Presynaptic enhancement by eugenol of spontaneous excitatory transmission in rat spinal substantia gelatinosa neurons is mediated by transient receptor potential A1 channels.

Eugenol, which is contained in several plants including clove, has been widely used as an analgesic and anti-inflammatory drug in the dental clinic. Eugenol also has anesthetic effects and produces sedation and the reduction of convulsion threshold. These benefits have been partly attributed to the effects of eugenol on neural tissues, such as inhibition of voltage-gated ion channels. As expected from the fact that eugenol is a vanilloid compound, this drug activates transient receptor potential (TRP) V1 channels in the peripheral nervous system (PNS). Although eugenol affects synaptic transmission in the central nervous system (CNS), this has not yet been fully examined. We investigated how eugenol affects spontaneous glutamatergic excitatory transmission in substantia gelatinosa (SG; lamina II of Rexed) neurons of adult rat spinal cord slices by use of the blind whole-cell patch-clamp technique. Bath-applied eugenol reversibly enhanced spontaneous excitatory transmission and produced an outward current at -70 mV in SG neurons. The former action was due to a large increase in the frequency of spontaneous excitatory postsynaptic current (sEPSC) with a small increase in the amplitude. These actions of eugenol were seen by its repeated application and resistant to a voltage-gated Na(+) channel blocker tetrodotoxin. The effect of eugenol on sEPSC frequency was concentration-dependent with an EC(50) value of 3.8 mM and unaffected by a TRPV1 antagonist capsazepine, whereas inhibited by a nonspecific TRP antagonist ruthenium red and a TRPA1 antagonist HC-030031. On the other hand, the eugenol-induced outward current was not affected by these TRP antagonists. It is concluded that eugenol activates TRPA1 channels in the SG, leading to an increase in the spontaneous release of L-glutamate to SG neurons, and that eugenol also produces a membrane hyperpolarization that is not mediated by TRP channels. Eugenol is suggested to activate different types of TRP channel between the PNS and CNS.

Effect of resiniferatoxin on glutamatergic spontaneous excitatory synaptic transmission in substantia gelatinosa neurons of the adult rat spinal cord.

The transient receptor potential (TRP) vanilloid type 1 (TRPV1) agonist, capsaicin, enhances glutamatergic spontaneous excitatory synaptic transmission in CNS neurons. Resiniferatoxin (RTX) has a much higher affinity for TRPV1 than capsaicin, but its ability to modulate excitatory transmission is unclear. We examined the effect of RTX on excitatory transmission using the whole-cell patch-clamp technique in substantia gelatinosa (SG) neurons of adult rat spinal cord slices. Bath-applied RTX dose-dependently increased the frequency, but not the amplitude, of spontaneous excitatory postsynaptic current (sEPSC), independent of its application time. In about a half of the neurons tested, this effect was accompanied by an inward current at -70 mV that was sensitive to glutamate-receptor antagonists. Repeated application of RTX did not affect excitatory transmission. RTX was more potent than capsaicin but showed similar efficacy. RTX activity could be blocked by capsazepine or SB-366791, a TRPV1 antagonist, but not tetrodotoxin, a Na(+)-channel blocker, and could be inhibited by pretreatment with capsaicin but not the TRPA1 agonist, allyl isothiocyanate. RTX enhances the spontaneous release of L-glutamate from nerve terminals with similar efficacy as capsaicin and produces a membrane depolarization by activating TRPV1 in the SG, with fast desensitization and slow recovery from desensitization. These results indicate a mechanism by which RTX can modulate excitatory transmission in SG neurons to regulate nociceptive transmission.

Proteinase-activated receptor-1 activation presynaptically enhances spontaneous glutamatergic excitatory transmission in adult rat substantia gelatinosa neurons.

