cells. PKCγ-neurons in this area are known to play a pivotal role in the polysynaptic pathway between tactile afferents and nociceptive projection cells that contributes to tactile allodynia. Therefore, pharmacological and/or genetic manipulations of HCN4-expressing neurons may provide a novel therapeutic strategy for the pain relief of tactile allodynia.

Defining a Spinal Microcircuit that Gates Myelinated Afferent Input: Implications for Tactile Allodynia.

Chronic pain presents a major unmet clinical problem. The development of more effective treatments is hindered by our limited understanding of the neuronal circuits underlying sensory perception. Here, we show that parvalbumin (PV)-expressing dorsal horn interneurons modulate the passage of sensory information conveyed by low-threshold mechanoreceptors (LTMRs) directly via presynaptic inhibition and also gate the polysynaptic relay of LTMR input to pain circuits by inhibiting lamina II excitatory interneurons whose axons project into lamina I. We show changes in the functional properties of these PV interneurons following peripheral nerve injury and that silencing these cells unmasks a circuit that allows innocuous touch inputs to activate pain circuits by increasing network activity in laminae I-IV. Such changes are likely to result in the development of tactile allodynia and could be targeted for more effective treatment of mechanical pain.

Noradrenaline modulates mechanically evoked responses in the rat spinal dorsal horn: an in vivo patch-clamp study.

Purpose: We investigated the effects of noradrenaline (NA) on physiologically evoked synaptic responses of substantia gelatinosa (SG) neurons using anesthetized animals. Methods: Male Sprague-Dawley rats (6-8 weeks, 200-300 g, n=21) were anesthetized. The lumbar spinal cord was exposed from L3 to L5; subsequently, the rats were fixed to a stereotaxic apparatus. The electrode was advanced at an angle of 30-45 degrees into the SG using a micromanipulator. We recorded excitatory post-synaptic currents (EPSC). Under these conditions, innocuous or noxious mechanical stimuli were applied to the receptive field of the ipsilateral hindlimb with or without NA, respectively. Results: NA (50 µM) pre-application induced three types of responses for pinch-evoked EPSCs. The number of neurons showing inhibition, facilitation, and no-effect was 15 (71.4%), 2 (9.5%), and 4 (19%), respectively (n=21). Pre-treatment with NA also induced three different types of responses for puff-evoked EPSC (n=21). The number of neurons showing inhibition, facilitation, and no-effect was 9 (42.9%), 9 (42.9%), and 3 (14.2%), respectively. Further, there was a significant difference in the rate distribution (inhibition, facilitation, and no change) between puff- and pinch-evoked responses. Conclusion: Our present data indicate that NA acts on noxious and innocuous mechanical transmission in the SG. Considering the distinct sensory inputs to the SG, the different actions of NA on the transmission of sensory information imply that NA exerts its analgesic effects in a manner more complicated than previously believed.

Essential roles of C-type lectin Mincle in induction of neuropathic pain in mice.

Increasing evidence indicates that pattern recognition receptors (PRRs) are involved in neuropathic pain after peripheral nerve injury (PNI). While a significant number of studies support an association between neuropathic pain and the innate immune response mediated through Toll-like receptors, a family of PRRs, the roles of other types of PRRs are largely unknown. In this study, we have focused on the macrophage-inducible C-type lectin (Mincle), a PRR allocated to the C-type lectin receptor family. Here, we show that Mincle is involved in neuropathic pain after PNI. Mincle-deficient mice showed impaired PNI-induced mechanical allodynia. After PNI, expression of Mincle mRNA was rapidly increased in the injured spinal nerve. Most Mincle-expressing cells were identified as infiltrating leucocytes, although the migration of leucocytes was also observed in Mincle-deficient mice. Furthermore, Mincle-deficiency affected the induction of genes, which are reported to contribute to neuropathic pain after PNI in the dorsal root ganglia and spinal dorsal horn. These results suggest that Mincle is involved in triggering sequential processes that lead to the pathogenesis of neuropathic pain.

