MiR-29b controls fetal mouse neurogenesis by regulating ICAT-mediated Wnt/ß-catenin signaling.

ß-Catenin has been widely implicated in the regulation of mammalian development and cellular homeostasis. However, the mechanisms by which Wnt/ß-catenin signaling components regulate physiological events during brain development remain undetermined. Inactivation of glycogen synthase kinase (GSK)-3ß leads to ß-catenin accumulation in the nucleus, where it couples with T-cell factor (TCF), an association that is disrupted by ICAT (inhibitor of ß-catenin and T cell factor). In this study, we sought to determine whether regulation of ICAT by members of the microRNA-29 family plays a role during neurogenesis and whether deregulation of ICAT results in defective neurogenesis due to impaired ß-catenin-mediated signaling. We found that miR-29b, but not miR-29a or 29c, is significantly upregulated in three-dimensionally cultured neural stem cells (NSCs), whereas ICAT is reduced as aged. Treatment with a miR-29b reduced the reporter activity of a luciferase-ICAT 3'-UTR construct whereas a control (scrambled) miRNA oligonucleotide did not, indicating that miR-29b directly targets the 3'-UTR of ICAT. We also found that treatment with miR-29b diminished NSC self-renewal and proliferation, and controlled their fate, directing their differentiation along certain cell lineages. Furthermore, our in vivo results showed that inhibition of miR-29b by in utero electroporation induced a profound defect in corticogenesis during mouse development. Taken together, our results demonstrate that miR-29b plays a pivotal role in fetal mouse neurogenesis by regulating ICAT-mediated Wnt/ß-catenin signaling.

Srg3, a mouse homolog of yeast SWI3, is essential for early embryogenesis and involved in brain development.

Srg3 (SWI3-related gene product) is a mouse homolog of yeast SWI3, Drosophila melanogaster MOIRA (also named MOR/BAP155), and human BAF155 and is known as a core subunit of SWI/SNF complex. This complex is involved in the chromatin remodeling required for the regulation of transcriptional processes associated with development, cellular differentiation, and proliferation. We generated mice with a null mutation in the Srg3 locus to examine its function in vivo. Homozygous mutants develop in the early implantation stage but undergo rapid degeneration thereafter. An in vitro outgrowth study revealed that mutant blastocysts hatch, adhere, and form a layer of trophoblast giant cells, but the inner cell mass degenerates after prolonged culture. Interestingly, about 20% of heterozygous mutant embryos display defects in brain development with abnormal organization of the brain, a condition known as exencephaly. Histological examination suggests that exencephaly is caused by the failure in neural fold elevation, resulting in severe brain malformation. Our findings demonstrate that Srg3 is essential for early embryogenesis and plays an important role in the brain development of mice.

Protection against beta-amyloid peptide toxicity in vivo with long-term administration of ferulic acid.

1. beta-Amyloid peptide (A beta), a 39 -- 43 amino acid peptide, is believed to induce oxidative stress and inflammation in the brain, which are postulated to play important roles in the pathogenesis of Alzheimer's disease. Ferulic acid is an antioxidant and anti-inflammatory agent derived from plants; therefore, the potential protective activity of ferulic acid against A beta toxicity in vivo was examined. 2. Mice were allowed free access to drinking water (control) or water containing ferulic acid (0.006%). After 4 weeks, A beta 1-42 (410 pmol) was administered via intracerebroventricular injection. 3. Injection of control mice with A beta 1-42 impaired performance on the passive avoidance test (35% decrease in step-through latency), the Y-maze test (19% decrease in alternation behaviour), and the water maze test (32% decrease in percentage time in platform-quadrant). In contrast, mice treated with ferulic acid prior to A beta 1-42 administration were protected from these changes (9% decrease in step-through latency; no decrease in alternation behaviour; 14% decrease in percentage time in platform-quadrant). A beta 1-42 induced 31% decrease in acetylcholine level in the cortex, which was tended to be ameliorated by ferulic acid. 4. In addition, A beta 1-42 increased immunoreactivities of the astrocyte marker glial fibrillary acidic protein (GFAP) and interleukin-1 beta (IL-1 beta) in the hippocampus, effects also suppressed by pretreatment with ferulic acid. 5. Administration of ferulic acid per se unexpectedly induced a transient and slight increase in GFAP and IL-1 beta immunoreactivity in the hippocampus on day 14, which returned to basal levels on day 28. A slight (8%) decrease in alternation behaviour was observed on day 14. 6. These results demonstrate that long-term administration of ferulic acid induces resistance to A beta 1-42 toxicity in the brain, and suggest that ferulic acid may be a useful chemopreventive agent against Alzheimer's disease.