Proteinase-activated receptors (PARs) have a unique activation mechanism in that a proteolytically exposed N-terminal region acts as a tethered ligand. A potential impact of PAR on sensory processing has not been fully examined yet. Here we report that synthetic peptides with sequences corresponding to PAR ligands enhance glutamatergic excitatory transmission in substantia gelatinosa (SG) neurons of adult rat spinal cord slices by using the whole cell patch-clamp technique. The frequency of spontaneous excitatory postsynaptic current (EPSC) was increased by PAR-1 agonist SFLLRN-NH2 (by 47% at 1 microM) with small increases by PAR-2 and -4 agonists (SLIGKV-NH2 and GYPGQV-OH, respectively; at >3 microM); there was no change in its amplitude or in holding current at -70 mV. The PAR-1 peptide action was inhibited by PAR-1 antagonist YFLLRNP-OH. TFLLR-NH2, an agonist which is more selective to PAR-1 than SFLLRN-NH2, dose-dependently increased spontaneous EPSC frequency (EC50=0.32 microM). A similar presynaptic effect was produced by PAR-1 activating proteinase thrombin in a manner sensitive to YFLLRNP-OH. The PAR-1 peptide action was resistant to tetrodotoxin and inhibited in Ca2+-free solution. Primary-afferent monosynaptically evoked EPSC amplitudes were unaffected by PAR-1 agonist. These results indicate that PAR-1 activation increases the spontaneous release of L-glutamate onto SG neurons from nerve terminals in a manner dependent on extracellular Ca2+. Considering that sensory processing within the SG plays a pivotal role in regulating nociceptive transmission to the spinal dorsal horn, the PAR-1-mediated glutamatergic transmission enhancement could be involved in a positive modulation of nociceptive transmission.

Tramadol, but not its major metabolite (mono-O-demethyl tramadol) depresses compound action potentials in frog sciatic nerves.

BACKGROUND AND PURPOSE: Although tramadol is known to exhibit a local anaesthetic effect, how tramadol exerts this effect is not understood fully. EXPERIMENTAL APPROACH: The effects of tramadol and its metabolite mono-O-demethyl-tramadol (M1) on compound action potentials (CAPs) were examined by applying the air-gap method to frog sciatic nerves, and the results were compared with those of other local anaesthetics, lidocaine and ropivacaine. KEY RESULTS: Tramadol reduced the peak amplitude of the CAP in a dose-dependent manner (IC50=2.3 mM). On the other hand, M1 (1-2 mM), which exhibits a higher affinity for mu-opioid receptors than tramadol, did not affect CAPs. These effects of tramadol were resistant to the non-selective opioid receptor antagonist naloxone and the mu-opioid receptor agonist, DAMGO, did not affect CAPs. This tramadol action was not affected by a combination of the noradrenaline uptake inhibitor, desipramine, and the 5-hydroxytryptamine uptake inhibitor, fluoxetine. Lidocaine and ropivacaine also concentration-dependently reduced CAP peak amplitudes with IC50 values of 0.74 and 0.34 mM, respectively. CONCLUSIONS AND IMPLICATIONS: These results indicate that tramadol reduces the peak amplitude of CAP in peripheral nerve fibres with a potency which is less than those of lidocaine and ropivacaine, whereas M1 has much less effect on CAPs. This action of tramadol was not produced by activation of mu-opioid receptors nor by inhibition of noradrenaline and 5-hydroxytryptamine uptake. It is suggested that the methyl group present in tramadol but not in M1 may play an important role in producing nerve conduction block.

Inhibition by endomorphin-1 and endomorphin-2 of excitatory transmission in adult rat substantia gelatinosa neurons.

