Rat Spinal Dorsal Horn Neurons Research Articles

Actions of remimazolam on inhibitory transmission of rat spinal dorsal horn neurons

Remimazolam is an ultra-short benzodiazepine that acts on the benzodiazepine site of γ-aminobutyric acid (GABA) receptors in the brain and induces sedation. Although GABA receptors are found localized in the spinal dorsal horn, no previous studies have reported the analgesic effects or investigated the cellular mechanisms of remimazolam on the spinal dorsal horn. Behavioral measures, immunohistochemistry, and in vitro whole-cell patch-clamp recordings of dorsal horn neurons were used to assess synaptic transmission. Intrathecal injection of remimazolam induced behavioral analgesia in inflammatory pain-induced mechanical allodynia (six rats/dose; p < 0.05). Immunohistochemical staining revealed that remimazolam suppressed spinal phosphorylated extracellular signal-regulated kinase activation (five rats/group, p < 0.05). In vitro whole-cell patch-clamp analysis demonstrated that remimazolam increased the frequency of GABAergic miniature inhibitory post-synaptic currents, prolonged the decay time (six rats; p < 0.05), and enhanced GABA currents induced by exogenous GABA (seven rats; p < 0.01). However, remimazolam did not affect miniature excitatory post-synaptic currents or amplitude of monosynaptic excitatory post-synaptic currents evoked by Aδ- and C-fiber stimulation (seven rats; p > 0.05). This study suggests that remimazolam induces analgesia by enhancing GABAergic inhibitory transmission in the spinal dorsal horn, suggesting its potential utility as a spinal analgesic for inflammatory pain.

Methylglyoxal activates transient receptor potential A1/V1 via reactive oxygen species in the spinal dorsal horn.

Methylglyoxal (MGO), a highly reactive dicarbonyl metabolite of glucose primarily formed during the glycolytic pathway, is a precursor of advanced glycation end-products (AGEs). Recently, numerous studies have shown that MGO accumulation can cause pain and hyperalgesia. However, the mechanism through which MGO induces pain in the spinal dorsal horn remains unclear. The present study investigated the effect of MGO on spontaneous excitatory postsynaptic currents (sEPSC) in rat spinal dorsal horn neurons using blind whole-cell patch-clamp recording. Perfusion of MGO increased the frequency and amplitude of sEPSC in spinal horn neurons in a concentration-dependent manner. Additionally, MGO administration increased the number of miniature EPSC (mEPSC) in the presence of tetrodotoxin, a sodium channel blocker. However, 6-cyano-7-nitroqiunocaline-2,3-dione (CNQX), an AMPA/kainate receptor antagonist, blocked the enhancement of sEPSC by MGO. HC-030031, a TRP ankyrin-1 (TRPA1) antagonist, and capsazepine, a TRP vanilloid-1 (TRPV1) antagonist, inhibited the action of MGO. Notably, the effects of MGO were completely inhibited by HC-030031 and capsazepine. MGO generates reactive oxygen species (ROS) via AGEs. ROS also potentially induce pain via TRPA1 and TRPV1 in the spinal dorsal horn. Furthermore, we examined the effect of MGO in the presence of N-tert-butyl-α-phenylnitrone (PBN), a non-selective ROS scavenger, and found that the effect of MGO was completely inhibited. These results suggest that MGO increases spontaneous glutamate release from the presynaptic terminal to spinal dorsal horn neurons through TRPA1, TRPV1, and ROS and could enhance excitatory synaptic transmission.

Neuropathic and inflammatory pain increases glutamatergic excitatory postsynaptic current on adult rat spinal dorsal horn neurons.

Neuropathic and inflammatory pain increases glutamatergic excitatory postsynaptic current on adult rat spinal dorsal horn neurons.

