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Neuronal hyperactivity in the spinal dorsal horn can amplify nociceptive input in diabetic neuropathic pain. The glutamate N-methyl-d-aspartate and α -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (NMDA receptors and AMPA receptors, respectively) are involved in spinal nociceptive transmission. It is unclear, however, whether painful diabetic neuropathy is associated with changes in the activity of synaptic NMDA receptors and AMPA receptors in spinal dorsal horn neurons. AMPA receptors lacking GluA2 are Ca 2+ -permeable (CP-AMPA receptors), and their currents display characteristic inward rectification. In this study, we showed that evoked excitatory postsynaptic currents (EPSCs), induced by streptozotocin, exhibited inward rectification in spinal dorsal neurons in diabetic rats. Presynaptic and postsynaptic NMDA receptor activity in the spinal dorsal horn was similar in diabetic and control rats. In the dorsal spinal cord, the membrane GluA2 protein level was significantly lower in diabetic than in control rats, whereas the cytosolic GluA2 level was greater in diabetic than in control rats. In contrast, the GluA1 subunit levels in the plasma membrane and cytosol did not differ between the two groups. Blocking CP-AMPA receptors significantly reduced the amplitude of EPSCs of dorsal horn neurons in diabetic but not in control rats. Furthermore, blocking spinal CP-AMPA receptors reduced pain hypersensitivity in diabetic rats but had no effect on nociception in control rats. Our study suggests that diabetic neuropathy augments CP-AMPA receptor activity in the spinal dorsal horn by causing intracellular retention of GluA2 and impairing GluA2 membrane trafficking. Increased prevalence of spinal CP-AMPA receptors sustains diabetic neuropathic pain. SIGNIFICANCE STATEMENT: This study demonstrates that the prevalence of synaptic calcium-permeable AMPA receptors is increased in the spinal dorsal horn, which mediates pain hypersensitivity in diabetic neuropathy. Thus, calcium-permeable AMPA receptors play an important role in glutamatergic synaptic plasticity in the spinal cord in painful diabetic neuropathy. This new knowledge improves our understanding of the mechanisms involved in central sensitization associated with diabetic neuropathic pain and suggests that calcium-permeable AMPA receptors are an alternative therapeutic target for treating this chronic pain condition., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

4-Aminopyridine (4-AP, fampridine) is used clinically to improve neuromuscular function in patients with multiple sclerosis, spinal cord injury, and myasthenia gravis. 4-AP can increase neuromuscular and synaptic transmission by directly stimulating high voltage-activated (HVA) Ca(2+) channels independent of its blocking effect on voltage-activated K(+) channels. Here we provide new evidence that the potentiating effect of 4-AP on HVA Ca(2+) channels depends on the specific combination of voltage-activated calcium channel α1 (Cavα1) and voltage-activated calcium channel β (Cavβ) subunits. Among the four Cavβ subunits examined, Cavβ3 was the most significant subunit involved in the 4-AP-induced potentiation of both L-type and N-type currents. Of particular note, 4-AP at micromolar concentrations selectively potentiated L-type currents reconstituted with Cav1.2, α2δ1, and Cavβ3. In contrast, 4-AP potentiated N-type currents only at much higher concentrations and had little effect on P/Q-type currents. In a phrenic nerve-diaphragm preparation, blocking L-type Ca(2+) channels eliminated the potentiating effect of low concentrations of 4-AP on end-plate potentials. Furthermore, 4-AP enhanced the physical interaction of Cav1.2 and Cav2.2 subunits to Cavβ3 and also increased their trafficking to the plasma membrane. Site-directed mutagenesis identified specific regions in the guanylate kinase, HOOK, and C-terminus domains of the Cavβ3 subunit crucial to the ability of 4-AP to potentiate L-type and N-type currents. Our findings indicate that 4-AP potentiates HVA Ca(2+) channels by enhancing reciprocal Cav1.2-Cavβ3 and Cav2.2-Cavβ3 interactions. The therapeutic effect of 4-AP on neuromuscular function is probably mediated by its actions on Cavβ3-containing L-type Ca(2+) channels., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Increased N-methyl-d-aspartate receptor (NMDAR) activity and phosphorylation in the spinal cord are critically involved in the synaptic plasticity and central sensitization associated with neuropathic pain. However, the mechanisms underlying increased NMDAR activity in neuropathic pain conditions remain poorly understood. Here we show that peripheral nerve injury induces a large GluN2A-mediated increase in NMDAR activity in spinal lamina II, but not lamina I, neurons. However, NMDAR currents in spinal dorsal horn neurons are not significantly altered in rat models of diabetic neuropathic pain and resiniferatoxin-induced painful neuropathy (postherpedic neuralgia). Inhibition of protein tyrosine kinases or protein kinase C has little effect on NMDAR currents potentiated by nerve injury. Strikingly, casein kinase II (CK2) inhibitors normalize increased NMDAR currents of dorsal horn neurons in nerve-injured rats. In addition, inhibition of calcineurin, but not protein phosphatase 1/2A, augments NMDAR currents only in control rats. CK2 inhibition blocks the increase in spinal NMDAR activity by the calcineurin inhibitor in control rats. Furthermore, nerve injury significantly increases CK2α and CK2β protein levels in the spinal cord. In addition, inhibition of CK2 or CK2β knockdown at the spinal level substantially reverses pain hypersensitivity induced by nerve injury. Our study indicates that neuropathic pain conditions with different etiologies do not share the same mechanisms, and increased spinal NMDAR activity is distinctly associated with traumatic nerve injury. CK2 plays a prominent role in the potentiation of NMDAR activity in the spinal dorsal horn and may represent a new target for treatments of chronic pain caused by nerve injury., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Clinically used calcineurin inhibitors, including tacrolimus (FK506) and cyclosporine A, can induce calcineurin inhibitor-induced pain syndrome (CIPS), which is characterized as severe pain and pain hypersensitivity. Increased synaptic N-methyl-D-aspartate receptor (NMDAR) activity in the spinal dorsal horn plays a critical role in the development of CIPS. Casein kinase II (CK2), a serine/threonine protein kinase, can regulate synaptic NMDAR activity in the brain. In this study, we determined whether spinal CK2 is involved in increased NMDAR activity and pain hypersensitivity caused by systemic administration of FK506 in rats. FK506 treatment caused a large increase in the amplitude of NMDAR-mediated excitatory postsynaptic currents (EPSCs) evoked by primary afferent stimulation and in the frequency of miniature EPSCs of spinal dorsal horn neurons. CK2 inhibition with either 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) or 4,5,6,7-tetrabromobenzotriazole (TBB) completely normalized the amplitude of evoked NMDAR-EPSCs of dorsal horn neurons in FK506-treated rats. In addition, DRB or TBB significantly attenuated the amplitude of NMDAR currents elicited by puff application of N-methyl-D-aspartate to dorsal horn neurons in FK506-treated rats. Furthermore, treatment with DRB or TBB significantly reduced the frequency of miniature EPSCs of spinal dorsal horn neurons increased by FK506 treatment. In addition, intrathecal injection of DRB or TBB dose-dependently reversed tactile allodynia and mechanical hyperalgesia in FK506-treated rats. Collectively, our findings indicate that CK2 inhibition abrogates pain hypersensitivity and increased pre- and postsynaptic NMDAR activity in the spinal cord caused by calcineurin inhibitors. CK2 inhibitors may represent a new therapeutic option for the treatment of CIPS.

The glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) are critically involved in the excitatory synaptic transmission, and blocking AMPARs at the spinal level reverses neuropathic pain. However, little is known about changes in the composition of synaptic AMPARs in the spinal dorsal horn after peripheral nerve injury. AMPARs lacking the GluA2 subunit are permeable to Ca(2+), and their currents show unique inward rectification. We found that AMPAR-mediated excitatory postsynaptic currents (AMPAR-EPSCs) of spinal dorsal horn neurons exhibited a linear current-voltage relationship in control rats, whereas AMPAR-EPSCs of dorsal horn neurons displayed inward rectification in rats with spinal nerve injury. In nerve-injured rats, compared with control rats, the GluA2 protein level was significantly less in the plasma membrane but was greater in the cytosolic vesicle fraction in the dorsal spinal cord. However, the GluA1 protein levels in these fractions did not differ significantly between nerve-injured and control rats. Blocking N-methyl-d-aspartate receptors (NMDARs) abolished inward rectification of AMPAR-EPSCs of dorsal horn neurons in nerve-injured rats. Furthermore, inhibition of calpain or calcineurin, but not protein kinase C, completely blocked nerve injury-induced inward rectification of AMPAR-EPSCs of dorsal horn neurons. In addition, blocking GluA2-lacking AMPARs at the spinal cord level reduced nerve injury-induced pain hypersensitivity. Our study suggests that nerve injury increases GluA2 internalization and the prevalence of GluA2-lacking AMPARs in the spinal dorsal horn to maintain chronic neuropathic pain. Increased prevalence of spinal GluA2-lacking AMPARs in neuropathic pain is mediated by NMDARs and subsequent stimulation of calpain and calcineurin signaling.

