Analyses of the Mode of Action of an Alpha-Adrenoceptor Blocker in Substantia Gelatinosa Neurons in Rats.

To elucidate why naftopidil increases the frequency of spontaneous synaptic currents in only some substantia gelatinosa (SG) neurons, post-hoc analyses were performed. Blind patch-clamp recording was performed using slice preparations of SG neurons from the spinal cords of adult rats. Spontaneous inhibitory and excitatory postsynaptic currents (sIPSCs and sEPSCs, respectively) were recorded. The ratios of the frequency and amplitude of the sIPSCs and sEPSCs following the introduction of naftopidil compared with baseline, and after the application of naftopidil, serotonin (5-HT), and prazosin, compared with noradrenaline (NA) were evaluated. First, the sIPSC analysis indicated that SG neurons reached their full response ratio for NA at 50 µM. Second, they responded to 5-HT (50 µM) with a response ratio similar to that for NA, but prazosin (10 µM) did not change the sEPSCs and sIPSCs. Third, the highest concentration of naftopidil (100 µM) led to two types of response in the SG neurons, which corresponded with the reactions to 5-HT and prazosin. These results indicate that not all neurons were necessarily activated by naftopidil, and that the micturition reflex may be regulated in a sophisticated manner by inhibitory mechanisms in these interneurons.

Nerve injury elevates functional Cav3.2 channels in superficial spinal dorsal horn.

Cav3 channels play an important role in modulating chronic pain. However, less is known about the functional changes of Cav3 channels in superficial spinal dorsal horn in neuropathic pain states. Here, we examined the effect of partial sciatic nerve ligation (PSNL) on either expression or electrophysiological properties of Cav3 channels in superficial spinal dorsal horn. Our in vivo studies showed that the blockers of Cav3 channels robustly alleviated PSNL-induced mechanical allodynia and thermal hyperalgesia, which lasted at least 14 days following PSNL. Meanwhile, PSNL triggered an increase in both mRNA and protein levels of Cav3.2 but not Cav3.1 or Cav3.3 in rats. However, in Cav3.2 knockout mice, PSNL predominantly attenuated mechanical allodynia but not thermal hyperalgesia. In addition, the results of whole-cell patch-clamp recordings showed that both the overall proportion of Cav3 current-expressing neurons and the Cav3 current density in individual neurons were elevated in spinal lamina II neurons from PSNL rats, which could not be recapitulated in Cav3.2 knockout mice. Altogether, our findings reveal that the elevated functional Cav3.2 channels in superficial spinal dorsal horn may contribute to the mechanical allodynia in PSNL-induced neuropathic pain model.

Inhibition by O-desmethyltramadol of glutamatergic excitatory transmission in adult rat spinal substantia gelatinosa neurons.

To reveal cellular mechanisms for antinociception produced by clinically used tramadol, we investigated the effect of its metabolite O-desmethyltramadol (M1) on glutamatergic excitatory transmission in spinal dorsal horn lamina II (substantia gelatinosa; SG) neurons. The whole-cell patch-clamp technique was applied at a holding potential of -70 mV to SG neurons of an adult rat spinal cord slice with an attached dorsal root. Under the condition where a postsynaptic action of M1 was inhibited, M1 superfused for 2 min reduced the frequency of spontaneous excitatory postsynaptic current in a manner sensitive to a µ-opioid receptor antagonist CTAP; its amplitude and also a response of SG neurons to bath-applied AMPA were hardly affected. The presynaptic effect of M1 was different from that of noradrenaline or serotonin which was examined in the same neuron. M1 also reduced by almost the same extent the peak amplitudes of monosynaptic primary-afferent Aδ-fiber and C-fiber excitatory postsynaptic currents evoked by stimulating the dorsal root. These actions of M1 persisted for >10 min after its washout. These results indicate that M1 inhibits the quantal release of L-glutamate from nerve terminals by activating µ-opioid but not noradrenaline and serotonin receptors; this inhibition is comparable in extent between monosynaptic primary-afferent Aδ-fiber and C-fiber transmissions. Considering that the SG plays a pivotal role in regulating nociceptive transmission, the present findings could contribute to at least a part of the inhibitory action of tramadol on nociceptive transmission together with its hyperpolarizing effect as reported previously.