Inhibiting the LPS-induced enhancement of mEPSC frequency in superficial dorsal horn neurons may serve as an electrophysiological model for alleviating pain

Pain is a major primary health care problem. Emerging studies show that inhibition of spinal microglial activation reduces pain. However, the precise mechanisms by which microglial activation contributes to nociceptive synaptic transmission remain unclear. In this study, we measured spontaneous synaptic activity of miniature excitatory postsynaptic currents (mEPSCs) in rat spinal cord superficial dorsal horn (SDH, laminae I and II) neurons. Lipopolysaccharide (LPS) and adenosine triphosphate (ATP) increased the frequency, but not amplitude, of mEPSCs in SDH neurons. Microglial inhibitors minocycline and paeonol, as well as an astrocyte inhibitor, a P2Y1 receptor (P2Y1R) antagonist, and a metabotropic glutamate receptor 5 (mGluR5) antagonist, all prevented LPS-induced enhancement of mEPSC frequency. In mouse behavioral testing, minocycline and paeonol effectively reduced acetic acid-induced writhing and LPS-induced hyperalgesia. These results indicate that LPS-activated microglia release ATP, which stimulates astrocyte P2Y1Rs to release glutamate, triggering presynaptic mGluR5 receptors and increasing presynaptic glutamate release, leading to an increase in mEPSC frequency and enhancement of nociceptive transmission in SDH neurons. We propose that these effects can serve as a new electrophysiological model for evaluating pain. Moreover, we predict that pharmacologic agents capable of inhibiting the LPS-induced enhancement of mEPSC frequency in SDH neurons will have analgesic effects.

The paracetamol metabolite N-acetylp-benzoquinone imine reduces excitability in first- and second-order neurons of the pain pathway through actions on KV7 channels.

Paracetamol (acetaminophen, APAP) is one of the most frequently used analgesic agents worldwide. It is generally preferred over nonsteroidal anti-inflammatory drugs because it does not cause typical adverse effects resulting from the inhibition of cyclooxygenases, such as gastric ulcers. Nevertheless, inhibitory impact on these enzymes is claimed to contribute to paracetamols mechanisms of action which, therefore, remained controversial. Recently, the APAP metabolites N-arachidonoylaminophenol (AM404) and N-acetyl-p-benzoquinone imine (NAPQI) have been detected in the central nervous system after systemic APAP administration and were reported to mediate paracetamol effects. In contrast to nonsteroidal anti-inflammatory drugs that rather support seizure activity, paracetamol provides anticonvulsant actions, and this dampening of neuronal activity may also form the basis for analgesic effects. Here, we reveal that the APAP metabolite NAPQI, but neither the parent compound nor the metabolite AM404, reduces membrane excitability in rat dorsal root ganglion (DRG) and spinal dorsal horn (SDH) neurons. The observed reduction of spike frequencies is accompanied by hyperpolarization in both sets of neurons. In parallel, NAPQI, but neither APAP nor AM404, increases currents through KV7 channels in DRG and SDH neurons, and the impact on neuronal excitability is absent if KV7 channels are blocked. Furthermore, NAPQI can revert the inhibitory action of the inflammatory mediator bradykinin on KV7 channels but does not affect synaptic transmission between DRG and SDH neurons. These results show that the paracetamol metabolite NAPQI dampens excitability of first- and second-order neurons of the pain pathway through an action on KV7 channels.

CB1 Receptors Mediated Inhibition of ATP-Induced [Ca2+]i Increase in Cultured Rat Spinal Dorsal Horn Neurons.

Spinal cannabinoid receptor 1 (CB1R) and purinergic P2X receptors (P2XR) play a critical role in the process of pathological pain. Both CB1R and P2XR are expressed in spinal dorsal horn (DH) neurons. It is not clear whether CB1 receptor activation modulates the function of P2X receptor channels within dorsal horn. For this reason, we observed the effect of CP55940 (cannabinoid receptor agonist) on ATP-induced Ca2+ mobilization in cultured rat DH neurons. The changes of intracellular calcium concentration ([Ca2+]i) were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator. 100μM ATP caused [Ca2+]i increase in cultured DH neurons. ATP-evoked [Ca2+]i increase in DH neurons was blocked by chelating extracellular Ca2+ and P2 purinoceptor antagonist PPADS. At the same time, ATP-γ-S (a non-hydrolyzable ATP analogue) mimicked the ATP action, while P2Y receptor agonist ADP failed to evoke [Ca2+]i increase in cultured DH neurons. These data suggest that ATP-induced [Ca2+]i elevation in cultured DH neurons is mediated by P2X receptor. Subsequently, we noticed that, in cultured rat DH neurons, ATP-induced Ca2+ mobilization was inhibited after pretreated with CP55940 with a concentration-dependent manner, which implies that the opening of P2X receptor channels are down-regulated by activation of cannabinoid receptor. The inhibitory effect of CP55940 on ATP-induced Ca2+ response was mimicked by ACEA (CB1R agonist), but was not influenced by AM1241 (CB2R agonist). Moreover, the inhibitory effect of CP55940 on ATP-induced Ca2+ mobilization was blocked by AM251 (CB1 receptor antagonist), but was not influenced by AM630 (CB2 receptor antagonist). In addition, we also observed that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a cell-permeable cAMP analog) reversed the inhibitory effect of CP55940, respectively. In a summary, our observations raise a possibility that CB1R rather than CB2R can downregulate the opening of P2X receptor channels in DH neurons. The reduction of cAMP/PKA signaling is a key element in the inhibitory effect of CB1R on P2X-channel-induced Ca2+ mobilization.

