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Glucocorticoids affect a variety of tissues to enable the organism to adapt to the stress. Hippocampal neurons contain glucocorticoid receptors and respond to elevated glucocorticoid levels by down-regulating the HPA axis. Chronically, however, stress is deleterious to hippocampal neurons. Chronically elevated levels of glucocorticoids result in a decrease in the number of dendritic spines, reduced axonal growth and synaptogenesis, and decreased neurogenesis in the hippocampus. Tolloid-like 1 (Tll-1) is a metalloprotease that potentiates the activity of the bone morphogenetic proteins (BMPs). Neurogenesis in the hippocampus of both developing and adult mammals requires BMPs. In this study, we demonstrate that Tll-1 expression is increased in mice that have increased neurogenesis. The Tll-1 promoter contains glucocorticoid response elements which are capable of binding to purified glucocorticoid receptor. Glucocorticoids decrease Tll-1 expression in vitro. Finally, prenatal stress leads to a decrease in Tll-1 mRNA expression in the hippocampus of adult female mice that is not observed in adult male mice indicating that Tll-1 expression is differentially regulated in males and females. The results of this study indicate that Tll-1 is responsive to glucocorticoids and this mechanism might influence neurogenesis in the hippocampus.

As estrogens have been implicated in altered cognitive function associated with menopause, the purpose of the present study was to determine the regulatory effects of different estrogen preparations on the expression of estrogen receptor subtypes in the hippocampus and cortex of ovariectomized rats. The expression of estrogen receptor mRNA and protein was determined with RT-PCR and immunohistochemistry, respectively. Two estrogen reagents, Premarin and Progynova, were used in the present study. Premarin, a conjugated equine estrogen, down-regulated ER alpha expression in the hippocampus and cortex of ovariectomized rats and had no effect on levels of ER beta expression in the same two regions. However, Progynova (valerate estradiol) was shown to up-regulate ER beta expression in the hippocampus and cortex and had no effect on the levels of ER alpha expression. Our present data suggest that different estrogen reagents used in estrogen replacement therapy could have different regulatory effects on the expression of estrogen receptor subtypes, which might, at least in part, explain why clinically, different estrogen preparations have distinct estrogenic effects on target organs.

Scutellariae radix is a Chinese herbal medicine that has been used to treat disease conditions accompanying inflammation and oxidative stress. In the present study, we examined the effect of Scutellariae radix extracts during acute ethanol exposure in N(2)a neuroblastoma. The Scutellariae radix extracts effectively inhibited ethanol-induced apoptosis and caspase-3/-7 activation. Ethanol induced the expression of caspase-11 that has been known as a dual regulator of pathological apoptosis and inflammatory response. The ethanol-induced caspase-11 expression was suppressed by pretreatment of the Scutellariae radix extracts. Furthermore, the activation of caspase-3/-7 and apoptosis were significantly inhibited in caspase-11-/- mouse embryonic fibroblasts following ethanol treatment. These results suggest that caspase-11 has a regulatory role in ethanol-induced apoptosis, and the suppression of caspase-11 may be a mechanism by which Scutellariae radix exerts its cytoprotective effect.

The mas-related gene (Mrg) family is a large family of G-protein-coupled receptors which are variable in number depending on species. The so-called sensory-neuron-specific receptors (SNSRs) make up a subset of the Mrg family, and several of these have been implicated in nociceptive processes. To verify their specific localization in sensory ganglia, we have determined the expression patterns of two of them, rMrgA and rMrgC, in a panel of rat tissues. The quantitative PCR results in the rat tissue panel indicate that, while several non-neuronal tissues contain significant levels of mRNA for both receptors, these two receptors are most highly expressed in dorsal root ganglia and trigeminal ganglia. Given this, we have examined the effects of spinal nerve ligation (SNL) on the expression of these genes. Peripheral neuropathy induced by ligation of spinal nerves at L5 and L6 resulted in a pronounced mechanical allodynia. These behavioral changes in tactile sensitivity were accompanied by significant decreases (10- to 100-fold) in the mRNA expression of both rMrgA and rMrgC exclusively in the L5 and L6 dorsal root ganglia ipsilateral to the SNL. In situ hybridization studies demonstrated that this decrease did not result from neuronal loss but rather from a reduction in the hybridization signals for rMrgC over small-to-medium diameter L5 and L6 dorsal root ganglia neurons. While the functional implications of the altered regulation of rMrgA and rMrgC in neuropathic pain models remain unclear, the results suggest that therapeutics targeting these receptors may have limited utility.

The aim of this study was to investigate the expression of cytokines, tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1beta), interleukin-6 (IL-6) and transforming growth factor-beta 1 (TGF-beta1) and chemokines, fractalkine, monocyte chemoattractant protein 1 (MCP-1) and stromal cell-derived factor 1 (SDF-1) in the dorsal motor nucleus of the vagus nerve (DMV) after right vagotomy. Results showed that the immunoreactivities of IL-1beta, IL-6, TGF-beta1, fractalkine and MCP-1 were upregulated in the DMV at 14 days and the upregulation persisted at least until 28 days following right vagotomy. Quantification analysis revealed significant increases in the number of their immunopositive cells in the right DMV at 14 and 28 days after right vagotomy. Moreover, the upregulation of TNF-alpha immunoreactivity and significantly increased number of TNF-alpha-immunopositive cells were observed in the injured DMV at 7 and 14 days, and the increase in SDF-1-immunopositive cells at 14 days, after right vagotomy. Real time RT-PCR analysis showed the significant increase in the mRNA expression of IL-1beta, fractalkine and MCP-1 at 7 days, and the upregulation of TNF-alpha mRNA expression at 1 day after vagotomy. However, the peak increase in TGF-beta1 mRNA expression was observed at 1 day and the significant increase persisted at least until 14 days following right vagotomy. Double immunofluorescence analysis showed co-localization of lectin, a marker for microglia with CX3CR1 but not with IL-1beta at 14 days following right vagotomy. This study suggests that cytokines and chemokines involved in neuroprotection and neurodestruction could be activated in the axotomized DMV. However, it warrants further investigation to understand the neurodestructive and neuroprotective mechanisms that determine the fate of the vagal motoneurons after vagotomy.

