Role of spinal voltage-dependent calcium channel alpha 2 delta-1 subunit in the expression of a neuropathic pain-like state in mice.

The present study was undertaken to investigate the role of spinal voltage-dependent calcium channel alpha(2)delta-1 subunit in the expression of a neuropathic pain-like state induced by partial sciatic nerve ligation in mice. In cultured spinal neurons, gabapentin (GBP), which displays the inhibitory effect of alpha(2)delta-1 subunit, suppressed the extracellular Ca(2+) influx induced by KCl, whereas it failed to inhibit the intracellular Ca(2+) release induced by inositol-1,4,5-triphosphate. Seven days after sciatic nerve ligation, the protein level of alpha(2)delta-1 subunit in the ipsilateral spinal cord was clearly increased compared to that observed in sham-operated mice. In addition, the mRNA level of alpha(2)delta-1 subunit was significantly increased in the dorsal root ganglion, but not in the spinal cord, of nerve-ligated mice. Under these conditions, a marked decrease in the latency of paw-withdrawal against a thermal stimulation and tactile stimulation, induced by sciatic nerve ligation was abolished by repeated intrathecal (i.t.) treatment with GBP. Additionally, the persistent reduction in the nociceptive threshold by i.t. treatment with GBP at the early stage of the neuropathic pain-like state was maintained for 7 days even after GBP withdrawal. It is of interest to note that a single i.t. post-injection of GBP showed a marked and transient inhibitory effect on the developed neuropathic pain-like state, whereas repeated i.t. post-treatment with GBP produced a persistent inhibitory effect during the treatment. In conclusion, we propose here that the neuropathic pain-like state with sciatic nerve ligation is associated with the increased level of the alpha(2)delta-1 subunit of Ca(2+) channels at the sensory nerve terminal in the spinal dorsal horn of mice. Furthermore, the present data provide evidence that the neuropathic pain may be effectively controlled by repeated treatment with GBP at the early stage.

Direct evidence for spinal cord microglia in the development of a neuropathic pain-like state in mice.

The present study was undertaken to further investigate the role of glial cells in the development of the neuropathic pain-like state induced by sciatic nerve ligation in mice. At 7 days after sciatic nerve ligation, the immunoreactivities (IRs) of the specific astrocyte marker glial fibrillary acidic protein (GFAP) and the specific microglial marker OX-42, but not the specific oligodendrocyte marker O4, were increased on the ipsilateral side of the spinal cord dorsal horn in nerve-ligated mice compared with that on the contralateral side. Furthermore, a single intrathecal injection of activated spinal cord microglia, but not astrocytes, caused thermal hyperalgesia in naive mice. Furthermore, 5-bromo-2'-deoxyuridine (BrdU)-positive cells on the ipsilateral dorsal horn of the spinal cord were significantly increased at 7 days after nerve ligation and were highly co-localized with another microglia marker, ionized calcium-binding adaptor molecule 1 (Iba1), but neither with GFAP nor a specific neural nuclei marker, NeuN, in the spinal dorsal horn of nerve-ligated mice. The present data strongly support the idea that spinal cord astrocytes and microglia are activated under the neuropathic pain-like state, and that the proliferated and activated microglia directly contribute to the development of a neuropathic pain-like state in mice.

Role of neuronal NR2B subunit-containing NMDA receptor-mediated Ca2+ influx and astrocytic activation in cultured mouse cortical neurons and astrocytes.