MiR-9 Mediates CALHM1-Activated ATP-P2X7R Signal in Painful Diabetic Neuropathy Rats.

In this study, we planned to illuminate the mechanisms of the expression and function of CALHM1 in painful diabetic neuropathy (PDN). PDN rat model was constructed. The expression of CALHM1 and miR-9 in rat spinal dorsal horn neurons was detected. The correlation between the level of CALHM1 mRNA and 50% PWT and the relationship between the expression of CALHM1 and miR-9 in rat spinal dorsal horn neurons were statistically analyzed. The effect of miR-9 and CALHM1 on each other's expression in PDN rat spinal dorsal horn neurons were tested by qRT-PCR or Western blot. The co-culture system of neurons and glias from PDN rat spinal dorsal horn was constructed. The concentration of calcium and ATP as well as the expression of P2X7 receptor regulated by CALHM1 and miR-9 in PDN rat spinal dorsal horn neurons was measured. The results showed that the expression of CALHM1 was increased in PDN rat compared with controls, while its mRNA level was negatively correlated with 50% PWT. miR-9, which was also upregulated in the spinal dorsal horn neurons of PDN rats, was positively correlated with the expression of CALHM1. The concentration of calcium and ATP as well as the expression of P2X7 receptor in glias was also increased in PDN rats. These increases could be reverted by inhibiting CALHM1 and/or miR-9. CALHM1 is involved in miR-9-mediated ATP-P2X7 pathway between neurons and glias in PDN rat.

High-frequency modulation of rat spinal field potentials: effects of slowly conducting muscle vs. skin afferents.

Long-term potentiation (LTP) in rat spinal dorsal horn neurons was induced by electrical high-frequency stimulation (HFS) of afferent C fibers. LTP is generally assumed to be a key mechanism of spinal sensitization. To determine the contribution of skin and muscle afferents to LTP induction, the sural nerve (SU, pure skin nerve) or the gastrocnemius-soleus nerve (GS, pure muscle nerve) were stimulated individually. As a measure of spinal LTP, C-fiber-induced synaptic field potentials (SFPs) evoked by the GS and by the SU were recorded in the dorsal horn. HFS induced a sustained increase of SFPs of the same nerve for at least 3 h, indicating the elicitation of homosynaptic nociceptive spinal LTP. LTP after muscle nerve stimulation (HFS to GS) was more pronounced (increase to 248%, P < 0.05) compared with LTP after skin nerve stimulation (HFS applied to SU; increase to 151% of baseline, P < 0.05). HFS applied to GS also increased the SFPs of the unconditioned SU (heterosynaptic LTP) significantly, whereas HFS applied to SU had no significant impact on the SFP evoked by the GS. Collectively, the data indicate that HFS of a muscle or skin nerve evoked nociceptive spinal LTP with large effect sizes for homosynaptic LTP (Cohen's d of 0.8-1.9) and small to medium effect sizes for heterosynaptic LTP (Cohen's d of 0.4-0.65). The finding that homosynaptic and heterosynaptic LTP after HFS of the muscle nerve were more pronounced than those after HFS of a skin nerve suggests that muscle pain may be associated with more extensive LTP than cutaneous pain.