As a member of family of cytosolic calcium binding proteins, Calbindin-D9k (CaBP-9k) is expressed in female reproductive system and regulated by steroid hormones, estrogen (E2) and progesterone (P4), but its expression and role in pituitary gland have not been elucidated yet. Thus, in this study, we elucidated the expression of CaBP-9k mRNA and protein in pituitary gland of rats. During estrous cycle of rats, pituitary CaBP-9k level fluctuated, and its mRNA was highly elevated during an E2-dominant stage (proestrus and estrus), whereas its level disappeared at a P4-dominant stage (metestrus and diestrus). In parallel with CaBP-9k mRNA, an increased level of CaBP-9k protein was observed during proestrus and estrus, suggesting that pituitary CaBP-9k may be up-regulated by E2. In addition, spatial CaBP-9k expression was attested by immunohistochemistry. Pituitary CaBP-9k protein was localized in the cytoplasm of a specific cell type in the anterior lobe, and the positive cells were abundant at proestrus and estrus. The CaBP-9k-positive cells were mainly localized in the acidophils producing growth hormones and prolactin. To verify hormonal regulation of pituitary CaBP-9k in this tissue, immature rats were treated with a physiological dose of E2 in the absence or presence P4 for 3 days. In a time-dependent experiment, pituitary CaBP-9k protein was induced at 48 h after the final E2 injection. A significant increase in CaBP-9k protein was caused by E2, whereas P4 antagonized E2-stimulated CaBP-9k expression as similarly observed in the uterus. Taken together, these results indicated for the first time that pituitary CaBP-9k expression is regulated during estrous cycle, and its expression is mainly controlled by E2 and antagonized by P4, suggesting that pituitary CaBP-9k in female rats may be involved in the central function of the reproduction system.

It has been reported that several of the effects induced by an octadecaneuropeptide (ODN), derived from an 86-amino-acid polypeptide termed diazepam-binding inhibitor, could be mediated by activation of a metabotropic receptor. In order to investigate the role and mechanism of action of ODN in the regulation of corticotropin-releasing factor (CRH) and neuropeptide Y (NPY) expression in the paraventricular nucleus and arcuate nucleus, respectively, we studied the effects of the acute intracerebroventricular administration of ODN (2 microg/rat) and the ODN antagonist to metabotropic receptor, cyclo(1-8)[Dleu5]OP (20 microg/rat), on the gene expression of the two neuropeptides in castrated male rat. ODN administration resulted in a 45% increase in CRH mRNA expression, an effect which was reversed by cyclo(1-8)[Dleu5]OP. When cyclo(1-8)[Dleu5]OP was administered alone, it induced a 19% decrease in CRH mRNA levels. ODN administration induced a 17% decrease in NPY mRNA expression while cyclo(1-8)[Dleu5]OP increased by 21% the hybridization signal. The administration of both ODN and ODN antagonist completely abolished the depressing effect of ODN on NPY mRNA. These data suggest that the effects of ODN on CRH and NPY mRNA might be mediated by interaction with metabotropic receptors. Moreover, since cyclo(1-8)[Dleu5]OP can by itself influence the expression of two peptide mRNAs, it might be suggested that ODN is exerting a tonic influence on NPY and CRH neurons.

Aquaporin (AQP) 4 is a member of the AQP gene family of water-selective transport proteins. We studied the effect of water deprivation on AQP4 gene expression in chickens. The nucleotide sequence of a chicken aquaporin 4 (AQP4) cDNA that encodes a protein of 335 amino acids showed high homology to mammalian AQP4. Using Northern blotting analysis, AQP4 mRNA in chickens was observed as a band of approximately 5.5 kb in several tissues in addition to the hypothalamus, proventriculus, kidney, and breast muscle. Quantitative analysis by real-time RT-PCR analysis showed that the mRNA expression of AQP4 in the hypothalamus significantly increased after dehydration. On the other hand, the mRNA expression of AQP4 in the kidney significantly decreased after dehydration. This suggests that AQP4 may play a pivotal role in osmoregulation in the chicken brain.

Behavioral studies have demonstrated that chronic food restriction augments the rewarding and motor-activating effects of centrally injected psychostimulants and direct dopamine (DA) receptor agonists. Recently, it has been shown that intracerebroventricular (i.c.v.) injection of the D-1 DA receptor agonist, SKF-82958, produces an enhanced locomotor-activating effect as well as increased activation of striatal ERK 1/2 MAP kinase, CaM kinase II, CREB, and c-fos in food-restricted (FR) relative to ad libitum fed (AL) rats. Striatal neurons that express the D-1 DA receptor coexpress dynorphin and substance P, and CREB is known to couple D-1 DA receptor stimulation to preprodynorphin (ppD) gene expression. The purpose of the present study was to examine possible genomic consequences of FR using real-time quantitative RT-PCR to measure striatal neuropeptide gene expression 3 h after i.c.v. injection of SKF-82958 (20 microg). Results indicate that, in nucleus accumbens (NAc), basal levels of ppD and preprotachykinin (ppT) mRNA are lower in FR than AL rats. This may reflect a decrease in tonic DA transmission during FR which precedes the compensatory upregulation of postsynaptic D-1 DA receptor-mediated cell signaling. In response to SKF-82958 challenge, however, FR subjects displayed greater levels of ppD and ppT mRNA in NAc than did AL subjects. A similar trend was seen in caudate-putamen (CPu). SKF-82958 also increased preproenkephalin (ppE) mRNA in Nac, but not CPu, with no difference between feeding groups. The present findings regarding ppD and ppT are consistent with prior findings of increased behavioral and cellular responses to acute D-1 DA agonist challenge in FR rats. The functional consequences of increased neuropeptide gene expression in response to acute drug challenge remain to be investigated but may include modulation of behavioral effects that emerge with repeated drug exposure, including sensitization, tolerance, and addiction.

