Megumi Shimoyama

Gabapentin affects glutamatergic excitatory neurotransmission in the rat dorsal horn

&NA; We investigated the effects of gabapentin (GBP) on glutamatergic synaptic transmission in the dorsal horn of the rat spinal cord. Patch clamp whole cell recordings were made from superficial and deep dorsal horn neurons of rat spinal cord slices. In the majority of neurons in the superficial lamina, GBP decreased the amplitudes of evoked excitatory postsynaptic currents (evoked EPSCs) mediated by either non‐NMDA or NMDA receptors. In contrast, neurons in the deep lamina showed variable effects, with a lower incidence of decrease in amplitude of evoked EPSCs and a subset of neurons showing an increase in amplitude of evoked NMDA receptor‐mediated EPSCs. No enhancement of evoked non‐NMDA receptor‐mediated EPSCs was observed in either lamina. To determine whether the observed effects of GBP are presynaptic and/or postsynaptic, spontaneous miniature excitatory postsynaptic currents (mEPSCs) were studied. In neurons that showed a decrease in its frequency of mEPSCs by GBP, no change in the amplitude or shape accompanied the effect. On the other hand, in neurons that showed an increase in the frequency of NMDA receptor‐mediated mEPSCs, the effect accompanied an increase in amplitude. These results suggest that GBP presynaptically inhibits glutamatergic synaptic transmission predominantly in the superficial lamina, while postsynaptically enhancing NMDA receptor‐mediated transmission in some neurons of the deep lamina. The antinociceptive effects of GBP may involve the inhibition of the release of excitatory amino acids from presynaptic terminals.

Spinal gabapentin is antinociceptive in the rat formalin test

Gabapentin is a novel anticonvulsant that may be of value for the relief of clinical pain. To determine whether gabapentin is antinociceptive after spinal administration, the drug was given via an intrathecal catheter in doses from 6 to 200 micrograms/rat 10 min prior to intraplantar formalin. Five percent formalin injected subcutaneously in the right hind paw produced a biphasic reaction consisting of flinching and licking behaviors (phase 1, 0-10 min; phase 2, 10-60 min). Gabapentin dose-dependently reduced the numbers of flinches and the duration of licking during phase 2 of the formalin test. The highest dose of gabapentin (200 micrograms/rat) did not affect the tail-flick response. These results demonstrate that spinal gabapentin is antinociceptive in the formalin test.

Gabapentin enhances the antinociceptive effects of spinal morphine in the rat tail-flick test

Abstract The antinociceptive effects of the combination of spinal morphine and gabapentin were evaluated in the tail‐flick test in rats. The intrathecal coadministration of a subantinociceptive dose of morphine at 0.2 &mgr;g and gabapentin at 300 &mgr;g produced significant antinociception. Pretreatment with spinal gabapentin at 300 &mgr;g shifted the dose‐response curve of spinal morphine to the left with a decrease in morphine ED50 value from 1.06 &mgr;g to 0.34 &mgr;g. The antinociceptive effects produced by the combination of a subantinociceptive dose of morphine and gabapentin were reversed by spinal naloxone at 30 &mgr;g but were not reversed by spinal bicuculline at 0.3 &mgr;g. Furthermore, the concurrent administration of spinal naloxone at 30 &mgr;g with the combination of morphine and gabapentin blocked antinociception, while the concurrent administration of spinal bicuculline at 0.3 &mgr;g failed to prevent antinociception. These results indicate that the combination of spinal gabapentin and morphine produces an enhancement of antinociception that appears to involve the spinal mu opioid receptors. Furthermore, repeated administration of gabapentin for 3 days did not affect the enhancing effect of gabapentin on the antinociceptive effect of morphine, indicating that tolerance did not develop to gabapentins ability to enhance morphine antinociception.

Persistent pain and stress activate pain-inhibitory orexin pathways

Orexins are synthesized by neurons in the hypothalamus and contribute to multiple physiological functions. Orexin fibers innervate many regions of the CNS, which include areas involved in descending control of pain. We examined the role orexins may play in endogenous modulation of pain transmission using prepro-orexin (precursor of orexin A and B) knockout mice. Baseline pain thresholds of knockout and wild type mice were not different. Knockout mice presented greater degree of hyperalgesia induced by peripheral inflammation and less stress-induced analgesia than wild type mice. Double staining of orexin and c-Fos in wild type mice revealed activation of orexin neurons under both conditions. These results suggest that persistent pain and stress activate orexin neurons, which act to inhibit pain transmission.