Nerve injury induces long-term changes in gene expression in the nociceptive circuitry and can lead to chronic neuropathic pain. However, the transcriptional mechanism involved in neuropathic pain is poorly understood. Nuclear factor of activated T-cells (NFATc) is a transcriptional factor regulated by the Ca(2+)-dependent protein phosphatase calcineurin. In this study, we determined nerve injury-induced changes in the expression of NFATc1-c4 in the dorsal root ganglia (DRG) and spinal cords and their role in the development of neuropathic pain. The mRNA of NFATc1-c4 was detected in the rat DRG and dorsal spinal cord. Nerve injury transiently elevated NFATc1-c3 mRNA levels and persistently increased NFATc4 and C-C chemokine receptor type 2 (CCR2) mRNA levels in the DRG. However, NFATc1-c4 mRNA levels in the spinal cord were not altered significantly by nerve injury. Nerve injury also significantly increased the protein level of dephosphorylated NFATc4 in the DRG. Intrathecal injection of the specific NFATc inhibitor 11R-VIVIT or the calcineurin inhibitor FK-506 (tacrolimus) early after nerve injury significantly attenuated the development of tactile allodynia. In addition, treatment with FK-506 or 11R-VIVIT significantly reduced the mRNA levels of NFATc4 and CCR2 but not large-conductance Ca(2+)-activated K(+) channels, in the DRG after nerve injury. Our findings suggest that peripheral nerve injury causes a time-dependent change in NFATc1-c4 expression in the DRG. Calcineurin-NFATc-mediated expression of pronociceptive cytokines contributes to the transition from acute to chronic pain after nerve injury.

Diabetic neuropathy is a common cause of chronic pain that is not adequately relieved by conventional analgesics. The α(2)-adrenoceptors are involved in the regulation of glutamatergic input and nociceptive transmission in the spinal dorsal horn, but their functional changes in diabetic neuropathy are not clear. The purpose of the present study was to determine the plasticity of presynaptic and postsynaptic α(2)-adrenoceptors in the control of spinal glutamatergic synaptic transmission in painful diabetic neuropathy. Whole-cell voltage-clamp recordings of lamina II neurons were performed in spinal cord slices from streptozotocin-induced diabetic rats. The amplitude of glutamatergic excitatory postsynaptic currents (EPSCs) evoked from the dorsal root and the frequency of spontaneous EPSCs (sEPSCs) were significantly higher in diabetic than vehicle-control rats. The specific α(2)-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline (UK-14304) (0.1-2 μM) inhibited the frequency of sEPSCs more in diabetic than vehicle-treated rats. UK-14304 also inhibited the amplitude of evoked monosynaptic and polysynaptic EPSCs more in diabetic than control rats. Furthermore, the amplitude of postsynaptic G protein-coupled inwardly rectifying K(+) channel (GIRK) currents elicited by UK-14304 was significantly larger in the diabetic group than in the control group. In addition, intrathecal administration of UK-14304 increased the nociceptive threshold more in diabetic than vehicle-control rats. Our findings suggest that diabetic neuropathy increases the activity of presynaptic and postsynaptic α(2)-adrenoceptors to attenuate glutamatergic transmission in the spinal dorsal horn, which accounts for the potentiated antinociceptive effect of α(2)-adrenoceptor activation in diabetic neuropathic pain.

Metabotropic glutamate receptors (mGluRs) are involved in the modulation of synaptic transmission and plasticity. Group II mGluRs in the spinal cord regulate glutamatergic input, but their functional changes in neuropathic pain are not clear. In this study, we determined the plasticity of spinal group II mGluRs in controlling excitatory and inhibitory synaptic transmission and nociception in neuropathic pain. Neuropathic pain was induced by spinal nerve ligation in rats, and whole-cell voltage-clamp recordings of glutamatergic excitatory postsynaptic currents (EPSCs) and spontaneous and miniature GABAergic and glycinergic inhibitory postsynaptic currents (sIPSCs and mIPSCs, respectively) were performed in spinal cord slices. The specific group II mGluR agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) had a similar inhibitory effect on monosynaptic EPSCs evoked from the dorsal root in sham and nerve-injured rats. However, DCG-IV produced a greater inhibitory effect on evoked polysynaptic EPSCs and the frequency of spontaneous EPSCs in nerve-injured rats than in control rats. Although DCG-IV similarly reduced the frequency of GABAergic sIPSCs and mIPSCs in both groups, it distinctly inhibited the frequency of glycinergic sIPSCs and mIPSCs only in nerve-injured rats. The DCG-IV effect was blocked by the group II mGluR antagonist but not by the N-methyl-D-aspartate receptor antagonist. Strikingly, intrathecal injection of DCG-IV dose-dependently attenuated allodynia and hyperalgesia in nerve-injured rats but produced hyperalgesia in control rats. Our study provides new information that nerve injury up-regulates group II mGluRs present on glutamatergic and glycinergic interneurons in the spinal cord. Activation of group II mGluRs reduces neuropathic pain probably by attenuating glutamatergic and glycinergic input to spinal dorsal horn neurons.