Intrathecally-administered endomorphin-1 and endomorphin-2 produce antinociceptive effects which are different from each other. In order to elucidate a cellular basis for this result, we examined the effects of endomorphin-1 and endomorphin-2 on holding currents and spontaneous glutamatergic excitatory transmission in substantia gelatinosa neurons of adult rat spinal cord slices by use of the whole-cell patch-clamp technique. In about half of the neurons examined, endomorphin-1 and endomorphin-2 produced an outward current having a similar amplitude (25-27 pA at 1 microM) at -70 mV with almost the same value of effective concentration producing half-maximal response (0.19-0.21 microM). Both of them reversed at a potential close to the equilibrium potential for K+, and had the slope conductance that was larger at negative (-120 to -140 mV) than positive potentials (-60 to -90 mV). The endomorphin-1 and endomorphin-2 currents were reduced in amplitude by K+-channel inhibitors, Ba2+ (100 microM) and 4-aminopyridine (1 mM), and also by mu-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (1 microM) to a similar extent. The endomorphin-2 but not endomorphin-1 current amplitude was increased by dipeptidyl peptidase IV inhibitor diprotin A (30 microM). One micromolar endomorphin-1 and endomorphin-2 reduced the frequency of spontaneous excitatory postsynaptic current with a similar time course and extent without altering its amplitude; these actions were not in the presence of D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (1 microM). We conclude that endomorphin-1 and endomorphin-2 hyperpolarize membranes by opening inwardly-rectifying K+ channels and attenuate the spontaneous release of L-glutamate from nerve terminals in the substantia gelatinosa, both of which are mediated by mu-opioid receptors, in a manner quantitatively similar to each other. The difference in antinociceptive effects between endomorphin-1 and endomorphin-2 could not be attributed to a distinction in their effects on excitatory transmission in substantia gelatinosa neurons, and may be explained by a difference in their enzymatic degradation.

Phospholipase A2 activation by melittin enhances spontaneous glutamatergic excitatory transmission in rat substantia gelatinosa neurons.

In order to know a role of phospholipase A2 in modulating nociceptive transmission, the effect of a secreted phospholipase A2 activator melittin on spontaneous glutamatergic excitatory transmission was investigated in substantia gelatinosa neurons of an adult rat spinal cord slice by using the whole-cell patch-clamp technique. Bath-applied melittin at concentrations higher than 0.5 microM increased both the amplitude and the frequency of spontaneous excitatory postsynaptic current in a manner independent of tetrodotoxin; the latter effect of which was examined in detail. In 80% of the neurons examined (n = 64), melittin superfused for 3 min gradually increased spontaneous excitatory postsynaptic current frequency (by 65+/-6% at 1 microM; n = 51) in a dose-dependent manner (effective concentration for half-maximal effect = 1.1 microM). This effect subsided within 3 min after washout. The spontaneous excitatory postsynaptic current frequency increase produced by melittin was reduced by the phospholipase A2 inhibitor 4-bromophenacryl bromide (10 microM) while being unaffected by the cyclooxygenase inhibitor indomethacin (100 microM) and the lipoxygenase inhibitor nordihydroguaiaretic acid (100 microM). A similar increase in spontaneous excitatory postsynaptic current frequency was produced by exogenous arachidonic acid (50 microM); this effect was also unaffected by the cyclooxygenase or lipoxygenase inhibitor. Melittin failed to increase spontaneous excitatory postsynaptic current frequency in a nominally Ca2+-free or La3+-containing Krebs solution. We conclude that melittin increases the spontaneous release of L-glutamate to substantia gelatinosa neurons by activating secreted phospholipase A2 and increasing Ca2+ influx through voltage-gated Ca2+ channels in nerve terminals, probably with an involvement of arachidonic acid but not its metabolites produced by cyclooxygenase and lipoxygenase. Considering that the substantia gelatinosa plays an important role in regulating nociceptive transmission, it is suggested that this transmission may be positively modulated by secreted phospholipase A2 activation in the substantia gelatinosa.

Modulation by adenosine of Adelta and C primary-afferent glutamatergic transmission in adult rat substantia gelatinosa neurons.

The present study examined the actions of adenosine on monosynaptic Adelta and C primary-afferent excitatory postsynaptic currents (EPSCs) recorded from substantia gelatinosa (SG) neurons of an adult rat spinal cord slice. In 67% of the neurons examined, adenosine reversibly decreased the amplitude of the Adelta-fiber EPSC, while in 13% of the neurons the amplitude was reduced or unaffected, which was followed by its increase persisting for several minutes after adenosine washout. The remaining neurons did not exhibit a change in the amplitude. The reduction in Adelta-fiber EPSC amplitude by adenosine was dose-dependent with an effective concentration for half-inhibition (EC50) value of 217 microM. When examined by using a paired-pulse stimulus, a ratio of the second to first Adelta-fiber EPSC amplitude under the reduction was larger than that of EPSC amplitude in the control, suggesting a presynaptic action of adenosine. In 69% of the neurons tested, the C-fiber EPSC was reversibly decreased in amplitude by adenosine (100 microM) by an extent comparable to that of Adelta-fiber EPSC; the remaining neurons were without adenosine actions. Similar inhibitory actions of adenosine were also seen in neurons where both Adelta-fiber and C-fiber EPSCs were elicited. Similar reduction in the Adelta-fiber or C-fiber EPSC amplitude was induced by an A1 adenosine-receptor agonist, N6-cyclopentyladenosine (1 microM), and the adenosine-induced reduction was not observed in the presence of an A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (1 microM). An A2a agonist, CGS 21680 (1 microM), did not significantly affect the Adelta-fiber EPSC amplitude. It is concluded that adenosine presynaptically inhibits monosynaptic Adelta-fiber and C-fiber transmission by a similar extent through the activation of the A1 receptor in many but not all SG neurons; this could contribute to at least a part of antinociception by intrathecally administered adenosine analogues and probably by endogenous adenosine.