Modulation of inhibitory and excitatory neurotransmissions by Zn2+ on the substantia gelatinosa neurons of the trigeminal subnucleus caudalis in mice.

The substantia gelatinosa of the trigeminal subnucleus caudalis has been considered to be an essential location for the transference of orofacial sensory signals. The co-localization of inhibitory and excitatory neurotransmitters in the same substantia gelatinosa (SG) neurons has demonstrated their essential part in the modification of nociceptive transmission. Zn2+ is particularly numerous in the mammalian central nervous system. There are proofs demonstrating the role of Zn2+ in the modulation of voltage- and ligand-gated ion channels. However, little is known about what roles Zn2+ may play in the modulation of signal transmission in the SG neurons of the trigeminal subnucleus caudalis (Vc). Therefore, in this study, we used the whole-cell patch clamp technique to find out the effect of Zn2+ on the responses of three main neurotransmitters (glycine, GABA, and glutamate) on SG neurons of the Vc in mice. We have proved that Zn2+ induces a big potentiation of glycine receptor-mediated response but attenuates GABA- and glutamate-induced responses at micromolar concentrations, however, enhances glutamate-induced response at nanomolar concentration. Taken together, these data demonstrated that Zn2+ can modulate glycine, GABA and glutamate-mediated actions on the SG neurons of the Vc and support an important mechanism in spinal sensory information signaling.

The endogenous agonist, ß-alanine, activates glycine receptors in rat spinal dorsal neurons.

ß-alanine is a structural analog of glycine and γ-aminobutyric acid (GABA) and is thought to be involved in the modulation of nociceptive information at the spinal cord. However, it is not known whether ß-alanine exerts its effect in substantia gelatinosa (SG) neurons of the spinal dorsal horn, where glycine and GABA play an important role in regulating nociceptive transmission from the periphery. Here, we investigated the effects of ß-alanine on inhibitory synaptic transmission in adult rat SG neurons using whole-cell patch-clamp. ß-alanine dose-dependently induced outward currents in SG neurons. Current-voltage plots revealed a reversal potential at approximately -70 mV, which was close to the equilibrium potential of Cl-. Pharmacological analysis revealed that ß-alanine activates glycine receptors, but not GABAA receptors. These results suggest that ß-alanine hyperpolarizes the membrane potential of SG neurons by activating Cl- channels through glycine receptors. Our findings raise the possibility that ß-alanine may modulate pain sensation through glycine receptors.

In vivo patch-clamp analysis of the antinociceptive actions of TRPA1 activation in the spinal dorsal horn.