The arterial baroreceptor is critically involved in the autonomic regulation of homoeostasis. The transient receptor potential vanilloid 1 (TRPV1) receptor is expressed on both somatic and visceral sensory neurons. Here, we examined the TRPV1 innervation of baroreceptive pathways and its functional significance in the baroreflex. Resiniferatoxin (RTX), an ultrapotent analog of capsaicin, was used to ablate TRPV1-expressing afferent neurons and fibers in adult rats. Immunofluorescence labeling revealed that TRPV1 immunoreactivity was present on nerve fibers and terminals in the adventitia of the ascending aorta and aortic arch, the nodose ganglion neurons, and afferent fibers in the solitary tract of the brainstem. RTX treatment eliminated TRPV1 immunoreactivities in the aorta, nodose ganglion, and solitary tract. Renal sympathetic nerve activity, blood pressure, and heart rate were recorded in anesthetized rats. The baroreflex was triggered by lowering and raising blood pressure through intravenous infusion of sodium nitroprusside and phenylephrine, respectively. Inhibition of sympathetic nerve activity and heart rate by the phenylephrine-induced increase in blood pressure was largely impaired in RTX-treated rats. The maximum gain of the baroreflex function was significantly lower in RTX-treated than vehicle-treated rats. Furthermore, blocking of TRPV1 receptors significantly blunted the baroreflex and decreased the maximum gain of baroreflex function in the high blood pressure range. Our findings provide important new information that TRPV1 is expressed along the entire baroreceptive afferent pathway. TRPV1 receptors expressed on baroreceptive nerve endings can function as mechanoreceptors to detect the increase in blood pressure and maintain the homoeostasis.

Removing transient receptor potential vanilloid type 1 (TRPV1)-expressing primary afferent neurons reduces presynaptic mu-opioid receptors but potentiates opioid analgesia. However, the sites and underlying cellular mechanisms for this paradoxical effect remain uncertain. In this study, we determined the presynaptic and postsynaptic effects of the mu-opioid receptor agonist [D-Ala(2),N-Me-Phe(4),Gly-ol(5)]-enkephalin (DAMGO) using whole-cell patch-clamp recordings of lamina II neurons in rat spinal cord slices. Treatment with the ultrapotent TRPV1 agonist resiniferotoxin (RTX) eliminated TRPV1-expressing dorsal root ganglion neurons and their central terminals in the spinal dorsal horn and significantly reduced the basal amplitude of glutamatergic excitatory postsynaptic currents (EPSCs) evoked from primary afferents. Although RTX treatment did not significantly alter the concentration-response effect of DAMGO on evoked monosynaptic and polysynaptic EPSCs, it causes a profound long-lasting inhibitory effect of DAMGO on evoked EPSCs. Subsequent naloxone treatment did not reverse the prolonged inhibitory effect of DAMGO on evoked EPSCs. Furthermore, brief application of DAMGO produced a sustained inhibition of miniature EPSCs in RTX-treated rats. However, the concentration response and the duration of the effects of DAMGO on G protein-coupled inwardly rectifying K+ currents in lamina II neurons were not significantly different between vehicle- and RTX-treated groups. These data suggest that stimulation of mu-opioid receptors on non-TRPV1 afferent terminals causes extended inhibition of neurotransmitter release to spinal dorsal horn neurons. The differential effect of mu-opioid receptor agonists on different phenotypes of primary afferents provides a cellular basis to explain why the analgesic action of opioids on mechanonociception is prolonged when TRPV1-expressing primary afferents are removed.

Glycine is an important inhibitory neurotransmitter in the spinal cord, but it also acts as a coagonist at the glycine site of N-methyl-d-aspartate (NMDA) receptors to potentiate nociceptive transmission. However, little is known about how increased nociceptive inflow alters synaptic glycine release in the spinal dorsal horn and its functional significance. In this study, we performed whole-cell recordings in rat lamina II neurons to record glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs). The transient receptor potential vanilloid receptor 1 agonist capsaicin caused a prolonged increase in the frequency of sIPSCs in 17 of 25 (68%) neurons tested. The potentiating effect of capsaicin on sIPSCs was blocked by ionotropic glutamate receptor antagonists or tetrodotoxin in most lamina II neurons examined. In contrast, the P2X agonist alphabeta-methylene-ATP increased sIPSCs in only two of 16 (12.5%) neurons. The glutamate transporter inhibitor l-trans-pyrrolidine-2,4-dicarboxylic acid either increased or reduced the basal frequency of sIPSCs but did not significantly alter the potentiating effect of capsaicin on sIPSCs. Furthermore, the groups II and III metabotropic glutamate receptor antagonists had no significant effect on the capsaicin-induced increase in the sIPSC frequency. Although capsaicin reduced the amplitude of evoked excitatory postsynaptic currents at high stimulation currents, it did not change the ratio of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/NMDA currents. This study provides the important new information that increased nociceptive inflow augments synaptic glycine release to spinal dorsal horn neurons through endogenous glutamate release. Potentiation of inhibitory glycinergic tone by stimulation of nociceptive primary afferents may function as a negative feedback mechanism to attenuate nociceptive transmission at the spinal level.