Supraspinal NMDA and non-NMDA receptors are differentially involved in the production of antinociception by morphine and beta-endorphin administered intracerebroventricularly in the formalin pain model.

Our previous studies have demonstrated that supraspinal glutamate receptors are differentially involved in the antinociception induced by morphine and beta-endorphin given intracerebroventricularly (i.c.v.) in the tail-flick and hot-plate tests. The formalin pain test was used in the present study. Injection of mice with formalin solution (2%, 10 microl) into the hindpaw intraplantarly produced the first (0-5 min) and second (20-40 min) phases of formalin responses. The formalin responses in the both phases were attenuated dose-dependently by morphine (0.125-1 microg) or beta-endorphin (0.125-1 microg) administered i.c.v. 5 min before. The antinociceptive effect of morphine was slightly more potent in the second phase whereas the effect of beta-endorphin was more pronounced in the first phase. MK-801 (0.1-1 microg), a non-competitive NMDA receptor antagonist, and CNQX (0.05-0.5 microg), a non-NMDA antagonist, given i.c.v., produced antinociceptive effect in the both phases, but only in a partial manner. Both MK-801 (0.05 microg) and CNQX (0.01 microg), at the dose which had no intrinsic effect, reversed the antinociceptive effect of beta-endorphin (1 microg) observed during the second, but not the first, phase partially but significantly. However, the antinociceptive effect of morphine (1 microg) was not affected by the same dose of MK-801 or CNQX given i.c.v. Our results indicate that, at the supraspinal level, both NMDA and non-NMDA receptors are involved in the production of antinociception induced by supraspinally administered beta-endorphin, but not morphine, in the formalin pain model.

Involvement of dynorphin in immobilization stress-induced antinociception in the mouse.

The effect of antiserum against [Met(5)]-enkephalin, [Leu(5)]-enkephalin, beta-endorphin, or dynorphin A-(1-13) administered intracerebroventricularly (i.c.v.) or intrathecally (i. t.) on immobilization-induced antinociception was studied in ICR mice. Antinociception was assessed by the tail-flick assay. Immobilization of the mouse increased inhibition of the tail-flick response at least 1 h. The i.c.v. or i.t. injection with antiserum against dynorphin A-(1-13) at the dose of 200 microg significantly attenuated immobilization-induced inhibition of the tail-flick response. However, antiserum against [Met(5)]-enkephalin, [Leu(5)]-enkephalin, or beta-endorphin did not affect the immobilization stress-induced antinociception. Furthermore, i.c.v. or i.t. injection with nor-binaltorphimine (Nor-BNI; from 1 to 20 microg) effectively inhibited immobilization stress-induced inhibition of the tail-flick response in a dose-dependent manner. However, beta-FNA (from 0.5 to 2 microg) or naltrindole (from 1 to 20 microg) administered i.c.v. or i.t. did not affect immobilization stress-induced antinociception. Our results suggest that supraspinally and spinally located dynorphin appears to be involved in the production of immobilization stress-induced antinociception via stimulating kappa-opioid receptors.

Regulation of c-fos gene expression by lipopolysaccharide and cycloheximide in C6 rat glioma cells.