Oral ketamine is antinociceptive in the rat formalin test : role of the metabolite, norketamine

The present study was designed to evaluate the oral efficacy and bioavailability of ketamine. Antinociceptive efficacy was determined with the rat formalin test and oral bioavailability by the measurement of plasma and brain concentrations of ketamine and its major metabolite, norketamine. Oral ketamine in a dose range from 30 to 180 mg/kg or saline was given prior to intraplantar formalin and the flinching behavior was measured. Oral ketamine dose-dependently reduced the flinching during phase 2, while flinching during phase 1 was reduced only with the highest dose given. Following oral ketamine at 100 mg/kg, blood and brain samples were obtained and plasma and brain ketamine and norketamine levels were measured using high-performance liquid chromatography (HPLC). The average concentration ratio of norketamine/ketamine, as expressed by the area under the curve (AUC) value, was 6.4 for plasma and 2.9 for brain. These results demonstrate that a significant amount of norketamine is formed by first pass biotransformation of ketamine and is distributed to the brain. Competition binding assays for the [3H]MK-801-labeled non-competitive site of the N-methyl-D-aspartate receptor (NMDA) receptor revealed that both norketamine and ketamine displaced [3H]MK-801 at low micromolar concentrations with Ki values of 2.5 and 0.3 mM in the forebrain, and 4.2 and 1.0 mM in the spinal cord, respectively. Spinal norketamine was approximately equipotent to ketamine in producing antinociceptive effects during phase 2 of the formalin test. Thus, norketamine appears to contribute to the antinociceptive effects of oral ketamine through its NMDA receptor antagonist activity.

A mouse model of neuropathic cancer pain

&NA; We developed a mouse model of neuropathic cancer pain by inoculating Meth A sarcoma cells to the immediate proximity of the sciatic nerve in BALB/c mice. The tumor grows predictably with time and gradually compresses the nerve, thereby causing nerve injury. Time courses of thermal hyperalgesia and mechanical sensitivity to von Frey hairs were determined and signs of spontaneous pain were evaluated. We compared this model with the chronic constriction injury (CCI) model, which is a neuropathic pain model widely utilized in the rat. Furthermore, to characterize the difference in nerve injury between the two models, we performed histological examination of the nerve of the two models by light and electron microscopy. Progressive compression of the sciatic nerve by growth of a tumor mass resulted in a gradual development of thermal hyperalgesia and mechanical allodynia in the ipsilateral hind paw. Signs of spontaneous pain, such as lifting of the paw, were also observed. However, further growth of the tumor reversed the mechanical hypersensitivity and produced mechanical hyposensitivity, while thermal hyperalgesia and signs of spontaneous pain still persisted. Histologically, gradual compression by the tumor resulted in a progressive damage to both myelinated and unmyelinated fibers. However, the severity of damage to the myelinated fibers was considerably less compared to that of the CCI mice. In the CCI mice, severe damage to myelinated fibers, especially large fibers, was observed and unmyelinated fibers were damaged to a lesser degree. These results suggest that gradual compression of a nerve by a malignant tumor results in nerve damage with a profile considerably different from that of chronic constriction injury produced by loose ligation of the nerve. Our new tumor model may be useful in studies of neuropathic cancer pain due to nerve compression by malignant tumors.

Multiple components of the defense response depend on orexin: Evidence from orexin knockout mice and orexin neuron-ablated mice

Stressor induces not only cognitive, emotional and behavioral changes but also autonomic changes. Although research on the neural circuits underlying such autonomic changes has implicated the hypothalamus in the defense response against stressors, neurotransmitters in this multifaceted and coordinated response have not been revealed. In this brief review, here we summarize our recent discovery using orexin knockout mice and orexin neuron-ablated mice of possible contribution of orexin in the defense response and discuss future directions.