The excitatory neurotransmitter glutamate has been shown to mediate such bidirectional communication between neurons and astrocytes. In the present study, we determined the role of N-methyl-D-aspartate (NMDA) receptors on glutamate-evoked Ca(2+) influx into neurons and astrocytes. Either a nonselective NMDA receptor antagonist (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) or selective NR2B subunit-containing NMDA receptor antagonists ifenprodil and (R,S)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperid inepropanol (Ro25-6981) significantly inhibited the glutamate-evoked Ca(2+) influx into neurons, but not into astrocytes. Furthermore, we investigated whether NR2B subunit-containing NMDA receptor antagonists could suppress the astrocytic activation, as detected by glial fibrillary acidic protein (GFAP; as a specific marker of astrocyte)-like immunoreactivities in mouse cortical astrocytes. Here, we demonstrated that the increases in the level of GFAP-like immunoreactivities induced by glutamate were markedly suppressed by cotreatment with ifenprodil in cortical neuron/glia cocultures, but not in purified astrocytes. These results suggest that NR2B subunit-containing NMDA receptor plays a critical role in not only glutamate-evoked Ca(2+) influx into neurons, but also glutamate-induced astrocytic activation. Thus, glutamate-mediated pathway via NR2B subunit-containing NMDA receptor may, at least in part, contribute to neuron-to-astrocyte signaling.

Protease-activated receptor-1 and platelet-derived growth factor in spinal cord neurons are implicated in neuropathic pain after nerve injury.

Recently, it has been reported that both thrombin-sensitive protease-activated receptor 1 (PAR-1) and platelet-derived growth factor (PDGF) are present not only in platelets, but also in the CNS, which indicates that they have various physiological functions. In this study, we evaluated whether PAR-1/PDGF in the spinal cord could contribute to the development of a neuropathic pain-like state in mice. Thermal hyperalgesia and tactile allodynia induced by sciatic nerve ligation were significantly suppressed by repeated intrathecal injection of hirudin, which is characterized as a specific and potent thrombin inhibitor. Furthermore, a single intrathecal injection of thrombin produced long-lasting hyperalgesia and allodynia, and these effects were also inhibited by hirudin in normal mice. In nerveligated mice, the increase in the binding of [35S]GTPgammaS to membranes of the spinal cord induced by thrombin and PAR-1-like immunoreactivity (IR) in the spinal cord were each greater than those in sham-operated mice. Thermal hyperalgesia and tactile allodynia induced by sciatic nerve ligation were also suppressed by repeated intrathecal injection of either the PDGF alpha receptor (PDGFRalpha)/Fc chimera protein or the PDGFR-dependent tyrosine kinase inhibitor AG17 [(3,5-di-tert-butyl-4-hydroxybenzylidene)-malononitrile]. Moreover, thermal hyperalgesia and tactile allodynia induced by thrombin in normal mice were virtually eliminated by intrathecal pretreatment with PDGFRalpha/Fc. In immunohistochemical studies, PAR-1-like IR-positive cells in the spinal dorsal horn were mostly colocated on PDGF-like IR-positive neuronal cells. These data provide novel evidence that PAR-1 and PDGF-A-mediated signaling pathway within spinal cord neurons may be directly implicated in neuropathic pain after nerve injury in mice.

[Basic studies on cancer pain control].

According to the World Health Organization (WHO) guidelines for patients with moderate or severe pain, morphine has been used as a "gold standard" treatment for cancer pain. Recent clinical experiences have demonstrated that when morphine is used to control pain in cancer patients, psychological dependence is not a major concern. However, undue anxiety about psychological dependence on morphine in cancer patients has caused physicians and patients to use inadequate doses of opioids. In basic research, we reported that the morphine-induced rewarding effects can be dramatically suppressed under a neuropathic pain-like state induced by sciatic nerve ligation and an inflammatory pain-like state produced by intraplantar injection of formalin or carrageenan in rodents. The use of morphine for the treatment of pain is sometimes accompanied with side effects such as emesis, constipation and drowsiness. We show that the lower doses of morphine produce emesis, whereas antinociceptive doses of morphine show no emetic responses in ferrets. These findings provide further evidence that an adequate dose of morphine is useful and safe in a clinical setting.

Effect of a selective GABA(B) receptor agonist baclofen on the mu-opioid receptor agonist-induced antinociceptive, emetic and rewarding effects.