We previously demonstrated that the brain, pituitary, and C6 glioblastoma cells express leptin. To determine the physiological role of brain-derived leptin, we sought to selectively silence its expression using RNA interference (RNAi) in vitro. One of four potential targets, siRNA L7, reduced leptin mRNA by 50% (P < 0.05) and protein by 55% (P < 0.0001) in C6 cells. RNAi also induced a twofold increase in cell death as seen by ethidium homodimer-1 (P < 0.015) and TUNEL (P < 0.005) staining. These data suggest that endogenous leptin may be a critical factor promoting cell survival in the brain.

The purpose of this study is to determine if calcium/calmodulin-dependent protein kinase-II (CaMKII) plays a role in neuronal cell death and if inhibition of this kinase affords some neuroprotection in the RGC-5 retinal ganglion cell line. The RGC-5 cells were treated with glutamate at various concentrations for increasing increments of time. Cytotoxicity was assayed by measuring the lactate dehydrogenase (LDH) leakage from non-viable cells and TUNEL assays. The involvement of caspase-3, Bcl-2 and caspase-8 in glutamate-induced cytotoxicity was determined by immunoblots and/or real time RT-PCR. In addition, the autocamtide-2-related inhibitory peptide (AIP), a specific inhibitor of CaMKII, was used to determine the involvement of CaMKII in glutamate-induced RGC-5 cell death. Application of increasing concentrations of glutamate to RGC-5 cells caused a dose-dependent increase in the level of cell death after 24 h. There was a glutamate-stimulated increase in the expression of caspase-8 and caspase-3 and a corresponding decrease in Bcl-2. The active fragment of caspase-3 increased in glutamate-treated cells. An early transient increase in the expression of CaMKIIalpha(B) gene and a corresponding CaMKIIalpha nuclear translocation was found in glutamate-treated cells. Treatment with AIP blocked the activation of caspase-3 and protected RGC from glutamate-mediated cell death but did not alter the glutamate-enhanced expression levels of caspase-8 or caspase-3. This report shows the likely involvement of a transcript of the CaMKIIalpha gene in the cytotoxicity response of RGC-5 cells similar to previous reports in the neural retina. AIP is shown to be a neuroprotectant for RGC-5 cells as was reported for the neural retina.

Neocarzinostatin (NCS), an enediyne antimitotic agent, induces cell death in both p75NTR neurotrophin receptor (NTR)-positive and p75NTR-negative PC12 cells in a concentration-dependent fashion. However, p75NTR-positive cells demonstrate a higher susceptibility to NCS-induced cell damage. Furthermore, treatment of p75NTR-positive cells with the p75NTR-specific ligand, MC192, resulted in apoptosis, while treatment of these cells with the TrkA-specific ligand, NGF-mAbNGF30, protected them from NCS-induced death, implying that both the naked and liganded p75NTR receptors have a pro-apoptotic effect on PC12 cells. Microarray studies aimed at examining differential gene expression between p75NTR-positive and p75NTR-negative cells suggested that enzymes of the cholesterol biosynthetic pathway are differentially expressed. We therefore tested the hypothesis that altered cholesterol biosynthesis contributes directly to the pro-apoptotic effects of p75NTR in this PC12 cell-NCS model. Subsequent Northern blotting studies confirmed that the expression of p75NTR is associated with the upregulation of cholesterol biosynthetic enzymes including 3-hydroxy-3-methylglutaryl CoA reductase (HMG CoA reductase), farnesyl-diphosphate synthase, and 7-dehydro-cholesterol reductase. Mevastatin, an HMG CoA reductase inhibitor, converts the apoptosis susceptibility of p75NTR-positive cells to that of p75NTR-negative cells. It does so at concentrations that do not themselves alter cell survival. These studies provide evidence that the pro-apoptotic effects of p75NTR in PC12 cells are related to the upregulation of cholesterol biosynthetic enzymes and consequent increased cholesterol biosynthesis.

Selective serotonin reuptake inhibitors (SSRIs) are widely used for the treatment of depressive mood disorders and well known to inhibit the reuptake of neurotransmitter serotonin into nerve terminals. Thus, it seems conceivable that these drugs may induce the outflow of serotonin from the synapse as a consequence of inhibiting the reuptake, resulting in the stimulation of glial cells surrounding nerve terminals. On this hypothesis, the effect of serotonin on steroid 5alpha-reductase type 1 (5alpha-R) gene expression in rat C6 glioma cells was examined as one of the in vitro model experiments for investigating the indirect influence of SSRIs on glial cells. Serotonin elevated 5alpha-R mRNA and protein levels through the stimulation of serotonin 5-HT2A receptors, and also elevated Egr-1 mRNA and protein levels prior to 5alpha-R gene expression in the glioma cells. Furthermore, serotonin failed to significantly increase 5alpha-R mRNA levels in the cells preloaded with the antisense oligodeoxynucleotide targeted on Egr-1 gene. These results indicate that serotonin may stimulate 5alpha-R gene expression via transcription factor Egr-1 in glial cells, thus suggesting that serotonin flowing out of the serotonergic synapse may be implicated in SSRI-induced changes in neurosteroid metabolism in brain.