Nociceptin inhibits excitatory but not inhibitory transmission to substantia gelatinosa neurones of adult rat spinal cord.

Although intrathecal administration of nociceptin, an endogenous ligand of the opioid receptor-like1 receptor, exhibits an antinociceptive effect in various pain models, cellular mechanisms underlying this action are still unknown. Here, we investigated the effects of nociceptin on excitatory and inhibitory synaptic transmission to substantia gelatinosa neurones of an adult rat spinal cord slice with an attached dorsal root by use of the blind whole-cell patch-clamp technique; this was done under the condition of a blockade of a hyperpolarising effect of nociceptin. In about 70% of the neurones examined, nociceptin (1 microM) reduced the amplitude of glutamatergic excitatory postsynaptic currents (EPSCs) which were monosynaptically evoked by stimulating Adelta- or C-afferent fibres; the inhibition of C-fibre EPSCs (50+/-6%, n=11) was larger than that of Adelta-fibre EPSCs (30+/-5%, n=23; P<0.05). Each of the nociceptin actions was dose-dependent in a concentration range of 0.1 to 1 microM, and was largely suppressed by a selective opioid receptor-like1 receptor antagonist, 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (3 microM). Nociceptin (1 microM) also decreased miniature EPSCs frequency by 22+/-6% (n=7) while not affecting their amplitude. Responses of substantia gelatinosa neurones to bath-applied alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (10 microM) were not changed by nociceptin. Both electrically evoked and miniature inhibitory postsynaptic currents, mediated by either the GABA(A) or glycine receptor, were unaffected by nociceptin. These results indicate that nociceptin suppresses excitatory but not inhibitory synaptic transmission to substantia gelatinosa neurones through the activation of the opioid receptor-like1 receptor; this action is pre-synaptic in origin. Considering that the substantia gelatinosa is the main part of termination of Adelta- and C-fibres transmitting nociceptive information, the present finding would account for at least a part of the inhibitory action of nociceptin on pain transmission. Nociceptin could inhibit more potently slow-conducting than fast-conducting pain transmission.

Nociceptin-induced outward current in substantia gelatinosa neurones of the adult rat spinal cord.