BACKGROUND: Transient receptor potential (TRP) channels are nonselective cation channels expressed in a variety of sensory structures, and are important molecular mediators of thermal, mechanical, cellular and chemical signals. We investigated the function of one key member of the TRP superfamily, TRPA1, in the spinal dorsal horn using in vivo patch-clamp recordings. RESULTS: The application of allyl isothiocyanate (AITC), a TRPA1 agonist, significantly increased the frequency and amplitude of inhibitory postsynaptic currents (IPSCs; holding potential (VH) = 0 mV) as well as excitatory postsynaptic currents (EPSCs; VH = -70 mV) in substantia gelatinosa (SG) neurons. The AITC-induced increases in EPSC frequency and amplitude were resistant to the Na(+) channel blocker tetrodotoxin (TTX). In the presence of the glutamate receptor antagonists CNQX and AP5, AITC did not generate any synaptic activity. The AITC-induced increases in IPSC frequency and amplitude were abolished by TTX or glutamate receptor antagonists. Moreover, the duration of IPSCs enhanced by TRPA1 activation were significantly longer than those of EPSCs enhanced by activation of this channel in the spinal dorsal horn. AITC induced hyperpolarization of the membrane potential of SG neurons in the spinal cord but depolarized the membrane potential in the presence of TTX. Furthermore, we examined the effects of mechanical stimuli to the skin during TRPA1 activation in the spinal dorsal horn in normal rats in both voltage-clamp and current-clamp modes. In the peripheral tissue stimuli test, AITC significantly suppressed EPSCs evoked by pinch or air puff stimulation of the skin. In current-clamp mode, AITC significantly suppressed excitatory postsynaptic potentials (EPSPs) evoked by pinch stimuli. CONCLUSIONS: TRPA1 appears to be localized not only at presynaptic terminals on SG neurons, enhancing glutamate release, but also in the terminals of primary afferents innervating spinal inhibitory interneurons, which have synaptic interactions with SG neurons. This study offers further insight into the mechanisms underlying the possible antinociceptive actions of TRPA1 activation in the spinal dorsal horn. Our findings suggest that pharmacological activation of spinal TRPA1 channels may have therapeutic potential for the treatment of pain.

Spontaneous L-glutamate release enhancement in rat substantia gelatinosa neurons by (-)-carvone and (+)-carvone which activate different types of TRP channel.

Transient receptor potential (TRP) channels in the spinal dorsal horn lamina II (substantia gelatinosa; SG), which are involved in the modulation of nociceptive transmission, have not yet been fully examined in property. Activation of the TRP channels by various plant-derived chemicals results in an increase in the spontaneous release of L-glutamate onto the SG neurons. We examined the effects of a monoterpene ketone (-)-carvone (contained in spearmint) and its stereoisomer (+)-carvone (in caraway) on glutamatergic spontaneous excitatory transmission in SG neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. (-)-Carvone and (+)-carvone increased the frequency of spontaneous excitatory postsynaptic current (sEPSC) in a reversible and concentration-dependent manner with a small increase in its amplitude. Half-maximal effective concentrations of (-)-carvone and (+)-carvone in increasing sEPSC frequency were 0.70 mM and 0.72 mM, respectively. The (-)-carvone but not (+)-carvone activity was inhibited by a TRPV1 antagonist capsazepine. On the other hand, the (+)-carvone but not (-)-carvone activity was inhibited by a TRPA1 antagonist HC-030031. These results indicate that (-)-carvone and (+)-carvone activate TRPV1 and TRPA1 channels, respectively, resulting in an increase in spontaneous L-glutamate release onto SG neurons, with almost the same efficacy. Such a difference in TRP activation between the stereoisomers may serve to know the properties of TRP channels in the SG.

Minocycline, a microglial inhibitor, blocks spinal CCL2-induced heat hyperalgesia and augmentation of glutamatergic transmission in substantia gelatinosa neurons.

