Kazuki Yabuuchi

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.

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.

In situ hybridization study of interleukin-1β mRNA induced by kainic acid in the rat brain

The distribution patterns of interleukin-1 beta (IL-1 beta) mRNA in various brain regions of saline- and kainic acid-treated rats were examined using in situ hybridization technique. In normal rat brain, the signals of IL-1 beta mRNA were observed in the cerebellar Purkinje cells and in dispersed cells in the hypothalamus. In the case of the kainic acid treatment, IL-1 beta mRNA was intensely induced in the olfactory bulb, lateral septum, thalamus, hypothalamus, polymorphic layers of hippocampus, piriform cortex, amygdala, entorhinal cortex and cerebral cortex at 2 h after the injection of kainic acid. In the hypothalamic region, we observed the induction of IL-1 beta mRNA around the paraventricular hypothalamic nucleus, anterior hypothalamic area, dorsomedial and ventromedial hypothalamic nucleus, mammillary regions and arcuate nucleus. The signal of IL-1 beta mRNA was still expressed 4 h after treatment with kainic acid, less intensely than at 2 h, but above the control level. In these regions, IL-1 beta mRNA was expressed mainly in the glial cells, which were densely stained by Cresyl violet and did not contain glial fibrillary acidic protein. These results suggest that IL-1 beta is produced by a certain type of glial cells, maybe microglia, and might have regulatory functions in the central nervous system.

An in situ hybridization study on interleukin-1β mRNA induced by transient forebrain ischemia in the rat brain

Expression of interleukin-1 beta (IL-1 beta) mRNA in the rat brain after transient forebrain ischemia was investigated by in situ hybridization histochemistry. Thirty min after the start of recirculation, IL-1 beta mRNA was induced in the several brain regions, including the olfactory bulb, cerebral cortex, hippocampus, striatum and thalamus where neuronal degeneration was reported to be observed after transient forebrain ischemia. The hybridization signals were observed both on the glial cells and around the vascular walls.

Molecular cloning and in situ hybridization histochemistry for rat μ-opioid receptor

Abstract We cloned a cDNA for the rat μ-opioid receptor from a rat thalamus cDNA library. The deduced amino-acid sequence of rat μ-opioid receptor consists of 398 residues with the features shared by the members of the G-protein coupled receptor family, and is 59% and 60% identical with those of rat κ-opioid and mouse δ-opioid receptors, respectively. Northern blot analysis showed that expression of μ-opioid receptor mRNA was intensive in the thalamus, striatum, hypothalamus and pons-medulla, moderate in the hippocampus and midbrain, and slight in the cerebral cortex and cerebellum. More detailed distribution of the mRNA in the rat brain was examined using the in situ hybridization technique. Intense expression of μ-opioid receptor mRNA was observed in the internal granular and glomerular layers of the olfactory bulb, caudate putamen, nucleus accumbens, medial raphe nucleus, inferior colliculus, parabrachial nucleus, locus coeruleus, nucleus solitary tract and ambiguus nucleus. Furthermore, μ-opioid receptor mRNA was moderately expressed in the hippocampus, globus pallidus, ventral pallidus, arcuate hypothalamic nucleus, supramammillary nucleus, superior colliculus, periacqueductal gray, and several nuclei of lower brain stem, including raphe magnus nucleus, reticular gigantocellular nucleus and lateral paragigantocellular nucleus.

In situ hybridization study of μ- and κ-opioid receptor mRNAs in the rat spinal cord and dorsal root ganglia

Abstract Distributions of μ- and κ-opioid receptor mRNAs in the lumbar spinal cord and dorsal root ganglia of the adult rat were examined using the in situ hybridization technique. In the lumbar spinal cord, μ-opioid receptor mRNA was expressed intensely in laminae I, II and VIII. On the other hand, κ-opioid receptor mRNA was expressed intensely in laminae I and II, and moderately throughout laminae III-VIII. In the dorsal root ganglia, μ-opioid receptor mRNA was intensely expressed and κ-opioid receptor mRNA was expressed in a smaller number of cells than μ-opioid receptor mRNA.