The effect of lipopolysaccharide (LPS) on the expression of immediate early genes, such as c-fos and c-jun, was examined in C6 rat glioma cells. LPS (1 microg/ml) alone did not affect c-fos mRNA level. LPS, however, transiently increased c-jun mRNA level. Cycloheximide (CHX, 20 microM), a protein synthesis inhibitor, alone caused increases of c-fos and c-jun mRNA levels. LPS showed a potentiating effect in the regulation of c-fos mRNA level, whereas LPS showed an additive action for the regulation of CHX-induced c-jun mRNA expression. To determine if CREB and mitogen-activated protein kinases (MAPKs) are involved in the regulation of c-fos mRNA expression by LPS and CHX, Western blot was carried out using the phosphorylated form of antibodies against ERK, JNK, p38, and CREB. LPS transiently increased the phosphorylation of p38-MAPK and CREB. In addition, LPS alone elevated phosphorylation of ERK (p44/p42) MAPK in a time-dependent manner. Furthermore, LPS plus CHX enhanced phosphorylation of ERK, p38, and CREB in a synergistic manner. Our results suggest that the phosphorylation of ERK, p38, and CREB may be involved in the regulation of synergistic c-fos mRNA expression induced by LPS plus CHX in C6 rat glioma cells.

Differential effects of cholera toxin and pertussis toxin on the c-fos and c-jun mRNA expression in rat C6 glioma cells.

Cholera toxin (CTX) increased c-fos mRNA level whereas it down-regulated the c-jun mRNA level in rat C6 glioma cells. In contrast to the action of CTX, pertussis toxin (PTX) did not affect either c-fos or c-jun mRNA level. The elevated c-fos mRNA level induced by CTX was significantly inhibited by the co-treatment with dexamethasone (DEX). However, DEX did not affect CTX-induced down-regulation of c-jun mRNA level. Cycloheximide (CHX) increased c-fos and c-jun mRNA levels. CHX caused a super-induction of CTX-induced c-fos mRNA level. Our results suggest that CTX-, but not PTX-, sensitive G-proteins may play an important role for c-fos mRNA up-regulation and c-jun mRNA down-regulation. In addition, DEX appears to have a selective inhibitory action against c-fos mRNA expression regulated by CTX. Ongoing protein synthesis inhibition is required for the superinduction of c-fos, but not c-jun, mRNA induced by CTX.

Modulatory role of ginsenosides injected intrathecally or intracerebroventricularly in the production of antinociception induced by kappa-opioid receptor agonist administered intracerebroventricularly in the mouse.

We examined the effects of ginseng total saponin and several ginsenosides injected intrathecally (i.t.) or intracerebroventricularly (i.c.v.) on the antinociception induced by U50, 488H (trans-3,4-dichloro-N-methyl-N-[2- (1-pyrrolidinyl)cyclohexyl]benzeocetamide; a kappa opioid receptor agonist) administered i.c.v. The tail-flick test was used as an analgesic assay. Total saponin fraction at doses of 0.1 to 20 micrograms, which when administered intrathecally (i.t.) or intracerebroventricularly (i.c.v.) alone did not affect the latencies of tail-flick threshold, attenuated dose-dependently the inhibition of the tail-flick response induced by U50, 488H (60 micrograms) administered i.c.v. The duration of antagonistic action of total saponin fraction against U50, 488H-induced antinociception lasted at least for 6 h. Various doses (from 0.1 to 1 microgram) of ginsenosides Rb1, Rb2, Rc, Rd, and Rg1, but not Re, injected i.t. dose-dependently attenuated antinociception induced by U50, 488H administered i.c.v. Furthermore, various doses (from 1 to 10 micrograms) of ginsenosides Rb2 and Re, but not Rb1, Rc, Rd, and Rg1, injected i.c.v. dose-dependently attenuated antinociception induced by U50, 488H administered i.c.v. In summary, ginsenosides Rb1, Rb2, Rc, Rd, and Rg1 administered spinally appear to be responsible for blocking the antinociception induced by U50, 488H administered supraspinally, whereas ginsenosides Rb2 and Re administered supraspinally appear to be responsible for blocking the antinociception induced by U50, 488H administered supraspinally.