BACKGROUND: Several lines of evidence suggest that CCL2 could initiate the hyperalgesia of neuropathic pain by causing central sensitization of spinal dorsal horn neurons and facilitating nociceptive transmission in the spinal dorsal horn. The cellular and molecular mechanisms by which CCL2 enhances spinal pain transmission and causes hyperalgesia remain unknown. The substantia gelatinosa (lamina II) of the spinal dorsal horn plays a critical role in nociceptive transmission. An activated spinal microglia, which is believed to release pro-inflammatory cytokines including TNF-α, plays an important role in the development of neuropathic pain, and CCL2 is a key mediator for spinal microglia activation. In the present study, we tested the hypothesis that spinal CCL2 causes the central sensitization of substantia gelatinosa neurons and enhances spinal nociceptive transmission by activating the spinal microglia and augmenting glutamatergic transmission in lamina II neurons. METHODS: CCL2 was intrathecally administered to 2-month-old male rats. An intrathecal injection of CCL2 induced heat hyperalgesia, which was assessed using the hot plate test. Whole-cell voltage-clamp recordings substantia gelatinosa neurons in spinal cord slices were performed to record glutamatergic excitatory postsynaptic currents (EPSCs) and GABAergic inhibitory postsynaptic currents (IPSCs). RESULTS: The hot plate test showed that 1 day after the intrathecal injection of CCL2 (1 µg), the latency of hind-paw withdrawal caused by a heat stimulus was significantly reduced in rats. One day after the intrathecal administration of CCL2, the amplitude of the evoked glutamatergic EPSCs and the frequency of spontaneous glutamatergic miniature EPSCs (mEPSCs) were significantly increased in outer lamina II neurons. Intrathecal co-injection of minocycline, a specific inhibitor of microglial activation, and CCL2 blocked the CCL2-induced reduction in the latency of hind-paw withdrawal and thermal hyperalgesia. Following intrathecal co-administration of CCL2 and minocycline, CCL2 failed to increase the frequency of glutamatergic mEPSCs and failed to promote glutamine release in lamina II neurons. Intrathecal co-injection of WP9QY, a selective TNF-α antagonist, and CCL2 completely inhibited CCL2-induced heat hyperalgesia and inhibited the increase in the frequency of glutamatergic mEPSCs in substantia gelatinosa neurons. CONCLUSION: In summary, our results suggest that an intrathecal injection of CCL2 causes thermal hyperalgesia by augmenting the excitatory glutamatergic transmission in substantia gelatinosa neurons through a presynaptic mechanism and facilitating nociceptive transmission in the spinal dorsal horn. Further studies show that intrathecal co-administration of minocycline, a specific inhibitor of microglial activation, or WP9QY, a selective TNF-α antagonist, completely inhibited CCL2 potentiation of glutamatergic transmission in substantia gelatinosa neurons and CCL2-induced heat hyperalgesia. The results of the present study suggest that peripheral nerve injury-induced upregulation of the spinal CCL2 level causes the central sensitization of substantia gelatinosa neurons by activating spinal microglia and that TNF-α mediates CCL2-induced thermal hyperalgesia and augmentation of glutamatergic transmission in lamina II neurons.

Dual effect of exogenous nitric oxide on neuronal excitability in rat substantia gelatinosa neurons.

Nitric oxide (NO) is an important signaling molecule involved in nociceptive transmission. It can induce analgesic and hyperalgesic effects in the central nervous system. In this study, patch-clamp recording was used to investigate the effect of NO on neuronal excitability in substantia gelatinosa (SG) neurons of the spinal cord. Different concentrations of sodium nitroprusside (SNP; NO donor) induced a dual effect on the excitability of neuronal membrane: 1 mM of SNP evoked membrane hyperpolarization and an outward current, whereas 10 µM induced depolarization of the membrane and an inward current. These effects were prevented by hemoglobin and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (c-PTIO) (NO scavengers), phenyl N-tert-butylnitrone (PBN; nonspecific reactive oxygen species scavenger), and through inhibition of soluble guanylyl cyclase (sGC). Pretreatment with n-ethylmaleimide (NEM; thiol-alkylating agent) also decreased effects of both 1 mM and 10 µM SNP, suggesting that these responses were mediated by direct S-nitrosylation. Charybdotoxin (CTX) and tetraethylammonium (TEA) (large-conductance Ca(2+)-activated K(+) channel blockers) and glybenclamide (ATP-sensitive K(+) channel blocker) decreased SNP-induced hyperpolarization. La(3+) (nonspecific cation channel blocker), but not Cs(+) (hyperpolarization-activated K(+) channel blocker), blocked SNP-induced membrane depolarization. In conclusion, NO dually affects neuronal excitability in a concentration-dependent manner via modification of various K(+) channels.

Transsynaptic inhibition of spinal transmission by A2 botulinum toxin.