Cyclooxygenase (COX) is the rate-limiting enzyme in the synthesis of prostaglandins (PGs) from arachidonic acid. Evidence suggests that neuronal COX-2 gene expression is mainly regulated by the transcription factor nuclear factor kappa-B (NF-kappaB). The present study was undertaken to determine whether there is a shared regulation of NF-kappaB or of nuclear factor of activated T-cells cytoplasmic (NFATc) with COX-2 and whether these transcription factors known to regulate COX-2 expression are altered in brain from COX-2 knockout (COX-2-/-) mice compared to wild type. We found a decrease in NF-kappaB DNA-protein binding activity, which was accompanied by a reduction of the phosphorylation state of both I-kappaBalpha and p65 proteins in the COX-2-/- mice. The mRNA and protein levels of p65 were also reduced in COX-2-/- mice, whereas total cytoplasmic I-kappaB protein level was not significantly changed. Taken together, these changes may be responsible for the observed decrease in NF-kappaB DNA binding activity. NF-kappaB DNA binding activity was selectively affected in the COX-2-/- mice compared to the wild type as there was no significant change in NFATc DNA binding activity. Overall, our data indicate that constitutive brain NF-kappaB activity is decreased in COX-2 deficient mice and suggest a reciprocal coupling between NF-kappaB and COX-2.

Nogo is a myelin-associated protein associated with neurite outgrowth and regeneration. A previous study has reported an association between an insertion/deletion polymorphism in schizophrenia. We tested for the distribution of the polymorphism and haplotypes of this and another insertion/deletion polymorphism in our population. We have also developed an assay combining allele-specific polymerase chain reaction (AS-PCR) and restriction fragment length polymorphism (RFLP) to simultaneously type these two insertion/deletion polymorphisms. There was a statistically significant difference at the allelic level for both the CAA (chi2 = 4.378, df = 1, P value = 0.036) and TATC (chi2 = 5.807, df = 1, P = 0.016) polymorphisms in the female subgroup, but not in males. With our genotyping method, we also determined the molecular haplotype. Within the female gender, odds ratio is at 1.57 (95% CI 1.05-2.37) for CAACAA-TATC and 1.40 (95% CI 0.55-3.60) for CAA-TATC, the two at-risk haplotypes. Odds ratio is 0.63 (95% CI 0.42-0.93) for the protective wildtype haplotype CAA-TATCTATC. Further study of these two polymorphisms to investigate functional significance and confirm gender-specific association should be carried out.

Epileptic seizures cause severe and long-lasting events on the architecture of the brain, including neuronal cell death, accompanied neurogenesis, reactive gliosis, and mossy fiber sprouting. However, it remains uncertain whether these functional and anatomical alterations are associated with the development of hyperexcitability, or as inhibitory processes. Neurotrophic factors are probable mediators of these pathophysiological events. The present study was designed to clarify the role of various neurotrophic factors on the pilocarpine model of seizures. At 4 h following pilocarpine-induced seizures, expression of NGF, BDNF, HB-EGF, and FGF-2 increased only in the mice manifesting tonic-clonic convulsions and not in mice without seizures. NT-3 expression decreased in pilocarpine-treated mice experiencing seizures, tonic-clonic or not, compared to mice with no seizures. Neuronal cell damage, which was evident by Fluoro-Jade B staining, was observed within 24 h in the mice exhibiting tonic-clonic seizures, followed by an increase in the number of BrdU-positive cells and glial cells, which were evident after 2 days. None of these pathophysiological changes occurred in the mice which showed no seizures, although they were injected with pilocarpine, nor in the activated epilepsy-prone EL mice, which experienced repeated severe seizures. Together, these results suggest that neuronal damage occurring in the brain of the mice manifesting tonic-clonic seizures is accompanied by neurogenesis. This sequence of events may be regulated through changes in expression of neurotrophic factors such as NGF, BDNF, HB-FGF, and NT-3.

Nicotine is known to induce the release of multiple neurotransmitters, including glutamate and dopamine, through activation of nicotinic receptors. Gene expression in the N-methyl-d-aspartate postsynaptic density (NMDA-PSD), as well as other functional groups, was compared in postmortem hippocampus of schizophrenic and nonmentally ill smokers and nonsmokers utilizing a microarray and quantitative RT-PCR approach. The expression of 277 genes was significantly changed between all smokers and nonsmokers. Specific gene groups, most notably genes expressed in the NMDA-PSD, were prevalent among these transcripts. Analysis of the interaction between smoking and schizophrenia identified several genes in the NMDA-PSD that were differentially affected by smoking in patients. The present findings suggest that smoking may differentially modulate glutamatergic function in schizophrenic patients and control subjects. The biological mechanisms underlying chronic tobacco use are likely to differ substantially between these two groups.

The effects of one or five daily intraperitoneal injections of a neurotensin (NT) receptor agonist NT69L (2 mg/kg, i.p.) on the expression of NT (NTS), dopamine 1 and 2 receptors, tyrosine hydroxylase, and DOPA decarboxylase using immunohistochemical and real-time PCR were investigated in rats. Except for the striatum, acute injection of NT69L did not affect neurotensin receptors as compared to saline control. However, 5 daily injections of NT69L resulted in down-regulation of both NTS-1 protein and mRNA levels in several brain regions with the striatum showing a dramatic decrease in NTS-1 expression (P<0.05). The down-regulation of NTS-1 in the striatum, hypothalamus, and substania nigra (SN) after 5 daily injections was confirmed by autoradiography. Acute injection of NT69L increased NTS-2 mRNA and protein level in prefrontal cortex (PFC). NTS-3 mRNA expression and protein levels were slightly down-regulated in hypothalamus, periaqueductal gray (PAG), and SN, though the difference was not significant. The results indicated a difference in the profile of NT receptors expression in response to NT69L. Tyrosine hydroxylase (TH) and DOPA decarboxylase (DDC) mRNA was significantly down-regulated in striatum but not in SN. Interestingly, Nurr 1, a transcriptional activator of TH, was dramatically up-regulated in striatum, but down-regulated in PFC, suggesting that different modulating mechanisms may participate in NT69L tolerance in different regions. The present results suggest that distinct NT receptors involved in the effects exerted by NT69L may contribute to the interactions of NT69L with both neural networks and cellular proteins.