Opioid receptor–triggered spinal mTORC1 activation contributes to morphine tolerance and hyperalgesia

The development of opioid-induced analgesic tolerance and hyperalgesia is a clinical challenge for managing chronic pain. Adaptive changes in protein translation in the nervous system are thought to promote opioid tolerance and hyperalgesia; however, how opioids drive such changes remains elusive. Here, we report that mammalian target of rapamycin (mTOR), which governs most protein translation, was activated in rat spinal dorsal horn neurons after repeated intrathecal morphine injections. Activation was triggered through μ opioid receptor and mediated by intracellular PI3K/Akt. Spinal mTOR inhibition blocked both induction and maintenance of morphine tolerance and hyperalgesia, without affecting basal pain perception or locomotor functions. These effects were attributed to the attenuation of morphine-induced increases in translation initiation activity, nascent protein synthesis, and expression of some known key tolerance-associated proteins, including neuronal NOS (nNOS), in dorsal horn. Moreover, elevating spinal mTOR activity by knocking down the mTOR-negative regulator TSC2 reduced morphine analgesia, produced pain hypersensitivity, and increased spinal nNOS expression. Our findings implicate the μ opioid receptor-triggered PI3K/Akt/mTOR pathway in promoting morphine-induced spinal protein translation changes and associated morphine tolerance and hyperalgesia. These data suggest that mTOR inhibitors could be explored for prevention and/or reduction of opioid tolerance in chronic pain management.

An Endogenous Pain Control System is Altered in Subjects with Interstitial Cystitis

Multiple studies have demonstrated that in healthy subjects, painful stimuli applied to one part of the body inhibit pain sensation in other parts of the body, a phenomenon referred to as conditioned pain modulation. Conditioned pain modulation is related to the presence of endogenous pain control systems. Studies have demonstrated deficits in conditioned pain modulation associated inhibition in many but not all chronic pain disorders. In this study we determine whether conditioned pain modulation is altered in subjects with interstitial cystitis/bladder pain syndrome. Female subjects with and without the diagnosis of interstitial cystitis/bladder pain syndrome were studied psychophysically using quantitative cutaneous thermal, forearm ischemia and ice water immersion tests. Conditioned pain modulation was assessed by quantifying the effects of immersion of the hand in ice water (conditioning stimulus) on threshold and tolerance of cutaneous heat pain (test stimulus) applied to the contralateral lower extremity. The conditioned pain modulation responses of the subjects with interstitial cystitis/bladder pain syndrome were statistically different from those of healthy control subjects for cutaneous thermal threshold and tolerance measures. Healthy control subjects demonstrated statistically significant increases in thermal pain tolerance whereas subjects with the diagnosis of interstitial cystitis/bladder pain syndrome demonstrated statistically significant reductions in thermal pain tolerance. An endogenous pain inhibitory system normally observed with conditioned pain modulation was altered in subjects with interstitial cystitis/bladder pain syndrome. This finding identifies interstitial cystitis/bladder pain syndrome as similar to several other chronic pain disorders such as fibromyalgia and irritable bowel syndrome, and suggests that a deficit in endogenous pain inhibitory systems may contribute to such chronic pain disorders.

Reactive oxygen species enhance excitatory synaptic transmission in rat spinal dorsal horn neurons by activating TRPA1 and TRPV1 channels

Central neuropathic pain (CNP) in the spinal cord, such as chronic pain after spinal cord injury (SCI), is an incurable ailment. However, little is known about the spinal cord mechanisms underlying CNP. Recently, reactive oxygen species (ROS) have been recognized to play an important role in CNP of the spinal cord. However, it is unclear how ROS affect synaptic transmission in the dorsal horn of the spinal cord. To clarify how ROS impact on synaptic transmission, we investigated the effects of ROS on synaptic transmission in rat spinal cord substantia gelatinosa (SG) neurons using whole-cell patch-clamp recordings. Administration of tert-butyl hydroperoxide (t-BOOH), an ROS donor, into the spinal cord markedly increased the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) in SG neurons. This t-BOOH-induced enhancement was not suppressed by the Na+ channel blocker tetrodotoxin. However, in the presence of a non-N-methyl-d-aspartate glutamate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, t-BOOH did not generate any sEPSCs. Furthermore, in the presence of a transient receptor potential ankyrin 1 (TRPA1) channel antagonist (HC-030031) or a transient receptor potential vanilloid 1 (TRPV1) channel antagonist (capsazepine or AMG9810), the t-BOOH-induced increase in the frequency of sEPSCs was inhibited. These results indicate that ROS enhance the spontaneous release of glutamate from presynaptic terminals onto SG neurons through TRPA1 and TRPV1 channel activation. Excessive activation of these ion channels by ROS may induce central sensitization in the spinal cord and result in chronic pain such as that following SCI.