Type A botulinum toxin blocks not only ACh release from motor nerve terminals but also central synaptic transmission, including glutamate, noradrenaline, dopamine, ATP, GABA and glycine. Neurotoxins (NTXs) are transported by both antero- and retrogradely along either motor or sensory axons for bidirectional delivery between peripheral tissues or the CNS. A newly developed type A2 NTX (A2NTX) injected into one rat foreleg muscle was transported to the contralateral muscle. This finding was consistent with the NTX traveling retrogradely via spinal neurons and then transsynaptically through motor neurons to the contralateral motor neurons within the spinal cord and on to the soleus muscle. In the present study we found that toxin injection into the rat left soleus muscle clearly induced bilateral muscle relaxation in a dose-dependent fashion, although the contralateral muscle relaxation followed the complete inhibition of toxin-injected ipsilateral muscles. The toxin-injected ipsilateral muscle relaxation was faster and stronger in A2NTX-treated rats than A1LL (BOTOX). A1LL was transported almost equally to the contralateral muscle via neural pathways and the bloodstream. In contrast, A2NTX was mainly transported to contralateral muscles via the blood. A1LL was more successfully transported to contralateral spinal neurons than A2NTX. We also demonstrated that A1LL and A2NTX were carried from peripheral to CNS and vice versa by dual antero- and retrograde axonal transport through either motor or sensory neurons.

Enhancement by interleukin-1ß of AMPA and NMDA receptor-mediated currents in adult rat spinal superficial dorsal horn neurons.

BACKGROUND: Proinflammatory cytokine interleukin-1ß (IL-1ß) 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-1ß 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. RESULTS: AMPA- and NMDA-induced currents were increased in peak amplitude by IL-1ß in a manner different from each other in SG neurons. These facilitatory actions of IL-1ß 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-1ß was recovered to control level 30 min after IL-1ß 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-1ß on AMPA- but not NMDA-induced currents, where minocycline itself depressed NMDA- but had not any effects on AMPA-induced currents. CONCLUSIONS: IL-1ß 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-1ß 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-1ß 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.

Activation of glycine and extrasynaptic GABA(A) receptors by taurine on the substantia gelatinosa neurons of the trigeminal subnucleus caudalis.

The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) has been known for the processing and transmission of orofacial nociceptive information. Taurine, one of the most plentiful free amino-acids in humans, has proved to be involved in pain modulation. In this study, using whole-cell patch clamp technique, we investigated the direct membrane effects of taurine and the action mechanism behind taurine-mediated responses on the SG neurons of the Vc. Taurine showed non-desensitizing and repeatable membrane depolarizations and inward currents which remained in the presence of amino-acid receptors blocking cocktail (AARBC) with tetrodotoxin, indicating that taurine acts directly on the postsynaptic SG neurons. Further, application of taurine at different doses (10 µM to 3 mM) showed a concentration dependent depolarizations and inward currents with the EC50 of 84.3 µM and 723 µM, respectively. Taurine-mediated responses were partially blocked by picrotoxin (50 µM) and almost completely blocked by strychnine (2 µM), suggesting that taurine-mediated responses are via glycine receptor (GlyR) activation. In addition, taurine (1 mM) activated extrasynaptic GABA(A) receptor (GABA(A)R)-mediated currents. Taken together, our results indicate that taurine can be a target molecule for orofacial pain modulation through the activation of GlyRs and/or extrasynaptic GABA(A)Rs on the SG neurons.

Cell type-specific postsynaptic effects of neuropeptide Y in substantia gelatinosa neurons of the rat spinal cord.