Nociceptin (NOC), also known as orphanin FQ, is a newly discovered endogenous ligand for the opioid receptor-like1 (ORL1) receptor. Although NOC has been shown to modulate nociceptive transmission, mechanisms for this action are still unknown. In the present study, actions of NOC on substantia gelatinosa (SG) neurones were examined in adult rat spinal cord slice preparations by using the whole-cell patch-clamp technique. NOC at a concentration of 1 microM induced an outward current having an amplitude of 26+/-5 pA (n=68) at a holding potential of -70 mV; this action was dose-dependent with an EC(50) value of 0.23 microM (Hill coefficient: 1.5). The NOC current reversed its polarity at a potential which was close to the equilibrium potential for K(+), as calculated by the Nernst equation (n=4). The NOC current had slope conductances of 0.80+/-0.15 nS and 0.50+/-0.13 nS (n=4) in voltage ranges of -120 to -140 mV and of -60 to -90 mV, respectively. The NOC current was inhibited by Ba(2+) (100 microM; by 56+/-8%, n=4) but not by 4-aminopyridine (4-AP; 1 mM; n=4) and tetraethylammonium (TEA; 5 mM; n=4). The NOC current was not affected by tetrodotoxin (TTX; 1 microM; n=4) and also by a non-specific opioid receptor antagonist, naloxone (1 microM; n=4). When examined using some inhibitors with respect to the ORL1 receptor, the NOC (1 microM) current was depressed in amplitude by a putative NOC precursor product, nocistatin (1 microM; by 18+/-4%, n=6) and also by a non-peptidyl ORL1 receptor antagonist, CompB (1 microM; by 64+/-10%, n=7) without a change in holding currents. On the other hand, a putative ORL1 receptor antagonist, [Phe(1)psi(CH(2)-NH)Gly(2)]nociceptin-(1-13)-NH(2) (1 microM; which is a derivative of NOC), by itself induced an outward current (7+/-3 pA, n=8), during which the NOC current was suppressed in amplitude by 56+/-8% (n=8). We conclude that NOC activates in SG neurones a K(+) channel exhibiting a mild inwardly rectification through the activation of ORL1 receptor; this hyperpolarising action of NOC might contribute to at least a part of its antinociceptive effect.

Voltage-clamp recordings of postsynaptic currents in substantia gelatinosa neurons in vitro and its applications to assess synaptic transmission.

We describe here procedures for recording postsynaptic currents in substantia gelatinosa neurons on a transverse spinal cord slice preparation with an attached dorsal root. At the holding potential of -70 mV, glutamatergic spontaneous excitatory postsynaptic currents (EPSCs) and dorsal root (A delta and/or C fiber) stimulation-evoked EPSCs could be observed. Whereas at the holding potential of 0 mV, spontaneous inhibitory postsynaptic currents (IPSCs) and dorsal root A delta fiber stimulation-evoked IPSCs could be encountered. The methods make it possible to evaluate synaptic transmission by analysing the postsynaptic currents on dorsal root attached spinal cord slice.

In vivo patch-clamp analysis of IPSCs evoked in rat substantia gelatinosa neurons by cutaneous mechanical stimulation.

To know a functional role of inhibitory synaptic responses in transmitting noxious and innoxious information from the periphery to the rat spinal dorsal horn, we examined inhibitory postsynaptic currents (IPSCs) elicited in substantia gelatinosa (SG) neurons by mechanical stimuli applied to the skin using the newly developed in vivo patch-clamp technique. In the majority (80%) of SG neurons examined, a brush stimulus applied to the ipsilateral hind limb produced a barrage of IPSCs that persisted during the stimulus, while a pinch stimulus evoked IPSCs only at its beginning and end. The pinch-evoked IPSCs may have been caused by a touch that occurs at the on/off time of the pinch. The evoked IPSCs were blocked by either a glycine-receptor antagonist, strychnine (4 microM), or a GABA(A)-receptor antagonist, bicuculline (20 microM). All SG neurons examined received inhibitory inputs from a wide area throughout the thigh and lower leg. When IPSCs were examined together with excitatory postsynaptic currents (EPSCs) in the same neurons, a brush evoked a persistent activity of both IPSCs and EPSCs during the stimulus while a pinch evoked such an activity of EPSCs but not IPSCs. It is suggested that innoxious mechanical stimuli activate a GABAergic or glycinergic circuitry in the spinal dorsal horn. This inhibitory transmission may play an important role in the modulation of noxious information in the SG.

Alteration in synaptic inputs through C-afferent fibers to substantia gelatinosa neurons of the rat spinal dorsal horn during postnatal development.