Muscarinic acetylcholine receptors (mAChRs) play an important role in the tonic regulation of nociceptive transmission in the spinal cord. However, how mAChR subtypes contribute to the regulation of synaptic glycine release is unknown. To determine their role, glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in lamina II neurons by using whole-cell recordings in spinal cord slices of wild-type (WT) and mAChR subtype knockout (KO) mice. In WT mice, the mAChR agonist oxotremorine-M dose-dependently decreased the frequency of sIPSCs in most neurons, but it had variable effects in other neurons. In contrast, in M3-KO mice, oxotremorine-M consistently decreased the glycinergic sIPSC frequency in all neurons tested, and in M2/M4 double-KO mice, it always increased the sIPSC frequency. In M2/M4 double-KO mice, the potentiating effect of oxotremorine-M was attenuated by higher concentrations in some neurons through activation of GABA(B) receptors. In pertussis toxin-treated WT mice, oxotremorine-M also consistently increased the sIPSC frequency. In M2-KO and M4-KO mice, the effect of oxotremorine-M on sIPSCs was divergent because of the opposing functions of the M3 subtype and the M2 and M4 subtypes. This study demonstrates that stimulation of the M2 and M4 subtypes inhibits glycinergic inputs to spinal dorsal horn neurons of mice, whereas stimulation of the M3 subtype potentiates synaptic glycine release. Furthermore, GABA(B) receptors are involved in the feedback regulation of glycinergic synaptic transmission in the spinal cord. This study revealed distinct functions of mAChR subtypes in controlling glycinergic input to spinal dorsal horn neurons.

Spinal muscarinic acetylcholine receptors (mAChRs) play an important role in the regulation of nociception. To determine the role of individual mAChR subtypes in control of synaptic GABA release, spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature IPSCs (mIPSCs) were recorded in lamina II neurons using whole-cell recordings in spinal cord slices of wild-type and mAChR subtype knockout (KO) mice. The mAChR agonist oxotremorine-M (3-10 microM) dose-dependently decreased the frequency of GABAergic sIPSCs and mIPSCs in wild-type mice. However, in the presence of the M2 and M4 subtype-preferring antagonist himbacine, oxotremorine-M caused a large increase in the sIPSC frequency. In M3 KO and M1/M3 double-KO mice, oxotremorine-M produced a consistent decrease in the frequency of sIPSCs, and this effect was abolished by himbacine. We were surprised to find that in M2/M4 double-KO mice, oxotremorine-M consistently increased the frequency of sIPSCs and mIPSCs in all neurons tested, and this effect was completely abolished by 4-diphenylacetoxy-N-methylpiperidine methiodide, an M3 subtype-preferring antagonist. In M2 or M4 single-KO mice, oxotremorine-M produced a variable effect on sIPSCs; it increased the frequency of sIPSCs in some cells but decreased the sIPSC frequency in other neurons. Taken together, these data strongly suggest that activation of the M3 subtype increases synaptic GABA release in the spinal dorsal horn of mice. In contrast, stimulation of presynaptic M2 and M4 subtypes predominantly attenuates GABAergic inputs to dorsal horn neurons in mice, an action that is opposite to the role of M2 and M4 subtypes in the spinal cord of rats.

The mu opioid agonist morphine has distinct effects on spinal dorsal horn neurons in the superficial and deep laminae. However, it is not clear if the inhibitory effect of morphine on dorsal horn projection neurons is secondary to its potentiating effect on inhibitory interneurons. In this study, we tested the hypothesis that removal of GABAergic and glycinergic inhibitory inputs attenuates the effect of morphine on dorsal horn projection neurons and the reduced spinal GABAergic tone contributes to attenuated morphine effect in neuropathic pain. Single-unit activity of deep dorsal horn projection neurons was recorded in anesthetized normal/sham controls and L(5) and L(6) spinal nerve-ligated rats. Spinal application of 10 microM morphine significantly inhibited the evoked responses of dorsal horn neurons in both normal/sham controls, and this effect was abolished by the specific mu opioid antagonist. However, the effect of morphine on dorsal horn projection neurons was significantly reduced in nerve-injured rats. Furthermore, topical application of the GABA(A) receptor antagonist bicuculline (20 microM) almost abolished the effect of morphine in normal/sham control rats but did not significantly attenuate the morphine effect in nerve-injured rats. On the other hand, the glycine receptor antagonist strychnine (4 microM) significantly decreased the effect of morphine in both nerve-injured and control animals. These data suggest that the inhibitory effect of opioids on dorsal horn projection neurons depends on GABAergic and glycinergic inputs. Furthermore, reduced GABAergic tone probably contributes to diminished analgesic effect of opioids in neuropathic pain.

Cholinergic circuitry and muscarinic receptors within the spinal cord have been proposed to contribute to the analgesic effects of systemic morphine. In this study, we determined whether the descending pathways are involved in the inhibitory effect of systemic morphine on dorsal horn projection neurons mediated by activation of the spinal cholinergic system. Single-unit activity of dorsal horn projection neurons was recorded in anesthetized rats. The neuronal responses to mechanical stimuli applied to the receptive field were determined before and after intravenous injection of morphine. The inhibitory effect of intravenous morphine on dorsal horn neurons was also tested before and after topical spinal application of the muscarinic antagonist atropine in both intact and spinally transected rats. Intravenous injection of 2.5 mg/kg morphine significantly inhibited the evoked response of dorsal horn neurons in both intact and spinally transected rats. Spinal topical application of the mu opioid antagonist H-d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP) completely blocked the effect of morphine on dorsal horn neurons. In addition, spinal application of 10 microM atropine significantly attenuated the effect of systemic morphine. In rats subjected to cervical spinal transection, atropine produced a similar attenuation of the inhibitory effect of systemic morphine on dorsal horn neurons. Data from this electrophysiological study suggest that systemic morphine inhibits ascending dorsal horn neurons through stimulation of spinal mu opioid receptors. Furthermore, activation of the local spinal cholinergic circuitry and muscarinic receptors is involved in the inhibitory effect of systemic morphine on dorsal horn projection neurons independent of descending pathways.