Cellular mechanisms of antinociceptive action of neuropeptide Y were investigated in substantia gelatinosa (SG) neurons in rat spinal cord slices. Somatic and synaptic effects of NPY were compared in two subpopulations of cells with different firing patterns, tonic (TFNs), and delayed firing (DFNs) neurons. For the study, TFNs were selected on morphological basis: they had appearance of central and radial but not islet cells, and are likely excitatory interneurons in dorsal horn networks. In their turn, DFNs were classified as radial and vertical cells. 0.3 µM NPY via Y1 receptors activated hyperpolarizing postsynaptic current of GIRK type in majority of TFNs (∼77%) but not DFNs (∼8%). Miniature synaptic currents in all neurons were seen as a mixture of excitatory (mEPSCs) and inhibitory (mIPSCs), the frequency of the former being ∼5 times greater. The mEPSCs were mediated by glutamate receptors of AMPA subtype, while the dominant part of mIPSCs--by glycine receptors. In all cell types, NPY moderately depressed the frequency of both mEPSCs and mIPSCs; the effects occurred via Y2 and Y1 receptors, respectively. The data suggest that behavioral NPY-evoked antinociception is achieved via postsynaptic hyperpolarization of majority of TFNs (assumingly, excitatory interneurons) via Y1 receptors and depression of the mEPSCs via Y2 receptors.

TRPV1 agonist piperine but not olvanil enhances glutamatergic spontaneous excitatory transmission in rat spinal substantia gelatinosa neurons.

We examined the effects of TRPV1 agonists olvanil and piperine on glutamatergic spontaneous excitatory transmission in the substantia gelatinosa (SG) neurons of adult rat spinal cord slices with the whole-cell patch-clamp technique. Bath-applied olvanil did not affect the frequency and amplitude of spontaneous excitatory postsynaptic current (sEPSC), and unchanged holding currents at -70 mV. On the other hand, superfusing piperine reversibly and concentration-dependently increased sEPSC frequency (half-maximal effective concentration: 52.3 µM) with a minimal increase in its amplitude. This sEPSC frequency increase was almost repetitive at an interval of more than 20 min. Piperine at a high concentration produced an inward current in some neurons. The facilitatory effect of piperine was blocked by TRPV1 antagonist capsazepine. It is concluded that piperine but not olvanil activates TRPV1 channels in the central terminals of primary-afferent neurons, resulting in an increase in the spontaneous release of l-glutamate onto SG neurons.

Zaltoprofen inhibits bradykinin-mediated enhancement of glutamate receptor activity in substantia gelatinosa neurons.

BACKGROUND: Zaltoprofen, a propionic acid derivative of nonsteroidal anti-inflammatory drugs, has been proposed to inhibit the nociception mediated by bradykinin. Here, I attempted to clarify the molecular mechanisms underlying the blocking effect of zaltoprofen on bradykinin-mediated enhancement of excitatory glutamatergic transmission in the superficial dorsal horn of the spinal cord. METHODS: The effects of zaltoprofen on the response to exogenous administration of α-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid (AMPA) currents were examined in lamina II neurons of adult rat spinal cord slices using the whole-cell patch-clamp technique. RESULTS: AMPA currents were significantly enhanced by preapplication of bradykinin (10 µM). However, zaltoprofen (1, 10 µM) and a nonselective cyclooxygenase (COX)-1 and COX-2 inhibitor, ibuprofen, blocked the bradykinin-mediated enhancing effect. The inhibitory effect of ibuprofen, but not zaltoprofen, was removed by adding prostaglandin E(2). Furthermore, the inhibitory effect of zaltoprofen was removed in the presence of protein kinase C (PKC) activator, whereas the effect of zaltoprofen was still present in the presence of phospholipase C activator. CONCLUSIONS: These findings suggest that the antinociceptive effect of zaltoprofen may block the augmenting effect of bradykinin on AMPA currents through inhibition of protein kinase C activation, without affecting COX in the superficial dorsal horn.

Mrgprd-expressing polymodal nociceptive neurons innervate most known classes of substantia gelatinosa neurons.