The change in synaptic inputs through primary afferent C- and A-fibers during postnatal development was examined in substantia gelatinosa neurons of a rat spinal cord slice with an attached L5 dorsal root by use of the blind whole-cell patch-clamp technique; the synaptic responses were compared between the slices obtained from immature (postnatal days 21-23) and mature (postnatal days 56-60) male rats. The mono- and/or polysynaptic afferent inputs were monitored by recording glutamatergic excitatory postsynaptic currents and potentials evoked by stimulating C- and A-fibers, the identification of which was based on the values of threshold stimulus intensity and of the conduction velocity of the fibers, determined by intracellular recordings from dorsal root ganglion neurons. Immature substantia gelatinosa neurons received synaptic inputs through Abeta-, Adelta- and C-afferents, with proportions of 51%, 46% and 36%, respectively. In mature substantia gelatinosa neurons, C- and Adelta-afferent inputs were increased in number (to 84% and 86%, respectively), while Abeta-inputs were decreased to 9%. In both immature and mature rats, repetitive stimulation of C-afferents did not elicit any slow responses, which are longer in duration than the monosynaptic excitatory postsynaptic currents, although C-fibers are known to contain not only excitatory amino acids, but also neuropeptides such as substance P, which is thought to be involved in the production of slow responses. These results indicate that both C- and Adelta-afferents innervating substantia gelatinosa neurons are reorganized following maturation, accompanied by a withdrawal or elimination of Abeta-fibers from the substantia gelatinosa, probably due to a competition among the fibers during development. In spite of the developmental increase in C-fiber inputs, mature as well as immature substantia gelatinosa neurons did not display any slow synaptic responses, which appear to be mediated by transmitters other than excitatory amino acids.

Capsaicin induces a slow inward current which is not mediated by substance P in substantia gelatinosa neurons of the rat spinal cord.

Whole-cell voltage-clamp techniques were employed to investigate a capsaicin-induced current in substantia gelatinosa (SG) neurons in the dorsal horn of adult rat spinal cord slices. Bath-applied capsaicin (2 microM) for 30 s activated a slow excitatory current having an amplitude of 21.3+/-6.3 pA and a duration of 93+/-13 s (n=10; V(H)=-70 mV). This capsaicin current was compared in amplitude under various conditions among different SG neurons. After either neonatal capsaicin treatment or sciatic-nerve transection, by which C-afferent fibers are known to degenerate, this capsaicin current was reduced in amplitude to 5.0+/-3.5 pA (n=8) or 4.5+/-2.3 pA (n=6), respectively. A non-N-methyl-D-aspartate (NMDA)-receptor antagonist, CNQX (10 microM), depressed greatly the capsaicin current to 4.0+/-1.3 pA (n=9). On the other hand, this current had an amplitude of 14.4+/-2.7 pA (n=10) in the presence of an NMDA-receptor antagonist, AP-5 (50 microM); this value was not significantly different from that in the control (P>0.05). Substance P (SP; 1-2 microM) superfused for 2 min had no detectable effect on all SG neurons examined (n=7). After SP washout, however, these cells exhibited a capsaicin current (22.8+/-12.1 pA); this current persisted in the presence of a neurokinin-1 receptor antagonist, L-732,138 (1 microM; 19.8+/-3.5pA, n=9). The capsaicin current was not abolished by an intracellular dialysis with GDP-beta-S (1 mM; 20. 2+/-2.4 pA, n=9) which inhibited a baclofen (10 microM) response mediated by the G-protein-coupled GABA(B) receptor. These results indicate that the capsaicin-induced current is mediated through the activation of C-fibers by non-NMDA receptors. This mechanism in SG neurons is different from that known in neurons in other laminae of the dorsal horn that is thought to be a direct action of SP released from C-fibers. This current in SG neurons would contribute to the pain sensation caused by capsaicin.

Mechanisms for ovariectomy-induced hyperalgesia and its relief by calcitonin: participation of 5-HT1A-like receptor on C-afferent terminals in substantia gelatinosa of the rat spinal cord.

Chronic treatment with calcitonin in osteoporotic patients alleviates the pain associated with this condition by an unknown mechanism. In ovariectomized rats that develop osteoporosis and hyperalgesia, we examined whether a functional change in serotonergic systems in the spinal dorsal horn was involved, using whole-cell recordings from substantia gelatinosa neurons in spinal cord slices and [(3)H]8-hydroxy-2(di-n-propylamino)tetralin ([(3)H]8-OH-DPAT) binding. Hyperalgesia could be attributed to the elimination of presynaptic inhibition by 5-HT of glutamatergic primary C-afferent terminals and an associated decrease in the density of [(3)H]8-OH-DPAT binding sites whose receptors are neither 5-HT(1A)- nor 5-HT(7)-subtype. These changes in serotonergic systems were restored after chronic treatment with calcitonin. Reversal of 5-HT receptor changes by calcitonin treatment may provide an explanation for its analgesic actions in patients.