Stimulation of spinal muscarinic acetylcholine receptors (mAChRs) produces potent analgesia. Both M(2) and M(4) mAChRs are coupled to similar G proteins (G(i/o) family) and play a critical role in the analgesic action of mAChR agonists. To determine the relative contribution of M(2) and M(4) subtypes to activation of G(i/o) proteins in the spinal cord, we examined the receptor-mediated guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding in M(2) and M(4) subtype knockout (KO) mice. Basal [(35)S]GTPgammaS binding in the spinal cord was similar in the wild-type controls, M(2) and M(4) single-KO, and M(2)/M(4) double-KO mice. The spinal [(35)S]GTPgammaS binding stimulated by either muscarine or oxotremorine-M was not significantly different among three groups of wild-type mouse strains. In M(2) single-KO and M(2)/M(4) double-KO mice, the agonist-stimulated [(35)S]GTPgammaS binding was completely abolished in the spinal cord. Furthermore, the agonist-stimulated [(35)S]GTPgammaS binding in the spinal cord of M(4) single-KO mice was significantly reduced ( approximately 15%), compared with that in wild-type controls. On the other hand, the spinal [(35)S]GTPgammaS binding stimulated by a mu-opioid agonist was not significantly different between wild-type and M(2) and M(4) KO mice. This study provides complementary new evidence that M(2) is the most predominant mAChR subtype coupled to the G(i/o) proteins in the spinal cord. Furthermore, these data suggest that a small but functionally significant population of M(4) receptors exists in the mouse spinal cord. The functional activity of these M(4) receptors seems to require the presence of M(2) receptors.

The spinal cholinergic system and muscarinic receptors are important for regulation of nociception. Activation of spinal muscarinic receptors produces analgesia and inhibits dorsal horn neurons through potentiation of GABAergic inputs. To determine the role of receptor subtypes in the muscarinic agonist-induced synaptic GABA release, spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in lamina II neurons using whole-cell voltage-clamp recordings in rat spinal cord slices. The muscarinic receptor agonist oxotremorine-M dose-dependently (1-10 microM) increased GABAergic sIPSCs but not miniature IPSCs. The potentiating effect of oxotremorine-M on sIPSCs was completely blocked by atropine. In rats pretreated with intrathecal pertussis toxin to inactive inhibitory G (i/o) proteins, 3 microM oxotremorine-M had no significant effect on sIPSCs in 31 of 55 (56%) neurons tested. In the remaining 24 (44%) neurons in pertussis toxin-treated rats, oxotremorine-M caused a small increase in sIPSCs, and this effect was completely abolished by subsequent application of 25 nM 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), a relatively selective M(3) subtype antagonist. Furthermore, himbacine (1 microM), a relatively specific antagonist for M(2) and M(4) subtypes, produced a large reduction in the stimulatory effect of oxotremorine-M on sIPSCs, and the remaining effect was abolished by 4-DAMP. Additionally, the M(4) receptor antagonist MT-3 toxin (100 nM) significantly attenuated the effect of oxotremorine-M on sIPSCs. Collectively, these data suggest that M(2) and M(4) receptor subtypes play a predominant role in muscarinic potentiation of synaptic GABA release in the spinal cord. The M(3) subtype also contributes to increased GABAergic tone in spinal dorsal horn by muscarinic agonists.

Opioids are potent analgesics, but the sites of their action and cellular mechanisms are not fully understood. The central nucleus of the amygdala (CeA) is important for opioid analgesia through the projection to the periaquaductal gray (PAG). In this study, we examined the effects of mu opioid receptor stimulation on inhibitory and excitatory synaptic inputs to PAG-projecting CeA neurons retrogradely labeled with a fluorescent tracer injected into the ventrolateral PAG of rats. Whole-cell voltage-clamp recordings were performed on labeled CeA neurons in brain slices. The specific mu opioid receptor agonist, [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO, 1 microM), significantly reduced the frequency of miniature inhibitory postsynaptic currents (mIPSCs) without altering the amplitude and decay constant of mIPSCs in 47.6% (10 of 21) of cells tested. DAMGO also significantly decreased the peak amplitude of evoked IPSCs in 69% (9 of 13) of cells examined. However, DAMGO did not significantly alter the frequency of miniature excitatory postsynaptic currents (EPSCs) and the amplitude of evoked EPSCs in 69% (9 of 13) and 83% (10 of 12) of labeled cells, respectively. The IPSCs were blocked by the GABA(A) receptor antagonist bicuculline, whereas the EPSCs were largely abolished by the non-N-methyl-d-aspartate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. The immunoreactivity of mu opioid receptors was colocalized with synaptophysin, a presynaptic marker, in close appositions to labeled CeA neurons. These results suggest that activation of mu opioid receptors on presynaptic terminals primarily attenuates GABAergic synaptic inputs to PAG-projecting neurons in the CeA.