Effects of remifentanil and fentanyl on N-methyl-D-aspartate receptor currents in rat spinal cord dorsal horn neurons

Objective To investigate the effect of remifentanil and fentanyl on N-methyl-D-aspartate (NMDA) receptor currents in rat spinal cord dorsal horn neurons.Methods Whole-cell patch-clamp technique was used to record the NMDA receptor currents.The primary cultured E14SD rat spinal cord dorsal horn neurons (DH cells) were randomly divided into 3 groups (n =10 each):remifentanil group (group R),fentanyl group (group F) and control group (group C).DH cells were perfused with 4 nmol/L remifentanil (group R) or 10 μmol/L fentanyl (group F) for 60 min followed by washout.NMDA receptor currents were recorded immediately after administration (T0),at 15,30,45 and 60 min of action of drugs (T1-4),and at 15 and 30 min (T5-6) after washout.Results Compared with group C,no significant change in the peak NMDA receptor current was found at each time point in group F and at T0 and T1 in group R (P ＞ 0.05),and the peak NMDA receptor current was significantly increased at T2-6 in group R (P ＜ 0.01).The peak NMDA receptor current was significantly higher at T2-6 than at T0,while lower at T2-4 and T6 than at T5 in group R (P ＜ 0.01).Conclusion Remifentanil can increase NMDA receptor function in rat spinal cord horn neurons,and the peak effect is reached after washout,but fentanyl dose not have the effect. Key words: Piperidines; Receptors, N-methyl-D-aspartate; Drug tolerance

Enhancement by Interleukin-1β of AMPA and NMDA Receptor-Mediated Currents in Adult Rat Spinal Superficial Dorsal Horn Neurons

BackgroundProinflammatory cytokine interleukin-1β (IL-1β) released from spinal microglia plays an important role in the maintenance of acute and chronic pain states. However, the cellular basis of this action remains poorly understood. Using whole-cell patch-clamp recordings, we examined the action of IL-1β on AMPA- and NMDA-receptor-mediated currents recorded from substantia gelatinosa (SG) neurons of adult rat spinal cord slices which are key sites for regulating nociceptive transmission from the periphery.ResultsAMPA- and NMDA-induced currents were increased in peak amplitude by IL-1β in a manner different from each other in SG neurons. These facilitatory actions of IL-1β were abolished by IL-1 receptor (IL-1R) antagonist (IL-1ra), which by itself had no detectable effects on AMPA- and NMDA-induced currents. The AMPA- but not NMDA-induced current facilitated by IL-1β was recovered to control level 30 min after IL-1β washout and largely depressed in Na+-channel blocker tetrodotoxin-containing or nominally Ca2+-free Krebs solution. Minocycline, a microglia inhibitor, blocked the facilitatory effect of IL-1β on AMPA- but not NMDA-induced currents, where minocycline itself depressed NMDA- but had not any effects on AMPA-induced currents.ConclusionsIL-1β enhances AMPA and NMDA responses in SG neurons through IL-1R activation; the former but not latter action is reversible and due to an increase in neuronal activity in a manner dependent on extracellular Ca2+ and minocycline. It is suggested that AMPA and NMDA receptors are positively modulated by IL-1β in a manner different from each other; the former but not latter is mediated by a neurotransmitter released as a result of an increase in neuronal activity. Since IL-1β contributes to nociceptive behavior induced by peripheral nerve or tissue injury, the present findings also reveal an important cellular link between neuronal and glial cells in the spinal dorsal horn.

Electrical Stimulation of Large Myelinated Afferents Inhibits Responses of Rat Spinal Dorsal Horn Neurons to Noxious and Innocuous Cutaneous Stimulation

Electrical Stimulation of Large Myelinated Afferents Inhibits Responses of Rat Spinal Dorsal Horn Neurons to Noxious and Innocuous Cutaneous Stimulation