Interleukin-27 controls basal pain threshold in physiological and pathological conditions.

Numerous studies have shown that pain sensation is affected by various immune molecules, such as cytokines, in tissues comprising the sensory pathway. Specifically, it has been shown that interleukin (IL)-17 promotes pain behaviour, but IL-10 suppresses it. IL-27 has been reported to have an anti-inflammatory effect through regulation of T cell differentiation, resulting in reduced IL-17 and induction of IL-10. Thus, we hypothesised that IL-27 would have some regulatory role in pain sensation. Here, we provide evidence that endogenous IL-27 constitutively controls thresholds for thermal and mechanical sensation in physiological and pathological conditions. Mice lacking IL-27 or its receptor WSX-1 spontaneously showed chronic pain-like hypersensitivity. Reconstitution of IL-27 in IL-27-deficient mice reversed thermal and mechanical hypersensitive behaviours. Thus, unlike many other cytokines induced by inflammatory events, IL-27 appears to be constitutively produced and to control pain sensation. Furthermore, mice lacking IL-27/WSX-1 signalling showed additional hypersensitivity when subjected to inflammatory or neuropathic pain models. Our results suggest that the mechanisms underlying hypersensitive behaviours caused by the ablation of IL-27/WSX-1 signalling are different from those underlying established chronic pain models. This novel pain control mechanism mediated by IL-27 might indicate a new mechanism for the chronic pain hypersensitivity.

Neuronal and glial expression of inward rectifier potassium channel subunits Kir2.x in rat dorsal root ganglion and spinal cord.

Inward rectifier K(+) channels of the Kir2.x subfamily play important roles in controlling the neuronal excitability. Although their cellular localization in the brain has been extensively studied, only a few studies have examined their expression in the spinal cord and peripheral nervous system. In this study, immunohistochemical analyses of Kir2.1, Kir2.2, and Kir2.3 expression were performed in rat dorsal root ganglion (DRG) and spinal cord using bright-field and confocal microscopy. In DRG, most ganglionic neurons expressed Kir2.1, Kir2.2 and Kir2.3, whereas satellite glial cells chiefly expressed Kir2.3. In the spinal cord, Kir2.1, Kir2.2 and Kir2.3 were all expressed highly in the gray matter of dorsal and ventral horns and moderately in the white matter also. Within the gray matter, the expression was especially high in the substantia gelatinosa (lamina II). Confocal images obtained using markers for neuronal cells, NeuN, and astrocytes, Sox9, showed expression of all three Kir2 subunits in both neuronal somata and astrocytes in lamina I-III of the dorsal horn and the lateral spinal nucleus of the dorsolateral funiculus. Immunoreactive signals other than those in neuronal and glial somata were abundant in lamina I and II, which probably located mainly in nerve fibers or nerve terminals. Colocalization of Kir2.1 and 2.3 and that of Kir2.2 and 2.3 were present in neuronal and glial somata. In the ventral horn, motor neurons and interneurons were also immunoreactive with the three Kir2 subunits. Our study suggests that Kir2 channels composed of Kir2.1-2.3 subunits are expressed in neuronal and glial cells in the DRG and spinal cord, contributing to sensory transduction and motor control.

A putative relay circuit providing low-threshold mechanoreceptive input to lamina I projection neurons via vertical cells in lamina II of the rat dorsal horn.