Increased glutamatergic input to spinal dorsal horn neurons constitutes an important mechanism for neuropathic pain. However, the role of group III metabotropic glutamate receptors (mGluRs) in regulation of nociception and dorsal horn neurons in normal and neuropathic pain conditions is not fully known. In this study, we determined the effect of the group III mGluR specific agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4) on nociception and dorsal horn projection neurons in normal rats and a rat model of neuropathic pain. Tactile allodynia was induced by ligation of L5/L6 left spinal nerves in rats. Allodynia was determined by von Frey filaments in nerve-injured rats. The nociceptive threshold was tested using a radiant heat and a Randall-Selitto pressure device in normal rats. Single-unit activity of ascending dorsal horn neurons was recorded from the lumbar spinal cord in anesthetized rats. An intrathecal (5-30 microg) L-AP4 dose-dependently attenuated allodynia in nerve-injured rats but had no antinociceptive effect in normal rats. Topical spinal application of 5 to 50 microM L-AP4 also significantly inhibited the evoked responses of ascending dorsal horn neurons in nerve-ligated but not normal rats. Furthermore, blockade of spinal group III mGluRs significantly decreased the withdrawal threshold and increased the evoked responses of dorsal horn neurons in normal but not nerve-injured rats. These data suggest that group III mGluRs play distinct roles in regulation of nociception and dorsal horn neurons in normal and neuropathic pain states. Activation of spinal group III mGluRs suppresses allodynia and inhibits the hypersensitivity of dorsal horn projection neurons associated with neuropathic pain.

Opioids have a selective effect on nociception with little effect on other sensory modalities. However, the cellular mechanisms for this preferential effect are not fully known. Two broad classes of nociceptors can be distinguished based on their growth factor requirements and binding to isolectin B4(IB4). In this study, we determined the difference in the modulation of voltage-gated Ca2+ currents by [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO, a specific mu opioid agonist) between IB4-positive and -negative small dorsal root ganglion (DRG) neurons. Whole-cell voltage-clamp recordings were performed in acutely isolated DRG neurons in adult rats. Both 1-10 microM DAMGO and 1 to 10 microM morphine had a greater effect on high voltage-activated Ca2+ currents in IB4-negative than IB4-positive cells. However, DAMGO had no significant effect on T-type Ca2+ currents in both groups. The N-type Ca2+ current was the major subtype of Ca2+ currents inhibited by DAMGO in both IB4-positive and -negative neurons. Although DAMGO had no effect on L-type and R-type Ca2+ currents in both groups, it produced a larger inhibition on N-type and P/Q-type Ca2+ currents in IB4-negative than IB4-positive neurons. Furthermore, double labeling revealed that there was a significantly higher mu opioid receptor immunoreactivity in IB4-negative than IB4-positive cells. Thus, these data suggest that N-and P/Q-type Ca2+ currents are more sensitive to inhibition by the mu opioids in IB4-negative than IB4-positive DRG neurons. The differential sensitivity of voltage-gated Ca2+ channels to the mu opioids in subsets of DRG neurons may constitute an important analgesic mechanism of mu opioids.

The rostral ventromedial medulla (RVM) is a major locus for the descending control of nociception and opioid analgesia. However, it is not clear how opioids affect synaptic inputs to RVM neurons. In this study, we determined the effect of mu-opioid receptor activation on excitatory and inhibitory synaptic transmission in spinally projecting RVM neurons. RVM neurons were retrogradely labeled with a fluorescent tracer injected into the dorsal horn of the spinal cord in rats. Whole-cell voltage-clamp recordings were performed on labeled RVM neurons in brain slices in vitro. The mu-receptor agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO, 1 microM) significantly decreased the amplitude of evoked excitatory postsynaptic currents (EPSCs) in 52% (9 of 17) of labeled cells. DAMGO also significantly reduced the amplitude of evoked inhibitory postsynaptic currents (IPSCs) in 69% (11 of 16) of cells examined. Furthermore, DAMGO significantly decreased the frequency of miniature EPSCs in 55% (15 of 27) of cells and significantly decreased the frequency of miniature IPSCs in all 12 cells studied. Although most EPSCs and IPSCs were mediated by glutamate and GABA, the nicotinic and glycine receptor antagonists attenuated EPSCs and IPSCs, respectively, in some labeled RVM neurons. Immunocytochemical labeling revealed that only 35% of recorded RVM neurons were tryptophan hydroxylase-positive, and 15% cells had GABA immunoreactivity. Thus, this study provides important functional evidence that activation of mu-opioid receptors decreases the release of both excitatory and inhibitory neurotransmitters onto most spinally projecting RVM neurons.