The management of excessive adverse effects of opioids is a major clinical problem. The present study was undertaken to investigate the effect of a selective gamma-aminobutyric acid (GABA)(B) receptor agonist baclofen on the mu-opioid receptor agonist-induced antinociceptive, emetic and rewarding effects. Either morphine or fentanyl produced a dose-dependent antinociceptive effect in both ferrets using Randall-Selitto test and mice using tail-flick test. Under these conditions, pretreatment of baclofen produced an additive antinociception induced by morphine or fentanyl. Furthermore, the augmentation of antinociception induced by systemic administration of baclofen with morphine or fentanyl was completely abolished by either i.c.v. or i.t. pretreatment with the selective GABA(B) receptor antagonist CGP 35348 in mice. We next investigated the emetic response induced by mu-opioid receptor agonist in ferrets. Morphine at lower doses than that used for antinociceptive assay produced both retching and vomiting, whereas fentanyl failed to produce the retching and vomiting in ferrets. Here we reported for the first time that baclofen significantly suppressed the retching and vomiting induced by morphine, indicating the involvement of GABA(B) receptor in emetic control pathway. Furthermore, baclofen also inhibited place preference elicited morphine or fentanyl in rats. Taken together, these results suggest that co-administration of baclofen with mu-opioid receptor agonist produced a potentiation of antinociceptive effect, whereas an untoward effect was completely blocked.

Involvement of spinal metabotropic glutamate receptor 5 in the development of tolerance to morphine-induced antinociception.

It is well known that prolonged exposure to morphine results in tolerance to morphine-induced antinociception. In the present study, we found that either intrathecal (i.t.) or subcutaneous (s.c.) injection of the selective metabotropic glutamate receptor 5 (mGluR5) antagonist, methyl-6-(phenylethynyl)-pyridine hydrochloride (MPEP), attenuated the development of tolerance to morphine-induced antinociception. Using the receptor binding assay, we found here that the number of mGluR5 in the mouse spinal cord was significantly increased by repeated treatment with morphine. Furthermore, repeated treatment with morphine produced a significant increase in the level of mGluR5 immunoreactivity in the dorsal horn of the mouse spinal cord. Double-labeling experiments showed that the increased mGluR5 was predominantly expressed in the neurons and sparsely expressed in the processes of astrocytes following repeated treatment with morphine. Consistent with these results, the response of Ca2+ to the selective group I mGluR agonist, 3,5-dihydroxyphenylglycine (DHPG), in cultured spinal cord neurons was potently enhanced by 3 days of in vitro treatment with morphine. These findings support the idea that the increased mGluR5 following repeated treatment with morphine leads to enhanced neuronal excitability and synaptic transmission in the dorsal horn of the spinal cord and, in turn, suppresses the morphine-induced antinociception in mice.

Treatment for psychological dependence on morphine: usefulness of inhibiting NMDA receptor and its associated protein kinase in the nucleus accumbens.

A growing body of evidence indicates that the mesolimbic dopaminergic (DAergic) pathway projecting from the ventral tegmental area (VTA) to the nucleus accumbens (N.Acc.) play a critical role in the initiation of psychological dependence on morphine. As well as DAergic system, the involvement of non-DAergic neurotransmitter and neuromodulator systems in rewarding effects induced by morphine has been recently documented. We previously demonstrated that the morphine-induced rewarding effect was dramatically suppressed by co-treatment with NMDA receptor antagonists, such as dizocilpine (MK-801), ketamine and ifenprodil. Therefore, we propose here that inhibiting the N-methyl-D-aspartate (NMDA) receptor and its associated protein kinase in the N.Acc. is useful for the treatment for psychological dependence on morphine. The following review provides a summary of recent our findings regarding the role of NMDA receptor and its associated protein kinase in the development of psychological dependence on morphine.

Long-lasting change in brain dynamics induced by methamphetamine: enhancement of protein kinase C-dependent astrocytic response and behavioral sensitization.