The Mas-related G-protein-coupled receptor D (Mrgprd) marks a distinct subset of sensory neurons that transmit polymodal nociceptive information from the skin epidermis to the substantia gelatinosa (SG, lamina II) of the spinal cord. Moreover, Mrgprd-expressing (Mrgprd(+)) neurons are required for the full expression of mechanical but not thermal nociception. While such anatomical and functional specificity suggests Mrgprd(+) neurons might synapse with specific postsynaptic targets in the SG, precisely how Mrgprd(+) neurons interface with spinal circuits is currently unknown. To study circuit connectivity, we genetically targeted the light-activated ion channel Channelrhodopsin-2-Venus (ChR2-Venus) to the Mrgprd locus. In these knock-in mice, ChR2-Venus was localized to nonpeptidergic Mrgprd(+) neurons and axons, while peptidergic CGRP(+) neurons were not significantly labeled. Dissociated Mrgprd(+) DRG neurons from mice expressing one or two copies of ChR2-Venus could be activated in vitro as evidenced by light-evoked currents and action potentials. In addition, illumination of Mrgprd-ChR2-Venus(+) axon terminals in spinal cord slices evoked EPSCs in half of all SG neurons. Within this subset, Mrgprd(+) neurons were monosynaptically connected to most known classes of SG neurons, including radial, tonic central, transient central, vertical, and antenna cells. This cellular diversity ruled out the possibility that Mrgprd(+) neurons innervate a dedicated class of SG neuron. Our findings set broad constraints on the types of spinal neurons that process afferent input from Mrgprd(+) polymodal nociceptors.

Inhibitory neurones of the spinal substantia gelatinosa mediate interaction of signals from primary afferents.

The spinal substantia gelatinosa (SG; lamina II) is a major synaptic zone for unmyelinated (C) primary afferents. Whereas a substantial proportion of intrinsic SG neurones are GABAergic inhibitory, their relationship to afferent activity is unknown. In spinal cord slices from a transgenic mouse in which certain GABAergic lamina II neurones are labelled with green fluorescent protein (GFP), we compared primary afferent input with local efferent connections made by inhibitory SG neurones. Simultaneous whole-cell recordings from characterized neurones establish that inhibitory SG neurones receive monosynaptic input from a subset of unmyelinated primary afferents and connect to other lamina II cells that have input from a different set of afferents, permitting interactions between distinctive afferent messages. Certain lamina II inhibitory cells were found to connect to one another by reciprocal links. Inhibitory lamina II connections appear arranged to modulate activity from different sets of peripheral unmyelinated fibres through neural circuitry that includes disinhibition.

Dopamine inhibition of glycine release in the rat trigeminal nucleus pars caudalis: possible involvement of trace amine receptors.

Dopamine (DA)-induced pre-synaptic inhibition of glycinergic transmission was studied from substantia gelatinosa (SG) neurons of the trigeminal nucleus pars caudalis using a conventional whole-cell patch clamp technique. The action potential-dependent glycinergic inhibitory post-synaptic currents (IPSCs) were recorded from SG neurons in the presence of 3 mM kynurenic acid and 10 µM 6-imino-3-(4-methoxyphenyl)-1(6H)-pyridazinebutanoic acid HBr (SR95531). In these conditions, bath applied DA (100 µM) reduced the amplitude of glycinergic IPSCs and increased the paired-pulse ratio, suggesting that DA acts pre-synaptically to reduce the probability of glycine release. However, the inhibitory action of DA on glycinergic IPSCs was not blocked by SCH23390 (10 µM) and spiperone (1 µM), selective D(1) - and D(2) -like receptor antagonists, respectively. In addition, either SKF38393 (100 µM), a selective D(1) -like receptor agonist, or quinpirole (100 µM), a selective D(2) -like receptor agonist, had no pre-synaptic effect on glycinergic IPSCs. The results suggest that both D(1) - and D(2) -like receptors are not involved in the DA-induced decrease in glycinergic IPSCs. On the other hand, tyramine (100 µM), one of representative trace amines, reduced the amplitude of glycinergic IPSCs and increased the paired-pulse ratio, suggesting that tyramine acts pre-synaptically to reduce the probability of glycine release. Considering that DA can activate trace amine (TA) receptors and that TA receptors are exclusively expressed on the trigeminal nucleus pars caudalis, DA might act on putative pre-synaptic TA receptors, rather than classical DA receptors, to inhibit glycinergic transmission onto SG neurons of the trigeminal nucleus pars caudalis.