BACKGROUND: Lamina I projection neurons respond to painful stimuli, and some are also activated by touch or hair movement. Neuropathic pain resulting from peripheral nerve damage is often associated with tactile allodynia (touch-evoked pain), and this may result from increased responsiveness of lamina I projection neurons to non-noxious mechanical stimuli. It is thought that polysynaptic pathways involving excitatory interneurons can transmit tactile inputs to lamina I projection neurons, but that these are normally suppressed by inhibitory interneurons. Vertical cells in lamina II provide a potential route through which tactile stimuli can activate lamina I projection neurons, since their dendrites extend into the region where tactile afferents terminate, while their axons can innervate the projection cells. The aim of this study was to determine whether vertical cell dendrites were contacted by the central terminals of low-threshold mechanoreceptive primary afferents. RESULTS: We initially demonstrated contacts between dendritic spines of vertical cells that had been recorded in spinal cord slices and axonal boutons containing the vesicular glutamate transporter 1 (VGLUT1), which is expressed by myelinated low-threshold mechanoreceptive afferents. To confirm that the VGLUT1 boutons included primary afferents, we then examined vertical cells recorded in rats that had received injections of cholera toxin B subunit (CTb) into the sciatic nerve. We found that over half of the VGLUT1 boutons contacting the vertical cells were CTb-immunoreactive, indicating that they were of primary afferent origin. CONCLUSIONS: These results show that vertical cell dendritic spines are frequently contacted by the central terminals of myelinated low-threshold mechanoreceptive afferents. Since dendritic spines are associated with excitatory synapses, it is likely that most of these contacts were synaptic. Vertical cells in lamina II are therefore a potential route through which tactile afferents can activate lamina I projection neurons, and this pathway could play a role in tactile allodynia.

Populations of inhibitory and excitatory interneurons in lamina II of the adult rat spinal dorsal horn revealed by a combined electrophysiological and anatomical approach.

Lamina II contains a large number of interneurons involved in modulation and transmission of somatosensory (including nociceptive) information. However, its neuronal circuitry is poorly understood due to the difficulty of identifying functional populations of interneurons. This information is important for understanding nociceptive processing and for identifying changes that underlie chronic pain. In this study, we compared morphology, neurotransmitter content, electrophysiological and pharmacological properties for 61 lamina II neurons recorded in slices from adult rat spinal cord. Morphology was related to transmitter content, since islet cells were GABAergic, while radial and most vertical cells were glutamatergic. However, there was considerable diversity among the remaining cells, some of which could not be classified morphologically. Transmitter phenotype was related to firing pattern, since most (18/22) excitatory cells, but few (2/23) inhibitory cells had delayed, gap or reluctant patterns, which are associated with A-type potassium (I(A)) currents. Somatostatin was identified in axons of 14/24 excitatory neurons. These had variable morphology, but most of those tested showed delayed-firing. Excitatory interneurons are therefore likely to contribute to pain states associated with synaptic plasticity involving I(A) currents. Although noradrenaline and serotonin evoked outward currents in both inhibitory and excitatory cells, somatostatin produced these currents only in inhibitory neurons, suggesting that its pro-nociceptive effects are mediated by disinhibition. Our results demonstrate that certain distinctive populations of inhibitory and excitatory interneuron can be recognised in lamina II. Combining this approach with identification of other neurochemical markers should allow further clarification of neuronal circuitry in the superficial dorsal horn.

Evidence against AMPA receptor-lacking glutamatergic synapses in the superficial dorsal horn of the rat spinal cord.

Pure NMDA receptor (NMDAr)-mediated EPSCs, thought to correspond to "silent" glutamatergic synapses that lack AMPA receptors (AMPArs), have been observed in superficial spinal dorsal horn of neonatal but not adult rats. Recent anatomical studies suggest that AMPArs are present at virtually all glutamatergic synapses in this region in adults. We used antigen retrieval to examine colocalization of AMPArs and PSD-95 (a marker for glutamatergic synapses) in laminae I-II of neonatal and adult rats. We found a high degree of colocalization in all cases, which suggests that AMPArs are present in the great majority of glutamatergic synapses even in neonatal animals. We therefore reexamined evidence for silent synapses by performing blind whole-cell recordings from superficial dorsal horn neurons in slices from neonatal or adult rats, with focal stimulation to activate glutamatergic synapses. On some occasions in both neonatal (10 of 109, 9%) and adult (9 of 77, 12%) slices, NMDAr-mediated EPSCs were observed when the holding potential was raised to +50 mV at a stimulus strength that had failed to evoke AMPAr-mediated EPSCs. However, in all cases tested, AMPAr-mediated EPSCs were then observed when the cell was returned to -70 mV; this and other properties of the EPSCs suggest that they do not represent genuine silent synapses. When compared with previous findings, our results indicate that the appearance of silent synapses depends on experimental protocol. This suggests that pure NMDAr-mediated EPSCs seen in previous studies do not correspond to AMPAr-lacking synapses but result from another mechanism, for example, loss of labile AMPArs from recently formed synapses.