It is well known that long-term exposure to psychostimulants induces neuronal plasticity. Recently, accumulating evidence suggests that astrocytes may actively participate in synaptic plasticity. In this study, we found that in vitro treatment of cortical neuron/glia co-cultures with either methamphetamine (METH) or morphine (MRP) caused the activation of astrocytes via protein kinase C (PKC). Purified astrocytes were markedly activated by METH, whereas MRP had no such effect. METH, but not MRP, caused a long-lasting astrocytic activation in cortical neuron/glia co-cultures. Furthermore, MRP-induced behavioral sensitization to hyper-locomotion was reversed by 2 months of withdrawal following intermitted MRP administration, whereas behavioral sensitization to METH-induced hyper-locomotion was maintained even after 2 months of withdrawal. Consistent with this cell culture study, in vivo treatment with METH, which was associated with behavioral sensitization, caused a PKC-dependent astrocytic activation in the cingulate cortex and nucleus accumbens of mice. These findings provide direct evidence that METH induces a long-lasting astrocytic activation and behavioral sensitization through the stimulation of PKC in the rodent brain. In contrast, MRP produced a reversible activation of astrocytes via neuronal PKC and a reversibility of behavioral sensitization. This information can break through the definition of drugs of abuse and the misleading of concept that morphine produces a long-lasting neurotoxicity.

Implication of cyclin-dependent kinase 5 in the development of psychological dependence on and behavioral sensitization to morphine.

In the present study, we investigated the role of cyclin-dependent kinase 5 (cdk5) in the brain dynamics changed by repeated in vivo treatment with morphine. The level of phosphorylated-cdk5 was significantly increased in the cingulate cortex of mice showing the morphine-induced rewarding effect. Under these conditions, roscovitine, a cdk5 inhibitor, given intracerebroventricularly (i.c.v.) caused a dose-dependent and significant inhibition of the morphine-induced rewarding effect. In addition, the dose-response effect of the morphine-induced rewarding effect was dramatically attenuated in cdk5 heterozygous (+/-) knockout mice. Furthermore, the development of behavioral sensitization by intermittent administration of morphine was virtually abolished in cdk5 (+/-) mice. These findings suggest that the induction and/or activation of cdk5 are implicated in the development of psychological dependence on morphine.

Implication of the descending dynorphinergic neuron projecting to the spinal cord in the (+)-matrine- and (+)-allomatrine-induced antinociceptive effects.

We previously reported that either (+)-matrine (matridin-15-one) or (+)-allomatrine (the C-6 epimer of matrine)-induced antinociceptive effect was attenuated by s.c. pretreatment with a kappa-opioid receptor (KOR) antagonist nor-binaltorphimine (nor-BNI), indicating the critical role of KORs in antinociceptive effects induced by these alkaloids. In the present study, we found that i.c.v. administration of either (+)-matrine- or (+)-allomatrine induced antinociceptive effects in the mouse tail-flick and warm-plate test, whereas these alkaloids when given spinally failed to induce antinociception. In the guanosine-5'-O-(3-[(35)S]thio)trisphosphate ([(35)S]GTPgammaS) binding assay, we demonstrated that neither (+)-matrine nor (+)-allomatrine produced the stimulation of [(35)S]GTPgammaS binding in the membranes of the spinal cord, indicating that (+)-matrine- and (+)-allomatrine-induced supraspinal antinociceptive actions was not due to a direct stimulation of KORs by these alkaloids. Therefore, we next investigated the involvement of dynorphin A (1-17) release at the spinal or supraspinal site in (+)-matrine- or (+)-allomatrine-induced antinociception. The i.c.v. pretreatment with an antiserum against dynorphin A (1-17) could not affect the antinociceptive effect induced by s.c. treatment of (+)-matrine. In contrast, the s.c.-administered (+)-matrine- and (+)-allomatrine-induced antinociceptive effect was significantly attenuated by i.t. pretreatment of an antiserum against dynorphin A (1-17). The present data suggest that either (+)-matrine or (+)-allomatrine when given i.c.v. may stimulate the descending dynorphinergic neuron, resulting in the stimulation of KORs in the spinal cord, and this phenomenon in turn produces the antinociception in mice.