Effect of melatonin on the regulation of proenkephalin and prodynorphin mRNA levels induced by kainic acid in the rat hippocampus.

The in vivo short-term effect of melatonin on kainic acid (KA)-induced proenkephalin (proENK) or prodynorphin (proDYN) mRNA, and on AP-1 protein levels in the rat hippocampus, were studied. Melatonin (5 mg/kg) or saline was administered intraperitoneally (i.p.) to rats 30 min prior to and immediately after i.p. injection of KA (10 mg/kg). Rats were sacrificed 1 and 3 h after KA injection. The proENK and proDYN mRNA levels were significantly increased 3 h after KA administration. The elevations of both proENK and proDYN mRNA levels induced by KA were significantly inhibited by the preadministration with melatonin. The increases of proENK and proDYN mRNA levels induced by KA were well-correlated with the increases of c-Fos, Fra-2, FosB, c-Jun, and JunB protein levels, which were significantly increased 3 h after KA administration and effectively inhibited by administration with melatonin. In an electrophoretic mobility shift assay, both AP-1 and ENKCRE-2 DNA binding activities were increased by KA, which were also attenuated by the administration of melatonin. In addition, cross-competition studies revealed that AP-1 or ENKCRE-2 DNA binding activity was effectively reduced by the 50x unlabeled cross-competitor. Therefore, these data suggest that melatonin has an inhibitory role in KA-induced gene expression, such as proENK and proDYN mRNA expression, and this may be due to a reduction of KA-induced AP-1 or ENKCRE-2 DNA binding activity.

Differential modulatory roles of cholera toxin and pertussis toxin in the regulation of pain responses induced by excitatory amino acids administered intrathecally in mice.

The present study was designed to characterize the possible roles of spinally located cholera toxin (CTX)- and pertussis toxin (PTX)-sensitive G-proteins in excitatory amino acids induced pain response. Intrathecal (i.t.) injection of glutamate (20 microg), N-methyl-D-aspartic acid (NMDA; 60 ng), alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA; 13 ng), and kainic acid (12 ng) showed pain response. Pretreatment with CTX (0.05 and 0.5 microg, i.t.) attenuated pain response induced by glutamate, NMDA, AMPA and kainic acid administered i.t. in a dose-dependent manner. On the other hand, i.t. pretreatment with PTX further increased the pain response induced by glutamate, NMDA, AMPA and kainic acid administered i.t., especially at the dose of 0.5 microg. Our results suggest that, at the spinal cord level, CTX- and PTX-sensitive G-proteins appear to play opposite roles in modulating the pain response induced by spinally administered. Furthermore, CTX- and PTX-sensitive G-proteins appear to modulate pain response induced by stimuli of both NMDA and non-NMDA glutamate receptors.

Cycloheximide increases proenkephalin and tyrosine hydroxylase gene expression in rat adrenal medulla.

The effect of cycloheximide (CHX; 5 mg/kg) on proenkephalin (proENK) and tyrosine hydroxylase (TH) mRNA expression in rat central and peripheral nervous systems was studied. CHX increased proENK and TH mRNA levels in the adrenal gland, but not in hippocampus, striatum, midbrain, brainstem, pituitary, and hypothalamus. The pretreatment with actinomycin D (0.5 mg/kg) significantly decreased CHX-induced proENK and TH mRNA expression, suggesting that the CHX-dependent increase of these mRNA levels may be caused by the increase of transcriptional activity rather than RNA stabilization. To investigate the factors involved in CHX-induced proENK and TH mRNA expression, the effect of CHX on activator protein-1 (AP-1), cAMP response element (CRE) binding protein (CREB), and glucocorticoid response element (GRE) was tested. In AP-1, the basal expression of Fra-2 and c-Jun proteins and AP-1 DNA binding activity in the adrenal medulla was higher than other tissues tested, but CHX reduced these protein levels and AP-1 DNA binding activity. In CREB, CHX time dependently increased the level of phospho-CREB without altering total CRE level and CRE DNA binding activity. Furthermore, phospho-CREB actively participated in CRE DNA binding activity. In GRE, although CHX increased plasma and adrenal corticosterone level, RU486 (10 mg/kg) reduced CHX-induced proENK, but not TH, mRNA level in a partial manner. These results suggest that the basal expression of proENK and TH mRNA transcription in the adrenal gland seems to be tonically inhibited by de novo protein synthesis. In addition, CHX-dependent increase of proENK and TH mRNA expression in the adrenal medulla is well correlated with phospho-CREB level, but not AP-1. Finally, glucocorticoid seems to be involved at least partially in CHX-dependent proENK, but not TH, mRNA expression in the adrenal medulla.