An Antisense Oligonucleotide to the N-Methyl-d-aspartate (NMDA) Subunit NMDAR1 Attenuates NMDA-Induced Nociception, Hyperalgesia, and Morphine Tolerance

We determined whether the i.t. administration of an 18-mer phosphodiester antisense oligodeoxynucleotide (ODN) that reduces the expression of the rat NMDAR1 subunit of the N-methyl-d-aspartate (NMDA) receptor would affect nociceptive behaviors and prevent the development of morphine tolerance. Rats received 5 μl of i.t. saline, 30 nM antisense, or mismatch ODN twice a day for 5 days (NMDA-induced nociception, NMDA-induced thermal hyperalgesia, NR1 mRNA, and ligand binding studies) or for 3 days (formalin study). For the tolerance study, 5 days of ODNs or saline were followed by 3 days of concurrent administration of ODNs or saline (twice a day) and i.t. morphine (three times a day). Antisense, but not mismatch, results in the reduction of formalin phase 2 flinching by 50%, the spinal cord dorsal horn levels of NMDAR1 mRNA by 30%, and ligand binding by 50%. The i.t. ED50 for NMDA-induced nociceptive behaviors is doubled, and thermal hyperalgesia is blocked by antisense treatment. The effects of antisense on NMDA-induced nociception and thermal hyperalgesia are completely reversed by discontinuing antisense. The coadministration of antisense with increasing doses of i.t. morphine for 3 days attenuates the development of morphine tolerance. These results demonstrate that an in vivo antisense targeting of the NMDAR1 subunit results in antihyperalgesic effects and a partial blockade of spinal morphine tolerance. They provide additional support for the critical role of the NMDA receptor in these forms of spinal nociception and in the development of morphine tolerance and suggest the potential therapeutic utility of this approach.

Change of dorsal horn neurochemistry in a mouse model of neuropathic cancer pain

&NA; We investigated some neurochemical changes that take place in the spinal cord dorsal horn in a mouse model of neuropathic cancer pain. The model was produced by inoculation of Meth‐A sarcoma cells to the vicinity of the sciatic nerve, which resulted in growth of a tumor mass embedding the nerve. Hind paw‐lifting, a behavioral sign of spontaneous pain, was at maximum on Day 18, but decreased thereafter. The decrease was likely caused by progression of motor paralysis. On Day 18, thermal and mechanical pain thresholds of the affected paw were significantly increased. Histologically, the sciatic nerve presented damages to both unmyelinated and myelinated fibers on Day 18, which were more pronounced on Day 25. In the spinal cord, c‐Fos‐positive cells were significantly increased in the superficial and deep layers on Day 18. The number of c‐Fos‐positive cells in the superficial layer correlated with the duration of paw‐lifting. The increase in c‐Fos‐positive cells was still present on Day 25 despite decreased paw‐lifting. Substance P and calcitonin gene‐related peptide were up‐regulated on Day 18 but down‐regulated on Day 25. A marked up‐regulation of dynorphin A (DynA) was present on Day 18 and persisted through Day 25. Our model caused progressive damage to the sciatic nerve and presented spontaneous pain‐behavior while the paw became hyposensitive to mechanical and thermal stimuli. Since the up‐regulation of DynA in the dorsal horn persisted and paralleled the increase in c‐Fos‐positive cells, the release of DynA may be associated with spontaneous pain in our model.

Morphine can produce analgesia via spinal kappa opioid receptors in the absence of mu opioid receptors.

Previous studies have demonstrated the virtual lack of analgesia in mu opioid receptor knockout mice after systemic administration of morphine. Thus, it has been suggested that analgesic actions of morphine are produced via the mu opioid receptor, despite its ability to bind to kappa and delta receptors in vitro. However, it is not clear whether the results of these studies reflect the effect of morphine in the spinal cord. In the present study, we report study of the analgesic actions of spinally-administered morphine and other opioid receptor agonists in mu opioid receptor knockout and wild type mice. Morphine produced a dose-dependent antinociceptive effect in the tail flick test in the knockout mice, although higher doses were needed to produce antinociception than in wild type mice. The antinociceptive effect of morphine was completely blocked by naloxone (a non-selective opioid antagonist) and nor-binaltorphimine (nor-BNI, a selective kappa-opioid receptor antagonist), but not by naltrindole (a selective delta-opioid receptor antagonist). U-50,488H (a selective kappa-opioid receptor agonist) also produced a dose-dependent antinociceptive effect in knockout mice but presented lower analgesic potency in knockout mice than in wild type mice. Analgesic effects of [d-Pen2,d-Pen5]enkephalin (DPDPE, a selective delta-opioid receptor agonist) were observed in wild type mice but abolished in knockout mice. SNC80 (a selective delta-opioid receptor agonist) was not antinociceptive even in wild type mice. The present study demonstrated that morphine can produce thermal antinociception via the kappa opioid receptor in the spinal cord in the absence of the mu opioid receptor. Lower potency of U50,488H in mu opioid receptor knockout mice suggests interaction between kappa and mu opioid receptors at the spinal level.