Detailed knowledge of the anatomical distribution of different GABA(A) receptor subunits is crucial for understanding the physiological actions of GABA in individual brain areas and for developing drugs acting through the individual GABA receptor subtypes. Since the amygdala is a key brain structure in the processing of emotional information with distinct functions in each nucleus, GABA(A) receptors in the amygdala are an important target of treatment for emotional disorders. In this study, we analyzed by quantitative RT-PCR the expression levels of all GABA(A) receptor subunits in distinct nuclei of the amygdala, the central (Ce) and the lateral/basolateral (LA/BLA) amygdala. We found the strongest expression of the gamma(2) subunit mRNA in both the Ce and LA/BLA, modest expressions of alpha(1), alpha(2) and alpha(3) mRNAs in the LA/BLA and alpha(2) and gamma(1) mRNAs in the Ce, and weak expressions of alpha(6), rho(2) and rho(3) mRNAs in both regions. We further revealed the significantly different expressions of alpha(1), alpha(3), alpha(5), gamma(1), gamma(2), delta, epsilon and theta subunit mRNAs in the Ce and LA/BLA. Differences in the expression levels of GABA(A) receptor subunits suggest different sensitivity to a variety of drugs including benzodiazepines and anesthetics in amygdala nuclei with distinct functions.

We analyzed gene expression profiles in embryonic day 12, 15, 18 and postnatal day 0 mouse brains by utilizing a GeneChip microarray. Significant differential expression was observed in 1413 of 12,422 (11.4%) represented on the chip. Then, 397 genes known to be related to neural development and functions were selected and analyzed in more detail. Clustering of the differentially expressed genes in terms of gene function and their temporal expression patterns indicated an aspect of the genetic foundation that underlies cellular events. Moreover, we identified a novel gene that encodes a putative protein kinase, Ebr kinase, which is differentially expressed in the developing brain.

There is increasing evidence that unilateral nerve injury evokes contralateral responses, but the underlying mechanisms are largely unknown. In the present investigation, we analyzed cytokine and chemokine gene induction in contralateral, non-lesioned nerves after sciatic nerve crush and chronic constriction injury (CCI) by quantitative reverse transcriptase polymerase chain reaction in mice. After sciatic nerve crush, contralateral changes in cytokine gene expression were restricted to interleukin (IL)-1beta, which showed a monophasic peak at the first postoperative day. Following CCI, contralateral transcripts for IL-1beta, IL-10 and monocyte chemoattractant protein-1 (MCP-1) were significantly increased already at day 1 and upregulation persisted over the next 4 weeks. In contrast, tumor necrosis factor alpha (TNF-alpha) levels remained unchanged. Contralateral gene induction was restricted to the homonymous opposite sciatic nerve, but spared the femoral nerve. NMDA receptor blockade completely abolished contralateral cytokine expression after CCI on the mRNA level. In contralateral dorsal root ganglia, only IL-10 mRNA levels were modified after nerve injury. Sham operation significantly increased the cytokine and chemokine gene expression at the ipsilateral side, but could not mediate contralateral effects. Our study confirms that nerve injury evokes contralateral responses and identifies NMDA-mediated signaling as one underlying mechanism.

The US11 gene product of herpes simplex virus is an RNA-binding protein with a C-terminal arginine-X-proline (RXP) repeating motif. We found that the RXP repeat mediates intercellular trafficking activity and accumulation in neuronal nuclei following in vivo transfection with the US11 gene, direct injection of the purified RXP-repeat fusion protein, or infection with herpes simplex virus. We discuss a possible therapeutic application of the US11 protein RXP repeat as a tactic against neurodegenerative disorders of the central nervous system.

The physiological effects of pineal melatonin are primarily mediated by melatonin receptors located in the brain and periphery. Even though there are a number of studies demonstrating the regulatory role of melatonin in the development of dopaminergic behaviors, such as psychostimulant-induced diurnal locomotor sensitization or drug seeking, little is known about the contribution of melatonin receptors (i.e., MT1) to this role. Therefore, as a first step in understanding the functional role of melatonin receptors in dopaminergic behaviors, we focused on determining the expression pattern of MT1 receptors in the dopaminergic system of the human and rodent brain. Regional (e.g., nucleus accumbens shell) and cellular (e.g., tyrosine hydroxylase immunopositive cells) expression of MT1 mRNA was characterized by applying the immuno-laser capture microdissection (immuno-LCM) technique coupled with nested RT-PCR. Moreover, employing quantitative Western immunoblotting and RT-PCR, we found that the mouse MT1 receptor expression presents diurnal variations (i.e., low mRNA and high protein levels at night, ZT21). The dopaminergic system-based presence of MT1 receptor proteins was not limited to rodents; we found these receptors in postmortem human brain as well. Further research is needed to understand the regional/cellular functional role of melatonin receptors in the regulation of dopaminergic behaviors, using models such as melatonin receptor knockout mice.

Tumor endothelial marker7 (TEM7) is a putative transmembrane protein that is highly expressed in the tumor endothelium. In the present study, the expression profile of TEM7 was investigated in TEM7-transfected human embryonic kidney (HEK) 293 cells and the rat brain. The extracellular secretion of the recombinant N-terminal ectodomain of TEM7, not full-length TEM7, was observed in the transiently transfected HEK 293 cells. The full-length TEM7 was found inside and membrane part of cells as demonstrated by confocal microscopy. In situ hybridization study revealed that TEM7 mRNA expressions were localized to specific neuronal areas, such as cerebellar Purkinje cells, the layer IV and V of cerebral cortex, hippocampal pyramidal cells and hypothalamic magnocellular nuclei. Immunohistochemical investigation of TEM expression with specific antibodies against TEM7 further supported the spatial expression patterns of TEM7 mRNA. The temporal expression of TEM7 mRNA in the cerebellar Purkinje cells demonstrated a postnatal developmental regulation of TEM7 expression. Our results indicate that TEM7 plays a role as a transmembrane receptor in some neuronal populations of the vertebrate brains.