Direct evidence for the involvement of brain-derived neurotrophic factor in the development of a neuropathic pain-like state in mice.

Thermal hyperalgesia and tactile allodynia induced by sciatic nerve ligation were completely suppressed by repeated intrathecal (i.t.) injection of a TrkB/Fc chimera protein, which sequesters endogenous brain-derived neurotrophic factor (BDNF). In addition, BDNF heterozygous (+/-) knockout mice exhibited a significant suppression of nerve ligation-induced thermal hyperalgesia and tactile allodynia compared with wild-type mice. After nerve ligation, BDNF-like immunoreactivity on the superficial laminae of the ipsilateral side of the spinal dorsal horn was clearly increased compared with that of the contralateral side. It should be noted that a single i.t. injection of BDNF produced a long-lasting thermal hyperalgesia and tactile allodynia in normal mice, and these responses were abolished by i.t. pre-treatment with either a Trk-dependent tyrosine kinase inhibitor K-252a or a selective protein kinase C (PKC) inhibitor Ro-32-0432. Supporting these findings, we demonstrated here for the first time that the increase in intracellular Ca2+ concentration by application of BDNF in cultured mouse spinal neurons was abolished by pre-treatment with either K-252a or Ro-32-0432. Taken together, these findings suggest that the binding of spinally released BDNF to TrkB by nerve ligation may activate PKC within the spinal cord, resulting in the development of a neuropathic pain-like state in mice.

Direct evidence for the involvement of the mesolimbic kappa-opioid system in the morphine-induced rewarding effect under an inflammatory pain-like state.

Recent clinical studies have demonstrated that when morphine is used to control pain in cancer patients, psychological dependence is not a major concern. The present study was undertaken to ascertain the modulation of psychological dependence on morphine under a chronic pain-like state in rats. The prototypical mu-opioid receptor agonist morphine (8 mg/kg, i.p.) induced a dose-dependent place preference. In the present study, we found that an inflammatory pain-like state following formalin injection significantly suppressed the morphine-induced rewarding effect. This effect was almost reversed by s.c. pretreatment with the kappa-opioid receptor antagonist nor-binaltorphimine (nor-BNI, 5 mg/kg). Furthermore, the morphine-induced increase in dopamine (DA) turnover in the limbic forebrain was significantly inhibited by treatment with formalin. This inhibition was also suppressed by pretreatment with nor-BNI. In addition, in vivo microdialysis studies clearly showed that the morphine-induced increase in the extracellular levels of DA and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, in the nucleus accumbens (N.Acc.) was significantly decreased in rats that had been pretreated with formalin. This effect was in turn reversed by the microinjection of a specific dynorphin A antibody into the N.Acc. These findings suggest that the inflammatory pain-like state induced by formalin injection may have caused a sustained activation of the kappa-opioidergic system within the N.Acc., resulting in suppression of the morphine-induced rewarding effect in rats. The present study provides further evidence of the clinical usefulness of morphine in patients suffering from severe pain.

Inhibition of the morphine-induced rewarding effect by direct activation of spinal protein kinase C in mice.

RATIONALE: We previously demonstrated that the morphine-induced rewarding effect was attenuated under a neuropathic pain-like state following partial sciatic nerve ligation in rodents. Furthermore, the up-regulation of protein kinase C (PKC) activity in the spinal cord is considered to be the key factor for induction of hyperalgesia following sciatic nerve ligation. However, little direct evidence is available for the involvement of activated PKC in the spinal cord in reduction of rewarding effects induced by morphine under chronic pain-like state. OBJECTIVE: The present study was to investigate whether direct activation of spinal PKC by intrathecal (IT) administration of a specific PKC activator, phorbol 12,13-dibutyrate (PDBu) could produce hyperalgesia and suppress the place preference induced by morphine in mice. METHOD: The morphine-induced rewarding effect was investigated using the conditioned place preference method. Conditioning sessions (three for morphine, three for saline) were started 24 h after IT injection of PDBu or saline and conducted once daily for 6 days. On the day after the final conditioning session, a post-conditioning test was performed. RESULTS: IT-administered PDBu produced a long-lasting thermal hyperalgesia. Under these conditions, the place preference induced by morphine was abolished by a single IT pretreatment with PDBu. The effect was reversed by concomitant IT treatment with the specific PKC inhibitor Ro-32-0432. In contrast, IT-administered PDBu failed to affect the hyperlocomotion and supraspinal antinociception induced by morphine. CONCLUSION: The present findings suggest that activated PKC in the spinal cord with chronic pain-like hyperalgesia may play a substantial role in the suppression of the morphine-induced rewarding effect in mice with chronic pain-like hyperalgesia.