Biphasic effects of intracerebroventricular interleukin-1 β on mechanical nociception in the rat

The effects of interleukin-1 beta on the mechanical nociceptive threshold in rat were examined using the paw-pressure test. An intracerebroventricular (i.c.v.) injection of interleukin-1 beta at doses of 10 and 100 pg/rat caused hyperalgesia to mechanical stimuli. Higher doses of interleukin-1 beta (1 and 10 ng/rat) induced an analgesic effect. The coadministration of the interleukin-1 receptor antagonist completely antagonized the hyperalgesic and analgesic effects of interleukin-1 beta. An i.c.v. injection of alpha-helical-corticotropin-releasing factor [9-41] 15 min prior to interleukin-1 beta administration completely blocked the hyperalgesic and analgesic effects of interleukin-1 beta. An i.c.v. injection of sodium salicylate 15 min prior to interleukin-1 beta administration inhibited the hyperalgesic effect of interleukin-1 beta, but not the analgesic effect. These results suggest that interleukin-1 beta produces biphasic effects on the mechanical nociceptive threshold through the interleukin-1 receptor in the brain and that a corticotropin-releasing factor-mediated pathway is involved. Furthermore, the hyperalgesic effect of interleukin-1 beta may be mediated by prostaglandins.

Participation of cAMP and cAMP-dependent protein kinase in β-adrenoceptor-mediated interleukin-1β mRNA induction in cultured microglia

Abstract We previously reported evidence of β-adrenoceptor-mediated induction of IL-1β mRNA in the rat hypothalamus. The present in vitro studies using northern blot analysis showed that the β-adrenoceptor agonist isoproterenol (1 × 10 −8 to 1 × 10 −5 M) caused a marked induction of IL-1β mRNA in microglia, but not in astrocytes. This induction was remarkably suppressed by pretreatment of cells with the β-adrenoceptor antagonist propranolol. These phenomena were confirmed by in situ hybridization with digoxigenin-labelled IL-1β RNA probe. Furthermore, dibutyryl cyclicAMP (dbcAMP) (5 × 10 −4 and 5 × 10 −5 M) markedly induced IL-1β mRNA in microglia. The intracellular level of cAMP in microglia was elevated in a dose-dependent manner when they were treated with isoproterenol, and this elevation was completely blocked by propranolol. The induction of IL-1β mRNA by either isoproterenol or dbcAMP was strongly inhibited by a cAMP-dependent protein kinase inhibitor, H8. These results, taken together, suggest that (1) microglia primarily induce IL-1β mRNA by stimulation of β-adrenoceptors, and (2) cAMP and cAMP-dependent protein kinase presumably participate in a signal transduction mechanism involved in the induction of IL-1β mRNA via β-adrenoceptors.

In situ hybridization study of κ-opioid receptor mRNA in the rat brain

Abstract Distribution of κ-opioid receptor mRNA in rat brain was examined by in situ hybridization technique. κ-Opioid receptor mRNA was expressed in various brain regions, especially intensely in the neocortex (layer V and VI), caudate-putamen, nucleus accumbens, preoptic area, paraventricular thalamic nucleus, amygdala, several nuclei of hypothalamus, ventral tegmental area and substantia nigra pars compacta.

Induction of interleukin-1β mRNA in the hypothalamus following subcutaneous injections of formalin into the rat hind paws

The induction of interleukin-1 beta (IL-1 beta) mRNA in the rat brain following subcutaneous injection of formalin into the hind paws was investigated by in situ hydridization. IL-1 beta mRNA was markedly induced in the hypothalamus after the injection of formalin into both hind paws. On the other hand, IL-1 beta mRNA was scarcely observed in the hypothalamus of saline-injected control rats. The type of cells expressing IL-1 beta mRNA was likely glia because their nuclei were densely stained by Cresyl violet and were relatively small. The present results suggest that IL-1 beta mRNA is induced in the glial cells of the hypothalamus by persistent pain which is caused by formalin injection.