Actions of midazolam on GABAergic transmission in substantia gelatinosa neurons of adult rat spinal cord slices.

BACKGROUND: Although intrathecal administration of midazolam has been found to produce analgesia, how midazolam exerts this effect is not understood fully at the neuronal level in the spinal cord. METHODS: The effects of midazolam on either electrically evoked or spontaneous inhibitory transmission and on a response to exogenous gamma-aminobutyric acid (GABA), a GABA(A)-receptor agonist, muscimol, or glycine were evaluated in substantia gelatinosa neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. RESULTS: Bath-applied midazolam (1 microM) prolonged the decay phase of evoked and miniature inhibitory postsynaptic currents (IPSCs), mediated by GABA(A) receptors, without a change in amplitudes, while not affecting glycine receptor-mediated miniature inhibitory postsynaptic currents in both the decay phase and the amplitude. Either GABA- or muscimol-induced currents were enhanced in amplitude by midazolam (0.1 microM) in a manner sensitive to a benzodiazepine receptor antagonist, flumazenil (1 microM); glycine currents were, however, unaltered by midazolam. CONCLUSIONS: Midazolam augmented both the duration of GABA-mediated synaptic current and the amplitude of GABA-induced current by acting on the GABA(A)-benzodiazepine receptor in substantia gelatinosa neurons; this would increase the inhibitory GABAergic transmission. This may be a possible mechanism for antinociception by midazolam.

Responsiveness of rat substantia gelatinosa neurones to mechanical but not thermal stimuli revealed by in vivo patch-clamp recording.

1. Synaptic responses of 46 substantia gelatinosa (SG) neurones in the spinal dorsal horn to cutaneous mechanical and/or thermal stimuli were investigated in an in vivo rat preparation with whole-cell patch-clamp recordings. The clamped neurones were identified as being in the SG based on either their morphological features by intrasomatic injection of biocytin or the depth of the neurones from the surface of the spinal cord. 2. In all SG neurones examined where spontaneous EPSCs occurred, pinch (noxious) and air (innocuous) stimuli applied to the ipsilateral hindlimb elicited a barrage of EPSCs (some of which initiated an action potential under current-clamp conditions), which subsided just after cessation of the stimuli without any residual slow current (or after-discharge). The spontaneous and evoked EPSCs were reversibly abolished by a non-N-methyl-D-aspartate (non-NMDA) receptor antagonist, CNQX (20 microM). 3. Noxious (>= 45 C) or innocuous (<= 40 C) thermal stimuli did not elicit any synaptic responses in all 18 SG neurones tested which were sensitive to mechanical stimuli. Noxious cold stimulation (<= 10 C) also failed to produce any responses (n = 6). 4. It is concluded that both noxious and innocuous mechanical information to SG neurones are transmitted primarily by activation of non-NMDA receptors, probably without any involvement of slow synaptic transmission, and that thermal information is conveyed to areas of the dorsal horn other than SG.

Actions of opioids on excitatory and inhibitory transmission in substantia gelatinosa of adult rat spinal cord.

1. The actions of opioid receptor agonists on synaptic transmission in substantia gelatinosa (SG) neurones in adult (6- to 10-week-old) rat spinal cord slices were examined by use of the blind whole-cell patch-clamp technique. 2. Both the mu-receptor agonist DAMGO (1 microM) and the delta-receptor agonist DPDPE (1 microM) reduced the amplitude of glutamatergic excitatory postsynaptic currents (EPSCs) which were monosynaptically evoked by stimulating Adelta afferent fibres. Both also decreased the frequency of miniature EPSCs without affecting their amplitude. 3. In contrast, the kappa-receptor agonist U-69593 (1 microM) had little effect on the evoked and miniature EPSCs. 4. The effects of DAMGO and DPDPE were not seen in the presence of the mu-receptor antagonist CTAP (1 microM) and the delta-receptor antagonist naltrindole (1 microM), respectively. 5. Neither DAMGO nor DPDPE at 1 microM affected the responses of SG neurones to bath-applied AMPA (10 microM). 6. Evoked and miniature inhibitory postsynaptic currents (IPSCs), mediated by either the GABAA or the glycine receptor, were unaffected by the mu-, delta- and kappa-receptor agonists. Similar results were also obtained in SG neurones in young adult (3- to 4-week-old) rat spinal cord slices. 7. These results indicate that opioids suppress excitatory but not inhibitory synaptic transmission, possibly through the activation of mu- and delta- but not kappa-receptors in adult rat spinal cord SG neurones; these actions are presynaptic in origin. Such an action of opioids may be a possible mechanism for the antinociception produced by their intrathecal administration.