Induction of c-fos expression in the mouse brain associated with hyperalgesia induced by intrathecal injection of protein kinase C activator.

Here, we found that a single intrathecal (i.t.) administration of a protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), induced pain-like behaviors in mice. Furthermore, i.t.-administered PDBu caused the increased c-fos-like immunoreactivity in the parafascicular nuclei (PF), amygdala and cingulate cortex (CG), but not hippocampus. These findings suggest that the stimulation of spinal PKC results in an enhancement of neuronal activity in the PF, amygdala and CG associated with hyperalgesia.

Molecular mechanism of changes in the morphine-induced pharmacological actions under chronic pain-like state: suppression of dopaminergic transmission in the brain.

In the present study, we demonstrated whether a neuropathic pain-like state induced by sciatic nerve ligation in rodents could cause a long-lasting change in intracellular signaling in both supraspinal and spinal cord related to the suppression of morphine's effect. Mice with sciatic nerve ligation exhibited a significant suppression of the morphine-induced antinociception. Under this condition, phosphorylated-conventional protein kinase C-like immunoreactivity (p-cPKC-IR) and phosphorylated-micro-opioid receptor (p-MOR)-IR were clearly increased on the ipsilateral side in the dorsal horn of the spinal cord of nerve-ligated mice. It is of interest to note that astroglial hypertrophy as well as its proliferation was also noted in this area of sciatic nerve-ligated mice. Like nerve injury, the increase in cPKC activities and astroglial hypertrophy/proliferation in this region was observed by repeated morphine treatment. These findings suggest that the phosphorylation of both cPKC and MOR in the dorsal horn of the spinal cord by sciatic nerve ligation may play a substantial role in the suppression of morphine-induced antinociception under a neuropathic pain-like state. Sciatic nerve injury also caused a significant inhibition of MOR-mediated G-protein activation onto GABAergic neurons and a dramatic reduction in ERK activities onto dopaminergic neurons in the ventral tegmental area (VTA) regulating the rewarding effect of opioids. Furthermore, we found that the inhibition of ERK cascade in the VTA by treatment with specific inhibitors suppressed the morphine-induced rewarding effect in normal mice. These findings provide evidence that the direct reduction in MOR function and the persistent decrease in ERK activity of dopaminergic neurons in the VTA may contribute to the suppression of the morphine-induced rewarding effect under a neuropathic pain-like state. Conclusively, our recent findings provide novel evidences for the mechanism underlying the less sensitivity to opioids under a neuropathic pain-like state.

Functional interaction among opioid receptor types: up-regulation of mu- and delta-opioid receptor functions after repeated stimulation of kappa-opioid receptors.