The frog (Xenopus laevis) retina has been an important model for the analysis of retinal circadian rhythms. In this paper, several isoforms of X. laevis casein kinase I (CKI) were analyzed to address whether they are involved in the phosphorylation and degradation of period protein (PER), as they are in the circadian oscillators of other species. cDNAs encoding two splice variants of CKI(delta) (a full-length form and deletion isoform, which is missing an exon that encodes a putative nuclear localization signal and two evolutionarily conserved protein kinase domains) were isolated and analyzed, together with a previously isolated CKI(epsilon) isoform. Both CKI(delta) and CKI(epsilon) were shown to be constitutively expressed in the photoreceptors of the retina, where a circadian clock has been localized. Both the full-length CKI(delta) and CKI(epsilon) were shown to have kinase activity in vitro, and the full-length CKI(delta) phosphorylated and degraded Drosophila PER when expressed in Drosophila S2 cells. The expression and biochemical characteristics of these CKIs are consistent with an evolutionarily conserved role for CKI in the Xenopus retinal clock. The CKI(delta) deletion isoform did not exhibit kinase activity and did not trigger degradation of PER. Subcellular localization of both CKI(delta) isoforms was cytoplasmic in several cell culture lines, but the full-length CKI(delta) , and not the deletion CKI(delta) isoform, was localized to both the nucleus and the cytoplasm in Drosophila S2 cells. These results indicate that the sequences missing in the deletion CKI(delta) isoform are important for the nuclear localization and kinase activity of the full-length isoform and that one or both of these features are necessary for degradation of Drosophila PER.

In mice, the MPTP-induced striatal dopaminergic denervation is followed by a spontaneous partial DAT recovery and by serotoninergic hyperinnervation. We show that IL-1RI-deficient mice have a higher DAT decrease in the ventromedial striatum after MPTP and a higher basal serotoninergic innervation of the whole striatum. These data point to a possible role of IL-1RI in the early MPTP-induced structural or functional remodeling of the nigrostriatal dopamine system.

The molecular messenger nitric oxide (NO) not only serves a number of physiologic functions, but is also involved in the pathophysiology of neurodegeneration. It is produced by the nitric oxide synthase (NOS) isoenzymes. One of the many players regulating NOS activity is the Protein Inhibitor of NOS, PIN. To gain further insight into the mechanisms of NOS regulation and NO-mediated cell death after nerve trauma, we examined PIN expression in a standard model of lesion-induced neurodegeneration, the rat optic nerve transsection model. In both the axotomized retinae and the control retinae PIN expression was predominantly observed in the retinal ganglion cell layer. Optic nerve lesion did neither change the amount of PIN mRNA, as determined by in situ hybridization and real-time RT-PCR, nor did it change the amount of PIN as determined by immunohistochemistry and Western blot analysis. These results suggest that in our model, NOS activity is not regulated by altered PIN levels, which contributes to our understanding of apoptotic mechanisms in injured neurons.

We isolated a fragment of the fukutin gene promoter from differentiated human NT2 cells using chromatin immunoprecipitation technique with an anti-CREB antibody. This fragment contained a CRE-like sequence and here we describe its functional validation. The results showed that the element was functional in vitro and in vivo and that CREB in neurons was involved in the transcriptional regulation of the fukutin gene. Moreover, its expression in neurons was regulated by cAMP and calcium ions, known triggers of CREB phosphorylation. To our knowledge, this is the first report on the regulation of fukutin gene by transcription factor CREB in response to the signals generated by synaptic activity. The true biological function of fukutin, the gene responsible for Fukuyama-type congenital muscular dystrophy and mental retardation, is at present not known. However, it has been suggested that it might possess glycosyltransferase activity and its intracellular localization within the Golgi structures is consistent with this function. As such, fukutin might play a significant role in post-translational modification of synaptic proteins in neuronal cells.

To characterize the gene activity that may be required for neuronal survival and regeneration, we used the Affymetrix GeneChip Mu74A to screen 12000 genes and expressed sequence tag (EST) mRNA from L4 and L5 mouse dorsal root ganglia (DRG) 12 h and 24 h after sciatic nerve transection. At 12 h, we found 17 upregulated transcripts, and at 24 h, 49 that met our criteria of a significant 2-fold increase in expression. The alterations included a total of eight transcription factors and several genes associated with TGF-beta- and IL-6-mediated signaling. Two of the changes, amphiregulin and plasminogen activator inhibitor-1 (PAI-1), were confirmed by real-time quantitative PCR (QPCR). Addition of amphiregulin (20 ng/ml) to organ-cultured DRG stimulated axonal outgrowth while PAI-1 (20 nM) inhibited migration of Schwann cells from the ganglia.

Evidence suggests that neuropeptide Y (NPY) and agouti-related protein (AgRP) in the arcuate nucleus (ARC) are modulated by glucoregulatory hormones and involved in maintaining normal eating patterns and glucose homeostasis in states of energy deficiency. This study investigated whether these peptides respond to glucose itself under conditions, e.g., before the nocturnal feeding cycle, when carbohydrate stores are low. After removal of food 3 h before dark onset, Sprague-Dawley rats were given a single, intraperitoneal (i.p.) injection of saline or 10% glucose (0.13 g/kg) and were sacrificed at different intervals, from 3.5 to 90 min later, for measurements of circulating hormones and metabolites or of NPY and AgRP mRNA in the ARC. With no change in insulin, leptin, or triglycerides, glucose injection produced a 1.8-mM rise in circulating glucose during the first 15 min, followed by a 30-60% reduction in NPY and AgRP mRNA at 30 and 60 min post-injection. A similar effect was observed with intraventricular administration of 5% glucose. At 90 min, however, this suppressive effect of i.p. glucose relative to saline was lost and actually reversed into a 50% increase in NPY and AgRP, possibly attributed to a decline in circulating glucose followed by a 50% rise in corticosterone at 60 min. These biphasic shifts over a 90-min period may reflect mechanisms underlying natural eating patterns at the onset of the nocturnal cycle, when spontaneous meals are approximately 90 min apart and rich in carbohydrate, glucose levels are low, and corticosterone and ARC peptides naturally peak.