Modulatory effect of ginseng total saponin on dopamine release and tyrosine hydroxylase gene expression induced by nicotine in the rat.

Several studies have demonstrated that behavioral activation induced by psychostimulants is prevented by ginseng total saponin (GTS), which has been known to act on the central dopaminergic system. In an attempt to investigate whether the effect of GTS is through its inhibitory action on the elevated dopaminergic transmission, we examined the effect of GTS on nicotine-induced dopamine (DA) release in the nucleus accumbens (NA) of freely moving rats using in vivo microdialysis. Systemic injection of nicotine (3 mg/kg; i.p.) produced a mild increase in extracellular DA of dialysates samples in the NA (132+/-13% over basal levels at the peak). GTS (100 mg/kg; i.p.) had no effect on resting levels of extracelluar DA. However, an increase in accumbens DA release produced by systemic nicotine was completely blocked by systemic pre-treatment with GTS (100 mg/kg; i.p.). In addition, the effect of GTS on nicotine-induced tyrosine hydroxylase (TH) and immediate early gene expression in ventral tegmental area (VTA) or NA regions was examined. A single injection of nicotine increased TH mRNA level at VTA region. GTS, which did not affect the basal TH mRNA expression, attenuated nicotine-induced TH mRNA expression. Nicotine slightly increased both c-fos and c-jun mRNA level and GTS, which did not affect the basal c-fos and c-jun mRNA expression, further enhanced nicotine-induced c-fos and c-jun mRNA level at both VTA and NA regions. Our results suggest that GTS may have an inhibitory action against nicotine-induced DA release in NA region and TH mRNA expression in VTA region. GTS may exert an potentiative effect on both c-fos and c-jun mRNA expression at NA region through inhibiting the release of DA in NA.

Stimulation of astrocyte-enriched culture with arachidonic acid increases proenkephalin mRNA: involvement of proto-oncoprotein and mitogen activated protein kinases.

In astrocyte-enriched cultures, arachidonic acid (AA, 100 microM) significantly increased the proenkephalin (proENK) mRNA level (4. 9-fold at 8 h). In addition, AA also increased several AP-1 proteins, such as c-Fos, Fra-1, Fra-2, JunB, JunD, and c-Jun, or AP-1 and ENKCRE-2 DNA-binding activity. As well as AP-1 proteins and their DNA-binding activities, proENK mRNA level induced by AA was reduced by the pretreatment with 15 microM of cycloheximide (CHX; 1.6-fold). AA-dependent increase of proENK mRNA is not mediated by cyclooxygenase- or lipoxygenase-dependent metabolites, or free radicals, because the AA-induced increase of proENK mRNA levels was not affected by indomethacin (10 microM), nordihydroguaiaretic acid (10 microM), or N-acetylcysteine. However, as well as proto-oncoprotein levels, such as Fra-1, Fra-2, c-Jun, JunB, but not JunD, AA-induced increase of proENK mRNA was significantly reduced by the pretreatment with 10 microM of PD98059 (1.3-fold) or 10 microM of SB203580 (1.8-fold). These results strongly suggest that AA rather than one of its metabolites is involved in the increase of proENK mRNA. In addition, the activation of both the p38 and ERK pathways appears to be involved in the AA-induced increase of proENK mRNA via activating the expression of proto-oncoprotein, such as Fra-1, Fra-2, c-Jun, and JunB.

