Masabumi Minami

Induction of Interleukin-1β mRNA in Rat Brain After Transient Forebrain Ischemia

Abstract: The expression of interleukin‐1β (IL‐1β) mRNA in the cerebral cortex, hippocampus, striatum, and thalamus of rats was studied after transient forebrain ischemia. IL‐1β mRNA was not detected in all these regions of sham‐operated control rats. IL‐1β mRNA was induced after transient forebrain ischemia and reached a detectable level in all regions examined 15 min after the start of recirculation. The induction of IL‐1β mRNA had a few peaks, that is, peaks were observed at 30 and 240 min in the four regions examined, and another peak was observed at 90 min in the striatum. One day after the start of recirculation, IL‐1β mRNA levels were markedly decreased, but even 7 days after that, IL‐1β mRNA was found at very low levels in all regions examined. The amounts of c‐fos and β‐actin mRNAs on the same blots were also examined. The induction of c‐fos mRNA was transient and had only one peak in all regions examined, whereas the levels of β‐actin mRNA in these regions were fairly constant throughout the recirculation period. Thus, we provide the first evidence for a characteristic expression of IL‐1β mRNA in several brain regions after transient forebrain ischemia.

Effects of kainic acid on messenger RNA levels of IL-1β, IL-6, TNFα and LIF in the rat brain

Summary We examined the kainic acid-induced changes of mRNA levels of several cytokines such as IL-1β, IL-6, TNFα and LIF in the rat brain regions using semiquantitative RT-PCR method. IL-1β mRNA was markedly increased in the cerebral cortex (CC), thalamus (THL) and hypothalamus (HT) 2 h after the injection of kainic acid in a convulsive dose (12 mg/kg i.p.), and tended to decrease 4 h after the injection. IL-6 mRNA was weakly induced in the hippocampus (HPP) 2 h after the injection of kainic acid and was markedly increased in the CC, HPP, THL, and HT at 4 h. The level of TNFα mRNA was highly elevated in the CC, HPP, striatum (STR), THL and HT at 2 and 4 h after the injection. LIF mRNA apparently expressed in the CC and HPP of control rats and was increased in the CC, HPP and HT by the treatment with kainic acid. These results indicate that mRNAs of several cytokines are increased in various brain regions with different time-courses by kainic acid.

Localization of fractalkine and CX3CR1 mRNAs in rat brain: does fractalkine play a role in signaling from neuron to microglia?

Localization of the mRNAs for fractalkine, a CX3C chemokine, and for its receptor CX3CR1 was investigated in the rat brain. In situ hybridization study revealed that fractalkine mRNA was dominantly expressed in neuronal cells particularly in the olfactory bulb, cerebral cortex, hippocampus, caudate putamen and nucleus accumbens. In vitro study using enriched neuronal or glial culture supported the dominant expression of fractalkine mRNA in neurons. On the other hand, CX3CR1 mRNA was dominantly expressed in glial cells throughout the whole brain. The in vitro study suggested the cells expressing CX3CR1 mRNA are microglia, not astrocytes or neurons. Fractalkine appears to function as a signal molecule from neuron to microglia.

Molecular biology of the opioid receptors: structures, functions and distributions

Opiates like morphine and endogenous opioid peptides exert their pharmacological and physiological effects through binding to their endogenous receptors, opioid receptors. The opioid receptors are classified into at least three types, mu-, delta- and kappa-types. Recently, cDNAs of the opioid receptors have been cloned and have greatly advanced our understanding of their structure, function and expression. This review focuses on the recent advances in the studies on opioid receptors using the cloned cDNAs. We describe the molecular cloning of the opioid receptor gene family and studies of the structure-function relationships, modes of coupling to second messenger systems, pharmacological effects of antisense oligonucleotide and anatomical distributions of opioid receptors.

Molecular pharmacology of the opioid receptors

We cloned kappa and mu opioid receptor cDNAs. Using these cDNAs, first, we examined the molecular mechanism for the subtype selectivity of opioid ligands, especially a mu-selective ligand DAMGO. Binding experiments using various chimera and mutated receptors revealed that DAMGO discriminates between mu and delta receptors by recognizing the difference in only one amino acid residue, that is, N(127) in mu and K(108) in delta, at the first extracellular loop, and that it distinguishes between mu and kappa receptors by the difference in four amino acid residues at the third extracellular loop. Second, we established the cell lines expressing the cloned mu, delta, or kappa receptor and elucidated the pharmacological properties, that is, binding affinity and agonistic activity of several opioid agonists. Third, distribution of the mRNAs for mu, delta, and kappa receptors in the brain, spinal cord, and DRG was examined by in situ hybridization histochemistry (ISHH). Double ISHH demonstrated that most of the substance P-producing DRG neurons express the micro receptor. Recently, we are interested in the emotional aspect of pain and its regulation by opioids. Behavioral and microdialysis studies showed that sustained pain evoked by the intraplanter injection of formalin induced conditioned place aversion through the increment of glutamate release followed by the activation of NMDA receptors in the basolateral nucleus of amygdala (BLA). Intra-BLA injection of morphine suppressed the place aversion by inhibiting the glutamate release.