The processes of differentiation and development of neurons involve the induction of neuron-specific genes by instructive signals with subsequent neurotrophic factor-driven survival and functional maturation. We have previously shown that bone morphogenetic protein-2 (BMP2) and retinoic acid synergistically induce the responsiveness of developing sympathetic neurons to neurotrophic factors, neurotrophin 3 (NT-3), and GDNF by upregulating corresponding receptors concomitantly with the induction of other neuron-specific genes including BRINP1, a neuron-specific cell-cycle regulatory protein. In the present study, we analyzed transcriptional mechanisms regulating the neuron-specific expression of TrkC/NT-3 receptor gene. TrkC gene contains at least four NRSE/RE-1 (neuron-restrictive silencing element/repressor element 1)-like elements (TrkC-NRSE A-D). Consequently, we found that in non-neuronal cells, neuron-restrictive silencing factor (NRSF) acts on TrkC-NRSE D located at the downstream of exon 3 to suppress the promoter activity of TrkC gene in a manner similar to the mechanism of NRSF suppressing BRINP1 transcription. In contrast, in neuronal cells, the biological activity of NRSF on TrkC was suppressed. From these observations, molecular mechanisms regulating the expression of neuron-specific genes via NRSE during neuronal differentiation are discussed.

Connexin 35 (Cx35) is a major component of electrical synapses in the central nervous system. Many gap junctions containing Cx35 are regulated by dopamine receptor pathways that involve protein kinase A (PKA). To study the mechanism of PKA regulation, we analyzed direct phosphorylation of Cx35 by PKA in vitro and studied the regulation of neurobiotin tracer coupling in HeLa cells expressing Cx35 or Cx35 mutants that lack phosphorylation sites. In Cx35-transfected cells, application of the PKA activator Sp-8-cpt-cAMPS caused a significant decline in coupling, while a PKA inhibitor, Rp-8-cpt-cAMPS, significantly increased tracer coupling. In vitro phosphorylation and mutagenic analysis showed that PKA phosphorylates Cx35 directly at two major sites, Ser110 in the intracellular loop and Ser276 in the carboxyl terminus. In addition, a minor phosphorylation site in the C-terminus was identified by truncation of the last 7 amino acids at Ser298. The mutations Ser110Ala or Ser276Ala significantly reduced regulation of coupling by the PKA activator while a combination of the two eliminated regulation. Truncation at Ser298 reversed the regulation such that the PKA activator significantly increased and the PKA inhibitor significantly decreased coupling. The activation was eliminated in the S110A, S276A, S298ter triple mutant. We conclude that PKA regulates Cx35 coupling in a complex manner that requires both major phosphorylation sites. Furthermore, the tip of the C-terminus acts as a "switch" that determines whether phosphorylation will inhibit or enhance coupling. Reliance on the combined states of three sites provides fine control over the degree of coupling through Cx35 gap junctions.

The aim of this study was to determine the distribution and function of G-substrate, a specific substrate of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)-cGMP-dependent protein kinase (PKG) signaling pathway, in normal rat retina and in G-substrate knockout mice. The retinas of adult wild-type rats and mice and G-substrate knockout mice were studied immunohistologically to characterize the upstream and downstream components of the NO-cGMP-PKG pathway. Immunoblot analysis showed that the molecular weight of retinal G-substrate was similar to that of cerebellar G-substrate. In adult rats and mice, retinal G-substrate was located in a subpopulation of amacrine cells and in C38-positive retinal ganglion cells (RGCs) but not in alpha RGCs. In addition, retinal G-substrate was co-expressed with other upstream and downstream signaling components of the NO-cGMP-PKG-G-substrate-phosphatase pathway in the adult retina. Electroretinographic (ERG) analysis demonstrated that there was no significant difference between the ERGs of wild-type and G-substrate knockout mice. These results suggest that retinal G-substrate plays a role as a downstream component of the NO-cGMP-PKG pathway. The co-localization of retinal G-substrate with protein Ser/Thr phosphatases suggests that it acts as an endogenous protein phosphatase inhibitor as in the cerebellum.

The Wnt signaling plays important roles in cell growth, differentiation, polarity formation, and neural development. In the canonical pathway, two DIX domain-containing proteins, Dishevelled (Dvl) and Axin, regulate the degradation of beta-catenin that activates Wnt target genes through TCF/LEF family transcription factors. Recently, we have isolated a third type of DIX domain-possessing protein, Coiled-coil-DIX1 (Ccd1). Ccd1 forms homomeric and heteromeric complexes with Dvl and Axin, and regulates the neural patterning in zebrafish embryos through Wnt pathway activation. Here, we report the isolation and characterization of mouse Ccd1. Fourteen putative mRNA isoforms are generated by different promoter usage and alternative splicing, and each isoform shows different expression patterns in various tissues. The predicted Ccd1 proteins are classified into three subtypes, and a novel form, termed Ccd1A, possesses an N-terminal calponin homology domain, suggesting an additional interaction of the isoform with actin or other proteins. When Ccd1 proteins were singularly expressed in Hela cells, they showed almost no activation of TCF-dependent reporter transcription on their own. However, when Dvl protein, at the level that did not activate Wnt pathway by itself, was co-expressed with Ccd1, the reporter transcription was greatly potentiated in Ccd1-dose-dependent manner. In addition, Ccd1- and Wnt3a-dependent activation of Wnt pathway was inhibited by Axin or a dominant negative Ccd1. These results indicate that mouse Ccd1 functions as a positive regulator of the Wnt/beta-catenin pathway. Furthermore, Ccd1 is highly expressed and co-localized with Wnt signaling molecules in the embryonic and adult brain, implicating the importance of Ccd1 in the Wnt-mediated neuronal development, plasticity, and remodeling.