Central injection of nitric oxide synthase inhibitors increases peripheral interleukin-6 and serum amyloid A: involvement of adrenaline from adrenal medulla.

1. Accumulating evidence suggests that plasma levels of interleukin-6 (IL-6), a major cytokine stimulating the synthesis of acute phase proteins, are intimately regulated by the central nervous system (CNS). 2. In the present study, effects of intracerebroventricular (i.c. v) injection of N(G)-nitro-L-arginine methyl ester (L-NAME) or 7-nitroindazole, nitric oxide synthase (NOS) inhibitors, on plasma IL-6 levels and peripheral IL-6 mRNA expression were examined in mice. 3. L-NAME (0.1 - 2 microg per mouse i.c.v.) and 7-nitroindazole (0.2 - 2 microg per mouse i.c.v.) induced a dose-dependent increase in plasma IL-6 levels and a subsequent increase in circulating serum amyloid A, a liver acute-phase protein. In contrast, an intraperitoneal (i.p.) injection of L-NAME up to the dose of 25 microg per mouse had no effect. 4. Pretreatment with yohimbine (alpha(2)-adrenergic antagonist; 1 mg kg(-1) i.p.), or ICI-118,551 (beta(2)-adrenergic antagonist; 2 mg kg(-1) i.p.), but not with prazosin (alpha(1)-adrenergic antagonist; 1 mg kg(-1) i.p.), nor betaxolol (beta(1)-adrenergic antagonist; 2 mg kg(-1) i.p.), significantly inhibited the central L-NAME-induced plasma IL-6 levels. 5. I.c.v. (50 microg per mouse) or i.p. (100 mg kg(-1)) pretreatment with 6-hydroxydopamine had no effect on central L-NAME-induced plasma IL-6 levels. However, intrathecal (i.t.) pretreatment with 6-hydroxydopamine (20 microg per mouse) markedly inhibited central L-NAME-induced plasma IL-6 levels. Both yohimbine (1.5 microg per mouse i.t.) and ICI-118,551 (1.5 microg per mouse i. t.) were effective in inhibition of central L-NAME-induced plasma IL-6 levels. 6. There was an elevation of base-line plasma IL-6 levels in adrenalectomized animals. The adrenalectomy-enhanced levels were not further increased by central L-NAME. 7. L-NAME (2 microg per mouse i.c.v.) induced an increase in IL-6 mRNA expression in liver, spleen, and lymph node. 8. These results suggest that NOS activity in the brain tonically down-regulates peripheral IL-6 by inhibiting adrenaline release from the adrenal medulla.

Identification and sequencing of pineal-specific enhancing region in the mouse tryptophan hydroxylase promoter.

Pineal-specific expression of the tryptophan hydroxylase (TPH) gene has been demonstrated by a number of studies. However, little is known about the regulatory mechanism for pineal-specific expression of the TPH gene. To identify the cis-acting region responsible for pineal-specific expression of the TPH gene, we investigated a 6.1-kb 5'-flanking region of the mouse TPH gene using an immortalized pineal cell line (PGT-beta) derived from transgenic mice. By deletion analysis, it was demonstrated that the pineal-specific enhancing region resides approximately between -6.1 and -4.7 kb upstream from the transcription initiation site of the mouse TPH gene. Additionally, nucleotide sequence analysis of this region showed that the (AC/TG)22 repetitive sequence is located approximately -5.78 kb upstream of the mouse TPH gene, and several known tissue-specific cis-acting elements, such as Pit-1 and the pituitary specific element (PSE), have also been identified in the region. We believe that the analysis of the sequence and several cis-acting elements in the pineal-specific enhancing region of the mouse TPH promoter would enhance our understanding of the precise mechanism of pineal-specific expression.

Differential potentiative effects of GABA receptor agonists in the production of antinociception induced by morphine and beta-endorphin administered intrathecally in the mouse.