Potentiation of excitatory transmission in substantia gelatinosa neurons of rat spinal cord by inhibition of estrogen receptor alpha.

BACKGROUND: It has been shown that estrogen is synthesized in the spinal dorsal horn and plays a role in modulating pain transmission. One of the estrogen receptor (ER) subtypes, estrogen receptor alpha (ERα), is expressed in the spinal laminae I-V, including substantia gelatinosa (SG, lamina II). However, it is unclear how ERs are involved in the modulation of nociceptive transmission. RESULTS: In the present study, a selective ERα antagonist, methyl-piperidino-pyrazole (MPP), was used to test the potential functional roles of spinal ERα in the nociceptive transmission. Using the whole-cell patch-clamp technique, we examined the effects of MPP on SG neurons in the dorsal root-attached spinal cord slice prepared from adult rats. We found that MPP increased glutamatergic excitatory postsynaptic currents (EPSCs) evoked by the stimulation of either Aδ- or C-afferent fibers. Further studies showed that MPP treatment dose-dependently increased spontaneous EPSCs frequency in SG neurons, while not affecting the amplitude. In addition, the PKC was involved in the MPP-induced enhancement of synaptic transmission. CONCLUSIONS: These results suggest that the selective ERα antagonist MPP pre-synaptically facilitates the excitatory synaptic transmission to SG neurons. The nociceptive transmission evoked by Aδ- and C-fiber stimulation could be potentiated by blocking ERα in the spinal neurons. Thus, the spinal estrogen may negatively regulate the nociceptive transmission through the activation of ERα.

TRPA1-expressing primary afferents synapse with a morphologically identified subclass of substantia gelatinosa neurons in the adult rat spinal cord.

The TRPA1 channel has been proposed to be a molecular transducer of cold and inflammatory nociceptive signals. It is expressed on a subset of small primary afferent neurons both in the peripheral terminals, where it serves as a sensor, and on the central nerve endings in the dorsal horn. The substantia gelatinosa (SG) of the spinal cord is a key site for integration of noxious inputs. The SG neurons are morphologically and functionally heterogeneous and the precise synaptic circuits of the SG are poorly understood. We examined how activation of TRPA1 channels affects synaptic transmission onto SG neurons using whole-cell patch-clamp recordings and morphological analyses in adult rat spinal cord slices. Cinnamaldehyde (TRPA1 agonist) elicited a barrage of excitatory postsynaptic currents (EPSCs) in a subset of the SG neurons that responded to allyl isothiocyanate (less specific TRPA1 agonist) and capsaicin (TRPV1 agonist). Cinnamaldehyde evoked EPSCs in vertical and radial but not islet or central SG cells. Notably, cinnamaldehyde produced no change in inhibitory postsynaptic currents and nor did it produce direct postsynaptic effects. In the presence of tetrodotoxin, cinnamaldehyde increased the frequency but not amplitude of miniature EPSCs. Intriguingly, cinnamaldehyde had a selective inhibitory action on monosynaptic C- (but not Adelta-) fiber-evoked EPSCs. These results indicate that activation of spinal TRPA1 presynaptically facilitates miniature excitatory synaptic transmission from primary afferents onto vertical and radial cells to initiate action potentials. The presence of TRPA1 channels on the central terminals raises the possibility of bidirectional modulatory action in morphologically identified subclasses of SG neurons.