Subunit-Specific Inhibition of Glycine Receptors by Curcumol

Emerging evidence has suggested that inhibitory glycine receptors (GlyRs) are an important molecular target in the treatment of numerous neurological disorders. Rhizoma curcumae is a medicinal plant with positive neurological effects. In this study, we showed that curcumol, a major bioactive component of R. curcumae, reversibly and concentration-dependently inhibited the glycine-activated current (IGly) in cultured rat hippocampal neurons. The inhibitory effect was neither voltage- nor agonist concentration-dependent. Moreover, curcumol selectively inhibited homomeric α2-containing, but not α1- or α3-containing, GlyRs. The addition of β subunit conferred the curcumol sensitivity of α3-containing, but not α1-containing, GlyRs. Site-directed mutagenesis analysis revealed that a threonine at position 59 of the α2 subunit is critical for the susceptibility of GlyRs to curcumol-mediated inhibition. Furthermore, paralleling a decline of α2 subunit expression during spinal cord development, the degree of IGly inhibition by curcumol decreased with prolonged culture of rat spinal dorsal horn neurons. Taken together, our results suggest that the GlyRs are novel molecular targets of curcumol, which may underlie its pharmaceutical effects in the central nervous system.

PI3-kinase/Akt Pathway-Regulated Membrane Insertion of Acid-Sensing Ion Channel 1a Underlies BDNF-Induced Pain Hypersensitivity

Central neural plasticity plays a key role in pain hypersensitivity. This process is modulated by brain-derived neurotrophic factor (BDNF) and also involves the type 1a acid-sensing ion channel (ASIC1a). However, the interactions between the BDNF receptor, tropomyosin-related kinase B (TrkB), and ASIC1a are unclear. Here, we show that deletion of ASIC1 gene suppressed the sustained mechanical hyperalgesia induced by intrathecal BDNF application in mice. In both rat spinal dorsal horn neurons and heterologous cell cultures, the BDNF/TrkB pathway enhanced ASIC1a currents via phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB/Akt) cascade and phosphorylation of cytoplasmic residue Ser-25 of ASIC1a, resulting in enhanced forward trafficking and increased surface expression. Moreover, in both rats and mice, this enhanced ASIC1a activity was required for BDNF-mediated hypersensitivity of spinal dorsal horn nociceptive neurons and central mechanical hyperalgesia, a process that was abolished by intrathecal application of a peptide representing the N-terminal region of ASIC1a encompassing Ser-25. Thus, our results reveal a novel mechanism underlying central sensitization and pain hypersensitivity, and reinforce the critical role of ASIC1a channels in these processes.

Role of δ-opioid receptor in remifentanil induced NMDA receptor miniature excitatory postsynaptic currents in rat spinal dorsal horn neurons

Objective To investigate the role of δ-opioid receptor in remifentanil-induced N-methyl-D-aspartate (NMDA) receptor miniature excitatory postsynaptic currents (mEPSCs) in rat spinal dorsal horn neurons.Methods Male 14-18 d old Wistar rats weighing 50-60 g were used in this study.The animals were anesthetized with intraperitoneal choral hydrate 400 mg/kg and sacrificed.Their lumbar segments of spinal cord (L1-S1 ) were immediately removed and sliced.Twenty-four slices were randomly divided into 4 groups ( n =6 each): control group (group C) ; glycine group (group G) ; remifentanil group (group R) and remifentanil + naltrindole(a δ receptor antagon) (group RN).Slices were cultured in artificial cerebrospinal fluid (ACSF) (group C) or incubated in ACSF containing glycine 0.24 μmol/L (group G) or remifentanil 4 nmol/L (group R) or remifentanil 4 nmol/L+ naltrindole 1 nmol/L (group RN) for 60 min.Whole cell patch clamp technique was used to measure NMDA receptor mEPSCs.Results The amplitude and frequency of mEPSCs were significantly higher in group R than in groups C and RN ( P ＜ 0.01).There were no significant differences in amplitude and frequency of mEPSCs among gorups C,G and RN ( P ＞ 0.05).Conclusion Activation of δ-receptor can enhance NMDA receptor function in spinal dorsal horn neurons in rats which may be the mechanism of remifentanil-induced hyperalgesia. Key words: Piperidines; Hyperalgesia; Receptors, N-Methyl-D-Aspartate; Receptors, opioid,delta; Spinal cord

Neurotensin inhibits background K+ channels and facilitates glutamatergic transmission in rat spinal cord dorsal horn