The effect of muscimol or baclofen injected intrathecally (i.t.) on the inhibition of the tail-flick response induced by morphine and beta-endorphin administered i.t. was studied in ICR mice. The i.t. injection of muscimol (100 ng) or baclofen (10 ng) alone did not affect the basal inhibition of the tail-flick response. Morphine (0.2 microg) and beta-endorphin (0.1 microg) caused only slight inhibition of the tail-flick response. Baclofen, but not muscimol, injected i.t. enhanced the inhibition of the tail-flick response induced by i.t. administered morphine. Both muscimol and baclofen injected i.t. significantly enhanced i.t. injected beta-endorphin-induced inhibition of the tail-flick response. Our results suggest that the GABA(B), but not GABA(A), receptors located in the spinal cord appear to be involved in enhancing the inhibition of the tail-flick response induced by morphine administered spinally. In addition, both GABA(A) and GABA(B) receptors are involved in enhancing the inhibition of the tail-flick response induced by beta-endorphin administered i.t.

Activation of adenylate cyclase results in down-regulation of c-jun mRNA expression in rat C6 glioma cells.

To investigate the possible mechanisms involved in forskolin-induced c-jun mRNA decrease in rat C6 glioma cells, we examined effects of a PKA inhibitor (H-89), a L-type Ca2+ channel blocker (nimodipine), a calmodulin activation inhibitor (calmidazolium chloride) and a Ca2+/calmodulin-dependent protein kinase II inhibitor (KN-62) on forskolin-induced c-jun mRNA down-regulation. H-89 caused a reversal of forskolin-induced c-jun mRNA decrease. Furthermore, nimodipine, KN-62 and calmidazolium chloride partially blocked forskolin-induced c-jun mRNA down-regulation. Our results suggest that activation of adenylate cyclase appears to be involved in a down-regulation of c-jun mRNA expression through a PKA pathway. In addition, L-type calcium channels, calmodulin and Ca2+/calmodulin-dependent protein kinase II may be partially involved in c-jun mRNA down-regulation induced by forskolin.

Central injection of nicotine increases hepatic and splenic interleukin 6 (IL-6) mRNA expression and plasma IL-6 levels in mice: involvement of the peripheral sympathetic nervous system.

Accumulating evidence suggests that plasma levels of interleukin 6 (IL-6), a major cytokine stimulating the synthesis of acute-phase proteins, are intimately regulated by the central nervous system. Nicotine, one of the major drugs abused by humans, has been shown to affect immunological functions. In the present study, effects of intracerebroventricular (i.c.v.) injection of nicotine on plasma IL-6 levels were investigated in mice. Nicotine administered i.c.v. dose-dependently increased plasma IL-6 levels; the lowest effective dose was 0.3 ng/mouse and the maximal effect was attained with the dose of 105 ng/mouse. The nicotine (105 ng/mouse, i.c.v.)-induced plasma IL-6 levels peaked at 3 h and approached basal levels 6 h after injection. Mecamylamine, a nicotinic receptor antagonist, blocked nicotine-induced plasma IL-6 levels. Depletion of peripheral norepinephrine with 6-hydroxydopamine [100 mg/kg, intraperitoneal (i. p.)] inhibited the nicotine-induced plasma IL-6 levels by 57%, whereas central norepinephrine depletion with 6-hydroxydopamine (50 microgram/mouse, i.c.v.) had no effect. Pretreatment with prazosin (alpha1-adrenergic antagonist; 1 mg/kg, i.p.), yohimbine (alpha2-adrenergic antagonist; 1 mg/kg, i.p.), and ICI-118,551 (beta2-adrenergic antagonist; 2 mg/kg, i.p.), but not with betaxolol (beta1-adrenergic antagonist; 2 mg/kg, i.p.), inhibited nicotine-induced plasma IL-6 levels. Among the peripheral organs, including the pituitary, adrenals, heart, lung, liver, spleen, and lymph nodes, nicotine (105 ng/mouse, i.c.v.) increased IL-6 mRNA expression only in the liver and spleen, which was inhibited by peripheral norepinephrine depletion. These results suggest that stimulation of central nicotinic receptors induces plasma IL-6 levels and IL-6 mRNA expression in the liver and spleen via the peripheral sympathetic nervous system, alpha1-, alpha2-, and beta2-adrenoreceptors being involved.