Spinally administered muscarinic receptor agonists or acetylcholinesterase inhibitors produce effective pain relief. Intrathecal injection of a small dose of neostigmine produces a profound antiallodynic effect in rats with diabetic neuropathy. However, the mechanisms of increased antinociceptive effect of cholinergic agents on diabetic neuropathic pain are not clear. In the present study, we tested the hypothesis that spinal muscarinic receptors are up-regulated in diabetes. The withdrawal threshold of the hindpaw in response to noxious heat and pressure stimuli was determined in streptozotocin-induced diabetic and age-matched normal rats. Muscarine-stimulated guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding was used to assess the change of functional muscarinic receptors in the spinal cord in diabetes. The [3H]AF-DX 384 membrane binding was performed to determine the number and affinity of spinal cord M2 muscarinic receptors in normal and diabetic rats. We found that the antinociceptive effect of intrathecal 2 to 12 mug muscarine in diabetic animals was potentiated significantly compared with that in normal animals. The maximal muscarine-stimulated [35S]GTPgammaS binding was 112.5 +/- 8.3% in normal rats and 168.8 +/- 12.1% (P < 0.05) in diabetic rats. Although the KD value (2.9 nM) was similar in both groups, the Bmax of [3H]AF-DX 384 membrane binding was significantly higher in diabetic than in normal rats (255.2 +/- 5.9 versus 165.9 +/- 3.5 fmol/mg protein, P < 0.05). Collectively, these data strongly suggest that the muscarinic receptor is up-regulated in the dorsal spinal cord in diabetic rats. This finding probably accounts for the increased efficacy of the antinociceptive effect of intrathecal muscarinic agonists in diabetic neuropathic pain.

Intrathecal [D-Pen2,D-Pen5]-enkephalin (DPDPE; a delta-opioid agonist) has a profound antinociceptive effect in neuropathic pain. Spinal nitric oxide (NO) has been implicated in the analgesic effect of several G protein-coupled receptor agonists. Little, however, is known about the role of spinal NO in the inhibitory effect of DPDPE on spinal dorsal horn neurons. In the present study, we determined the role of NO in the inhibitory effect of DPDPE on ascending dorsal horn neurons in normal rats and in a rat model of diabetic neuropathic pain. Single-unit activity of ascending dorsal horn neurons was recorded in anesthetized rats. The responses of dorsal horn neurons to graded mechanical stimuli and von Frey filaments were determined before and after local spinal application of 0.1 to 5 microM DPDPE. The influence of an NO synthase inhibitor, 1-(2-trifluoromethylphenyl) imidazole (TRIM; 30 microM), on the effect of DPDPE was then studied in separate groups of dorsal horn neurons in normal and diabetic rats. DPDPE inhibited the response of dorsal horn neurons in both normal and diabetic rats in a concentration-dependent fashion. The inhibitory effect of 1 microM DPDPE was abolished by 1 microM naltrindole, a delta-opioid antagonist. Furthermore, the inhibitory effect of DPDPE on the evoked response of dorsal horn neurons was largely eliminated by TRIM in normal and diabetic rats. These data suggest that DPDPE has a profound inhibitory effect on dorsal horn neurons in normal and diabetic rats. Spinal endogenous NO is essential for the inhibitory effect of DPDPE on ascending dorsal horn neurons in both normal and diabetic rats.

Increased glutamate availability in the spinal cord and primary afferent nerves plays an important role in acute and chronic pain. Afferent ectopic discharges from the site of nerve injury constitute a source of abnormal sensory input to the spinal dorsal horn. The ectopic afferent activity is largely responsible for the development of hypersensitivity of dorsal horn neurons and neuropathic pain. Inhibition of glutamate carboxypeptidase II (GCP II) reduces glutamate release generated from N-acetyl-aspartyl-glutamate in nerve tissues and may have an analgesic effect on neuropathic pain. In the present study, we determined the effect of a GCP II inhibitor, 2-(phosphono-methyl)-pentanedioic acid (2-PMPA), on allodynia and ectopic afferent discharges in an animal model of neuropathic pain. Neuropathic pain was induced by partial ligation of the left sciatic nerve in rats. Tactile allodynia was assessed using von Frey filaments applied to the plantar surface of the injured hindpaw. Single-unit activity of ectopic discharges was recorded from the sciatic nerve proximal to the site of ligation. Intravenous injection of 50 to 100 mg/kg 2-PMPA significantly reduced allodynia in a dose-dependent manner. Furthermore, 2-PMPA dose-dependently attenuated the ectopic discharge activity of injured sciatic afferent nerves. At a dose of 100 mg/kg, 2-PMPA significantly inhibited the ectopic activity from 14.7 +/- 2.1 to 4.4 +/- 0.5 impulses/s without altering the conduction velocity of afferent nerves. Therefore, these data suggest that the antiallodynic effect of 2-PMPA may be mediated, at least in part, by inhibition of ectopic afferent discharges at the site of nerve injury.