It has been widely accepted that repeated administration of kappa-opioid receptor agonists leads to the development of antinociceptive tolerance. The present study was designed to investigate the effect of repeated administration of a selective kappa-opioid receptor agonist (1S-trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide hydrochloride ((-)U-50,488H) on the mu- and delta-opioid receptor agonist-induced antinociception and G-protein activation in mice. The mice were injected either subcutaneously (s.c.) or intracerebroventricularly (i.c.v.) pretreated with saline or (-)U-50,488H once a day for seven consecutive days. Two hours after the last injection, the mice were challenged by either mu- or delta-opioid receptor agonist for the antinociceptive assay. Repeated treatment with (-)U-50,488H (s.c. or i.c.v.) significantly enhanced antinociceptive effect of both mu-opioid receptor agonist (morphine) and delta-opioid receptor agonists ([d-Ala2]deltorphin (DELT) and (+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dime thyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC-80) compared to saline-treated groups. Under these conditions, repeated s.c. injection of (-)U-50,488H significantly enhanced both mu- and delta-opioid receptor agonist-stimulated [35S]GTPgammaS binding in the membrane of the thalamus. On the contrary, either repeated administration of morphine (s.c. or i.c.v.) or SNC-80 failed to affect the kappa-opioid receptor agonist-induced antinociception and G-protein activation. Taken together, these results suggest that repeated stimulation of kappa-opioid receptor markedly increases the functional mu- and delta-opioid receptors, whereas repeated stimulation of either mu- or delta-opioid receptor had no direct effect on kappa-opioidergic function in mice.

Neuronal protein kinase C gamma-dependent proliferation and hypertrophy of spinal cord astrocytes following repeated in vivo administration of morphine.

Repeated administration of morphine induced a time-dependent inhibition of the morphine-induced antinociceptive action, indicating the development of tolerance to morphine. We demonstrated that mice tolerant to morphine exhibited a significant increase in the level of protein kinase Cgamma-like immunoreactivity (PKCgamma-IR) in the dorsal horn of the spinal cord. The PKCgamma-IR was exclusively colocalized with the neuron-specific markers neuronal nuclei (NeuN) and microtubule associated protein 2ab (MAP2ab). Here we found a dramatic increase in reactive astrocytes in the dorsal horn of the spinal cord following repeated treatment with morphine, as characterized by the increase and morphological changes in glial fibrillary acidic protein (GFAP)-positive cells. Furthermore, transgenic mice that express enhanced green fluorescent protein (EGFP) under the control of the mouse GFAP promoter displayed enhanced levels of EGFP expression after repeated treatment with morphine. Under these conditions, mice lacking the PKCgamma gene failed to show any changes in astroglial hypertrophy or proliferation after repeated treatment with morphine. These findings strongly support the idea that the sustained activation of neuronal PKCgamma is implicated in the increased levels of reactive astrocytes in the dorsal horn of the spinal cord following repeated treatment with morphine. This neuron-glia communication may lead to the development of tolerance to morphine-induced antinociception.

Increased phosphorylated-mu-opioid receptor immunoreactivity in the mouse spinal cord following sciatic nerve ligation.

The present study was designed to determine whether a state of neuropathic pain induced by sciatic nerve ligation could alter phosphorylated-mu-opioid receptor-like immunoreactivity in the superficial dorsal horn of the mouse spinal cord. Mice with sciatic nerve ligation exhibited a significant suppression of the morphine-induced antinociception. Under this condition, phosphorylated-mu-opioid receptor-like immunoreactivity was clearly increased on the ipsilateral side in the superficial laminae of the L5 lumbar spinal dorsal horn in nerve-ligated mice. These findings suggest that the phosphorylation of the mu-opioid receptor in the spinal cord under a neuropathic pain-like state may, at least in part, contribute to the reduction in the antinociceptive effect produced by morphine in the mouse.

Change in the expression of c-fos in the rat brain following sciatic nerve ligation.

A little or none is known about the direct evidence for the possible change in the expression of c-fos at the supraspinal level after nerve injury. Therefore, the present study was designed to investigate the level of c-fos in some brain regions following sciatic nerve ligation in the rat. Immunoblot analysis clearly showed that the levels of c-fos in the rat frontal cortex, thalamus and periaqueductal gray matter were significantly increased, whereas it was significantly decreased in the nucleus accumbens and ventral tegmental area. Under these conditions, the levels of c-fos in the rat amygdala, hippocampus and hypothalamus were not changed. These results provide direct evidence that the neuropathic pain-like state causes a substantial change in the expression of c-fos in the rat brain.