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.

Cell-type-specific excitatory and inhibitory circuits involving primary afferents in the substantia gelatinosa of the rat spinal dorsal horn in vitro.

The substantia gelatinosa (SG) of the spinal dorsal horn shows significant morphological heterogeneity and receives primary afferent input predominantly from A delta- and C-fibres. Despite numerous anatomical and physiological studies, correlation between morphology and functional connectivity, particularly in terms of inhibitory inputs, remains elusive. To compare excitatory and inhibitory synaptic inputs on individual SG neurones with morphology, we performed whole-cell recordings with Neurobiotin-filled-pipettes in horizontal slices from adult rat spinal cord with attached dorsal roots. Based on dendritic arborization patterns, four major cell types were confirmed: islet, central, radial and vertical cells. Dorsal root stimulation revealed that each class was associated with characteristic synaptic inputs. Islet and central cells had monosynaptic excitatory inputs exclusively from C-afferents. Islet cells received primary-afferent-evoked inhibitory inputs only from A delta-fibres, while those of central cells were mediated by both A delta- and C-fibres. In contrast, radial and vertical cells had monosynaptic excitatory inputs from both A delta- and C-fibres and inhibitory inputs mediated by both fibre types. We further characterized the neurochemical nature of these inhibitory synaptic inputs. The majority of islet, central and vertical cells exhibited GABAergic inhibitory inputs, while almost all radial cells also possessed glycinergic inputs. The present study demonstrates that SG neurones have distinct patterns of excitatory and inhibitory inputs that are related to their morphology. The neurotransmitters responsible for inhibitory inputs to individual SG neurones are also characteristic for different morphological classes. These results make it possible to identify primary afferent circuits associated with particular types of SG neurone.

Direct GABAergic and glycinergic inhibition of the substantia gelatinosa from the rostral ventromedial medulla revealed by in vivo patch-clamp analysis in rats.

Stimulation of the rostral ventromedial medulla (RVM) is believed to exert analgesic effects through the activation of the serotonergic system descending to the spinal dorsal horn; however, how nociceptive transmission is modulated by the descending system has not been fully clarified. To investigate the inhibitory mechanisms affected by the RVM, an in vivo patch-clamp technique was used to record IPSCs from the substantia gelatinosa (SG) of the spinal cord evoked by chemical (glutamate injection) and electrical stimulation (ES) of the RVM in adult rats. In the voltage-clamp mode, the RVM glutamate injection and RVM-ES produced an increase in both the frequency and amplitude of IPSCs in SG neurons that was not blocked by glutamate receptor antagonists. Serotonin receptor antagonists were unexpectedly without effect, but a GABAA receptor antagonist, bicuculline, or a glycine receptor antagonist, strychnine, completely suppressed the RVM stimulation-induced increase in IPSCs. The RVM-ES-evoked IPSCs showed fixed latency and no failure at 20 Hz stimuli with a conduction velocity of >3 m/s (3.1-20.7 m/s), suggesting descending monosynaptic GABAergic and/or glycinergic inputs from the RVM to the SG through myelinated fibers. In the current-clamp mode, action potentials elicited by noxious mechanical stimuli applied to the receptive field of the ipsilateral hindlimb were suppressed by the RVM-ES in more than half of the neurons tested (63%; 10 of 16). These findings suggest that the RVM-mediated antinociceptive effects on noxious inputs to the SG may be exerted preferentially by the direct GABAergic and glycinergic pathways to the SG.