Action of capsaicin on dorsal root-evoked synaptic transmission to substantia gelatinosa neurons in adult rat spinal cord slices.

An action of capsaicin was investigated on dorsal root-evoked synaptic transmission to substantia gelatinosa (SG) neurons in adult rat spinal cord slices by use of the whole-cell voltage-clamp technique. In 79% of neurons examined, superfusing capsaicin (1 microM) for 30 s depressed a C-fiber-evoked excitatory synaptic current in a manner sensitive to a capsaicin-receptor antagonist, capsazepine (10 microM). On the contrary, Adelta-fiber-evoked excitatory and inhibitory synaptic currents were unaffected by capsaicin in all of cells tested. It is concluded that capsaicin specifically acts on C-afferents, resulting in an inhibition of evoked excitatory transmission to the SG; this may contribute to, at least in part, an acute analgesic action of capsaicin.

Capsaicin facilitates excitatory but not inhibitory synaptic transmission in substantia gelatinosa of the rat spinal cord.

Actions of capsaicin were examined on synaptic transmissions in the substantia gelatinosa (SG) of adult rat spinal cord slices using the whole-cell patch-recording technique. Bath-applied capsaicin at a concentration of 2 microM activated a slow inward current (having an amplitude of 33 pA at -70 mV), which was accompanied by an increase in the frequency of glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs; by 234%); these actions were blocked by a capsaicin-receptor antagonist, capsazepine (10 microM). The capsaicin-induced increase in sEPSC frequency was resistant to tetrodotoxin (0.5-1 microM). On the other hand, capsaicin (2 microM) did not affect either glycine- or gamma-aminobutyric acid-mediated spontaneous synaptic transmission. The results indicate that capsaicin enhances excitatory but not inhibitory synaptic transmission, possibly through a direct action on primary afferent terminals in the SG. As the SG has been thought to participate in nociceptive pathway, it is suggested that such a presynaptic action of capsaicin contributes to nociceptive transmissions.

Phenotypic characterization of septal neurons in culture: immunohistochemistry of GABA, calbindin D-28k and choline acetyltransferase, and histochemistry of acetylcholinesterase.

Phenotypes of septal neurons, dissociated from 19-day-old fetal rat brains and then cultured in a medium containing nerve growth factor for 4 weeks, were examined using gamma-aminobutyric acid (GABA), calbindin D-28k, parvalbumin and choline acetyltransferase immunohistochemistry, and acetylcholinesterase histochemistry. There were primarily four groups of neurons identified in this septal culture: the first group (12.7% of 212 neurons examined) displayed a cholinergic, but not GABAergic, phenotype and had an average diameter of 13.6 +/- 2.7 microm (mean +/- S.D.); the second group (31.6%) displayed both cholinergic and GABAergic phenotypes and had a diameter of 12.2 +/- 2.8 microm; the third group (31.0%) displayed only a GABAergic phenotype and had a diameter of 10.4 +/- 2.3 microm; and the fourth group (24.7%) displayed neither a GABAergic nor cholinergic phenotype and had a diameter of 10.4 +/- 2.1 microm. Neurons in the first two groups described were significantly larger than those in the second two groups; neurons in the third and fourth groups were the same size. Calbindin D-28k was expressed in some neurons of each group (31.3%, 18.8%, 9.6% and 15.7%, respectively). These results demonstrate that septal neurons have the ability to express a variety of phenotypes when grown in vitro. This culture will be a useful tool for studying mechanisms of phenotype expression in septal neurons.