Differential involvement of central and peripheral norepinephrine in the central lipopolysaccharide-induced interleukin-6 responses in mice.

Intracerebroventricular injection of lipopolysaccharide (LPS) induces a marked increase in circulating interleukin (IL)-6 levels and in IL-6 mRNA expression in brain and peripheral organs. Recently, it was reported that intraperitoneal administration of alpha-adrenoceptor antagonists inhibits centrally injected LPS-induced increases in plasma IL-6 levels, suggesting the involvement of the norepinephrine (NE) system in the central LPS-induced IL-6 response. However, the localization (either central or peripheral) of NE involvement in the central LPS-induced IL-6 response has not been characterized. In the present study, mice were pretreated with 6-hydroxydopamine (6-OHDA) administered intracerebroventricularly or intraperitoneally to deplete central or peripheral stores of NE, respectively. Intracerebroventricular LPS (50 ng/mouse) markedly increased plasma IL-6 levels and IL-6 mRNA expression in choroid plexus, hypothalamus, pituitary, adrenals, heart, liver, spleen, and lymph nodes, but with minimal effect in lung, kidney, and testis, as revealed by RT-PCR. Pretreatment with intracerebroventricular 6-OHDA (50 microg/mouse) decreased the LPS-induced plasma IL-6 levels by 39% and the LPS-induced IL-6 mRNA expression in liver, spleen, and lymph nodes, but not in choroid plexus, hypothalamus, pituitary, adrenals, and heart. Pretreatment with intraperitoneal 6-OHDA (100 mg/kg) decreased the LPS-induced plasma IL-6 levels by 36% and the LPS-induced IL-6 mRNA expression in all the peripheral organs displaying increased IL-6 mRNA. Central LPS-induced increase in plasma corticosterone levels was decreased slightly by central but not by peripheral NE depletion. These results suggest that central NE and peripheral NE are differentially involved in the central LPS-induced IL-6 mRNA expression in peripheral organs.

Effects of ginsenosides injected intrathecally or intracerebroventricularly on antinociception induced by beta -endorphin administered intracerebroventricularly in the mouse.

The effect of total saponin fraction of ginseng injected intrathecally (i.t.) or intracerebroventricularly (i.c.v.) on the antinociception induced by beta-endorphin administered i.c.v. was studied in ICR mice in the present study. The antinociception was assessed by the tail-flick test. Total saponin fraction at doses 0.1 to 1.0 microgram, which administered i.t. alone did not affect the latencies of tail-flick threshold, attenuated dose-dependently the inhibition of the tail-flick response induced by i.c.v. administered beta-endorphin (1 microgram). However, total saponin fraction at doses 1 to 20 microgram, which administered i.c.v. alone did not affect the latencies of the tail-flick response, did not affect i.c.v. administered beta-endorphiun (1 microgram)-induced antinociception. The duration of antagonistic action of total saponin fraction against beta-endorphin-induced antinociception lasted at least for 6 h. Various doses (from 0.1 to 1 microgram) of ginsenoside R(c), but not R(b2), R(d), Rg(1), R(b1)and R(e)injected i.t. dose-dependently attenuated antinociception induced by beta-endorphin administered i.c.v. Our results indicate that total saponin fraction injected spinally appears to have antagonistic action against the antinociception induced by supraspinally applied beta-endorphin. Ginsenoside R(c)appears to be responsible for blocking i.c.v. administered beta-endorphin-induced antinociception. On the other hand, total ginseng fraction, at supraspinal sites, may not exert an antagonistic action against the antinociception induced by supraspinally administered beta-endorphin.