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.

Electrophysiological mapping of the nociceptive inputs to the substantia gelatinosa in rat horizontal spinal cord slices.

To study the functional projection patterns of the primary afferents in the spinal cord, the postsynaptic responses of substantia gelatinosa (SG) neurones evoked by L5 dorsal root stimulation (DRS) were examined from the neurones located at L2 to S1 in horizontal slices of the adult rat spinal cord using a blind whole-cell patch-clamp technique. In the voltage-clamp mode, the L5 DRS evoked the Adelta- and C-afferent-mediated excitatory postsynaptic currents (EPSCs) in more than 70% of the neurones tested at the L5 level. Both Adelta- and C-afferent EPSCs were also recorded in more than 50% of the neurones at L4. At L3 and L6, the number of neurones receiving the C-afferent EPSCs (> 40%) was significantly greater than that of Adelta-afferent EPSCs (< 20%). On the other hand, the Adelta- and C-afferent-mediated inhibitory postsynaptic currents (IPSCs) elicited by L5 DRS were almost equally observed from L2 to S1. In the current-clamp mode, L5 DRS evoked Adelta- and C-afferent-mediated EPSPs, some of which initiated action potentials (APs). Most of the Adelta-afferent-mediated APs were limited at the L5 level, while C-afferent-mediated APs were observed at L5 and L4. As the L2 DRS-evoked APs in the L2 SG neurones were suppressed by L5 DRS, the widespread distribution of the inhibitory inputs was considered to be functional. These findings suggest that the excitatory projection of the C afferents to the SG neurones was thus spread more rostrocaudally than that of the Adelta afferents, thereby contributing to more diffuse pain transmission. In addition, the widespread distribution of the inhibitory inputs may thus play a role as a lateral inhibitory network and thereby prevent the expansion of the excitatory inputs of noxious stimuli.

Actions of noradrenaline on substantia gelatinosa neurones in the rat spinal cord revealed by in vivo patch recording.

To elucidate the mechanisms of antinociception mediated by the descending noradrenergic pathway in the spinal cord, the effects of noradrenaline (NA) on noxious synaptic responses of substantia gelatinosa (SG) neurones, and postsynaptic actions of NA were investigated in rats using an in vivo whole-cell patch-clamp technique. Under urethane anaesthesia, the rat was fixed in a stereotaxic apparatus after the lumbar spinal cord was exposed. In the current-clamp mode, pinch stimuli applied to the ipsilateral hindlimb elicited a barrage of EPSPs, some of which initiated an action potential. Perfusion with NA onto the surface of the spinal cord hyperpolarized the membrane (5.0-9.5 mV) and suppressed the action potentials. In the voltage-clamp mode (V(H), -70 mV), the application of NA produced an outward current that was blocked by Cs(+) and GDP-beta-S added to the pipette solution and reduced the amplitude of EPSCs evoked by noxious stimuli. Under the blockade of postsynaptic actions of NA, a reduction of the evoked and spontaneous EPSCs of SG neurones was still observed, thus suggesting both pre- and postsynaptic actions of NA. The NA-induced outward currents showed a clear dose dependency (EC(50), 20 microM), and the reversal potential was -88 mV. The outward current was mimicked by an alpha(2)-adrenoceptor agonist, clonidine, and suppressed by an alpha(2)-adrenoceptor antagonist, yohimbine, but not by alpha(1)- and beta-antagonists. These findings suggest that NA acts on presynaptic sites to reduce noxious stimuli-induced EPSCs, and on postsynaptic SG neurones to induce an outward current by G-protein-mediated activation of K(+) channels through alpha(2)-adrenoceptors, thereby producing an antinociceptive effect.