Neurotensin (NT) is a neuropeptide involved in the modulation of nociception. We have investigated the actions of NT on cultured postnatal rat spinal cord dorsal horn (DH) neurons. NT induced an inward current associated with a decrease in membrane conductance in 46% of the neurons and increased the frequency of glutamatergic miniature excitatory synaptic currents in 37% of the neurons. Similar effects were observed in acute slices. Both effects of NT were reproduced by the selective NTS1 agonist JMV449 and blocked by the NTS1 antagonist SR48692 and the NTS1/NTS2 antagonist SR142948A. The NTS2 agonist levocabastine had no effect. The actions of NT persisted after inactivation of G(i/o) proteins by pertussis toxin but were absent after inactivation of protein kinase C (PKC) by chelerythrine or inhibition of the MAPK (ERK1/2) pathway by PD98059. Pre- and postsynaptic effects of NT were insensitive to classical voltage- and Ca(2+) -dependent K(+) channel blockers. The K(+) conductance inhibited by NT was blocked by Ba(2+) and displayed no or little inward rectification, despite the presence of strongly rectifying Ba(2+) -sensitive K(+) conductance in these neurons. This suggested that NT blocked two-pore domain (K2P) background K(+) -channels rather than inwardly rectifying K(+) channels. Zn(2+) ions, which inhibit TRESK and TASK-3 K2P channels, decreased NT-induced current. Our results indicate that in DH neurons NT activates NTS1 receptors which, via the PKC-dependent activation of the MAPK (ERK1/2) pathway, depolarize the postsynaptic neuron and increase the synaptic release of glutamate. These actions of NT might modulate the transfer and the integration of somatosensory information in the DH.

Effect of eugenol on glutamatergic spontaneous excitatory synaptic transmission in adult rat spinal dorsal horn neurons

Effect of eugenol on glutamatergic spontaneous excitatory synaptic transmission in adult rat spinal dorsal horn neurons

&lt;b&gt;Effect of galanin on excitatory and inhibitory synaptic &lt;/b&gt;&lt;b&gt;transmission in substantia gelatinosa neurons &lt;/b&gt;&lt;b&gt;of rat spinal cord slices &lt;/b&gt;

Galanin (Gal), a neuropeptide composed of 29 / 30 amino acid residues, is thought to play a pivotal role in various physiological functions including nociception. There is much evidence showing that Gal plays a role in modulating nociceptive transmission at the rat spinal cord level. Gal and three types (GalR1-3) of receptor for this are expressed in rat dorsal root ganglion neurons and spinal dorsal horn neurons. Behavioral studies have demonstrated that intrathecal administration of Gal modulates nociceptive transmission in rats. Cellular mechanisms for this Gal action, however, have not been fully examined yet. We have previously reported that Gal facilitates the spontaneous release of L-glutamate from nerve terminals by activating GalR2/R3 without a change in L-glutamate-receptor sensitivity and also produces a membrane hyperpolarization through GalR1 activation and/or depolarization in rat substantia gelatinosa (SG) neurons which play a crucial role in regulating nociceptive transmission through primary-afferent Aδ and C fibers from the periphery. In order to further know a role of Gal in regulating nociceptive transmission, in neurons where the postsynaptic response of Gal was not seen, we examined the action of Gal (0.1 µM) on dorsal root-evoked monosynaptic Aδ-fiber and C-fiber glutamatergic excitatory synaptic transmission, where the blind whole-cell patch-clamp technique was applied to SG neurons of an adult rat spinal cord slice attached with a dorsal root. Since SG neurons receive not only excitatory but also inhibitory inputs, the effect of Gal (0.1 µM) on inhibitory synaptic transmission was also examined. Gal reduced more effectively the peak amplitude of monosynaptic Aδ fiber than C-fiber excitatory postsynaptic current (EPSC) recorded at a holding potential of -70 mV; this was so in a neuron where both Aδ-fiber and C-fiber EPSCs were evoked. These Gal effects were mimicked by a selective GalR2/R3 agonist galanin (2-11) but not a GalR1 agonist M617. On the other hand, spontaneous and focally-evoked (GABAergic and glycinergic) inhibitory synaptic transmission was not affected by Gal (0.1 µM). These results indicate that Gal reduces the evoked release of L-glutamate onto SG neurons from primary-afferent Aδ-fiber and C-fiber central terminals, possibly by activating GalR2/R3. This Gal effect could contribute to its regulation of nociceptive transmission.