Cloning and expression of a cDNA for the rat k-opioid receptor

We cloned a cDNA for the rat K‐opioid receptor from a rat thalamus cDNA library. The deduced amino acid sequence consists of 380 residues with features shared by members of the G protein‐coupled receptor family. The specific binding of [3H]bremazocine to the membrane of COS‐7 cells transfected with the cDNA was displaced by k‐specific opioid ligands, but not by μ‐ and δ‐specific ligands. Xenopus oocytes injected with the in vitro transcribed mRNA responded to opioid ligands with the same subtype specificity. Northern blot analysis demonstrated that k‐opioid receptor mRNA is expressed in a regionally specific manner in rat brain.

Immobilization stress induces interleukin-1β mRNA in the rat hypothalamus

Abstract Immobilization stress induced interleukin-1β (IL-1β) mRNA in the rat hypothalamus. IL-1β mRNA was induced at 30 min after the start of immobilization, reached a maximum at 60 min and then was still detected with a decreased level at 120 min. However, 240 min after the start of the immobilization, IL-1β mRNA became hardly detectable despite the continuance of immobilization. Distinct expression of IL-1β mRNA was not detected in any other brain region examined at 30 and 60 min after the start of immobilization. These results demonstrate that the immobilization stress induces the expression of IL-1β mRNA solely in the hypothalamus and suggest that IL-1β is involved in the response to stress there.

Enhanced production of monocyte chemoattractant protein-1 in the dorsal root ganglia in a rat model of neuropathic pain: possible involvement in the development of neuropathic pain.

Chemokines are a family of peptides originally identified as the factors regulating the migration of leukocytes in inflammatory and immune responses. Recently, they have been shown to be produced in the central and peripheral nervous systems under various pathological conditions and act on neuronal and glial cells. In this study, we examined the production of monocyte chemoattractant protein-1 (MCP-1), a well-characterized chemokine, in dorsal root ganglia (DRG) in a rat model of neuropathic pain. Partial ligation of the sciatic nerve induced mechanical allodynia in the ipsilateral hindpaw with weaker allodynia in the contralateral one. Immunohistochemical analyses revealed that the number of MCP-1 immunoreactivity (ir)-positive cells was increased in the ipsilateral DRG. The increase started by 4h after the ligation, peaked at 24h and continued to at least 48 h. The weaker but significant increase was observed in the contralateral DRG. Double immunofluorescent staining demonstrated that almost all of the MCP-1ir-positive cells were neuronal cells. In situ hybridization histochemistry showed that MCP-1 mRNA expression was markedly upregulated in the ipsilateral DRG with weaker increase in the contralateral one at 24 h after the ligation, indicating that the elevation in MCP-1ir detected by immunohistochemistry was due to an upregulation of MCP-1 production by the DRG neurons themselves. Furthermore, intrathecal administration of MCP-1 induced mechanical allodynia. These results suggest that MCP-1 produced in the DRG neurons is involved in the development of mechanical allodynia induced by nerve injury.

Convulsants induce interleukin-1β messenger RNA in rat brain

Abstract The effects of systemic administration of kainic acid and pentylenetetrazol on interleukin-1β gene expression in the rat brain was studied. After the administration of kainic acid in a convulsive dose (10 mg/kg i.p.), Interleukin-1β mRNA was induced intensely in the cerebral cortex, thalamus and hypothalamus, moderately in the hippocampus and weakly in the striatum, but not in the midbrain, pons-medulla and cerebellum. Pentylenetetrazol induced Interleukin-1β mRNA in the cerebral cortex, hypothalamus, and hippocampus with a faster time-course than kainic acid. Diazepam suppressed both the convulsion and the induction of Interleukin-1β mRNA produced by kainic acid. Dexamethasone suppressed the induction of Interleukin-1β mRNA, but did neither the convulsion nor the induction of c-fos mRNA following the injection of kainic acid. These results provide the first evidence that intensive neuronal excitation induces Interleukin-1β mRNA in particular regions of the brain.

Double in situ hybridization study on coexistence of μ-, δ- and κ-opioid receptor mRNAs with preprotachykinin A mRNA in the rat dorsal root ganglia

Coexistence of the mRNA for each subtype of opioid receptor (OPR) with the mRNA for preprotachykinin A (PPTA), a precursor protein of substance P (SP), in the rat dorsal root ganglia was examined by double in situ hybridization technique. About 90% and 30% of PPTA mRNA-positive neurons expressed mu- and kappa-OPR mRNAs at high level, respectively. However, only about 3% of PPTA mRNA-positive neurons expressed delta-OPR mRNA at high level. These results suggest that mu- and kappa-OPRs exist on most of and a part of the primary afferent terminals containing SP, respectively. On the other hand, among the neurons which highly expressed mu-, delta- or kappa-OPR mRNA, PPTA mRNA was not expressed in about 58%, 95% or 24% of those neurons, respectively. These findings suggest the possibility that OPRs co-exist with other neurotransmitters and/or neuromodulators than SP in the primary afferent neurons.