p55 is a membrane-associated guanylate kinase (MAGuK) family member that consists of a single PDZ followed by SH3, HOOK and guanylate kinase (GuK or GK) domains. We investigated rat p55 (r-p55) in the brain. r-p55 mRNA was expressed widely in various tissues and in various regions of the brain. r-p55 protein was also expressed widely in various rat tissues, including brain and erythrocytes. The protein was enriched in the synaptic plasma membrane and postsynaptic density (PSD) fractions of the forebrain. An immunocytochemical study using cultured cortical neurons suggested postsynaptic localization of r-p55 protein. Pull-down assay showed that r-p55 protein interacted with r-p55 itself and various PSD proteins, such as PSD-95, SAP97, GKAP, CASK, GRIP, neuroligin, cadherin, tubulin, actin, alpha-internexin, neurofilament-L and Ca(2+)/calmodulin-dependent protein kinase II, through its PDZ, SH3, HOOK or GK domains. The interaction with PSD-95 was found to occur between the PDZ domains of PSD-95 and the HOOK and GK domains of r-p55 protein. These findings, together with the presence of r-p55 puncta in a period of early synaptogenesis, suggest that r-p55 protein functions as one of postsynaptic scaffold component in an early stage of synaptogenesis in the brain. r-p55 protein may form a basic structure, which interlinks diverse functional molecules of the PSD necessary for postsynaptic signaling and synaptic adhesion.

To investigate the effects of repeated immobilization-stress challenge on HPA axis, genomic transcriptome in the hypothalamus of immobilization-stressed mouse was analyzed by using cDNA microarray. With the 1.5-fold cutoff of arbitrary criteria, the expression levels of 108 genes out of 6016 genes were significantly modulated in the hypothalamus by the stress. Energy metabolism-, lipid metabolism-, and apoptosis- and signal transduction-related genes were activated while DNA repair-, protein biosynthesis-, and structure integrity-related genes were down-regulated in the hypothalamus. Eighteen genes among them were selected for RT-PCR analysis to confirm the change of their expression levels on agarose gels. Besides, dozens of novel genes, which have not been previously reported, were screened to be modulated by the immobilization stress through the transcriptome analysis. These genes are related to apoptosis, tumor-suppression, DNA-binding and protein folding, and thus may be used as potential targets for the development of therapeutics of chronic stress or depressant.

The thalamus degenerates following cerebral infarction in the territory supplied by the middle cerebral artery (MCA), and apoptosis is suspected to be the mechanism of this phenomenon. The author studied the role of the growth arrest and DNA damage-inducible gene (GADD) 153 in this thalamic degeneration. The MCA was occluded in stroke-prone spontaneously hypertensive rats. The expression of GADD 153 and Bcl-2, and the release of cytochrome c from the mitochondria to cytosol, were examined in the thalamus until 7 days after ischemia using in situ hybridization, immunoblot, immunohistochemistry and RT-PCR analyses. Gadd153 mRNA expression and GADD153 protein increased transiently at 2, 3, 5 and 7 days, and at 3 and 5 days after ischemia. Bcl-2 mRNA expression and Bcl-2 protein decreased at 3 and 5 days. The release of cytochrome c from the mitochondria was detected at 5 days. These results suggest that increased GADD 153 suppresses Bcl-2 expression, which causes the release of cytochrome c from the mitochondria and leads to thalamic degeneration.

The neuroprotective effects of Delta(9)-tetrahydrocannabinol (THC) were examined using an in vitro model in which the AF5 CNS cell line was exposed to toxic levels of N-methyl-d-aspartate (NMDA), an agonist of the NMDA glutamate receptor. NMDA toxicity was reduced by THC, but not by the more specific cannabinoid receptor agonist, WIN55,212-2. Addition of dibutyryl cAMP (dbcAMP) to the culture medium did not alter the neuroprotective effect of THC and did not unmask a neuroprotective effect of WIN55,212-2. The cannabinoid antagonist SR141716A did not inhibit the neuroprotection induced by THC or alter the response to WIN55,212-2, even in the presence of dbcAMP, indicating that the neuroprotective effect of THC was cannabinoid receptor-independent. On the other hand, both THC and WIN55,212-2 produced cellular toxicology at higher dosages, an effect which was blocked in part by SR141716A. Capsaicin, an antioxidant and vanilloid receptor agonist, also produced a protective effect against NMDA toxicology. The protective effect of capsaicin was blocked by co-application of ruthenium red, but was not blocked by the specific vanilloid receptor antagonist capsazepine, and the transient receptor potential vanilloid type 1 (TRPV1) and ANKTM1 transcripts were not detected in AF5 cells. Thus, the neuroprotective effects of THC and capsaicin did not appear to be mediated by TRP ion channel family receptors. The antioxidant alpha-tocopherol prevented neurotoxicity in a dose-dependent manner. Therefore, THC may function as an antioxidant to increase cell survival in NMDA-induced neurotoxicity in the AF5 cell model, while higher dosages produce toxicity mediated by CB1 receptor stimulation.

Choline is an essential nutrient and an important methyl donor. Choline deficiency alters fetal development of the hippocampus in rodents and these changes are associated with decreased memory function lasting throughout life. Also, choline deficiency alters global and gene-specific DNA methylation in several models. This gene expression profiling study describes changes in cortical neural precursor cells from embryonic day 14 mice, after 48 h of exposure to a choline-deficient medium. Using Significance Analysis of Microarrays, we found the expression of 1003 genes to be significantly changed (from a total of 16,000 total genes spotted on the array), with a false discovery rate below 5%. A total of 846 genes were overexpressed while 157 were underexpressed. Classification by gene ontology revealed that 331 of these genes modulate cell proliferation, apoptosis, neuronal and glial differentiation, methyl metabolism, and calcium-binding protein classes. Twenty-seven genes that had changed expression have previously been reported to be regulated by promoter or intron methylation. These findings support our previous work suggesting that choline deficiency decreases the proliferation of neural precursors and possibly increases premature neuronal differentiation and apoptosis.

Neurons of the basal forebrain (BF) possess unique combinations of voltage-gated membrane currents. Here, we describe subtypes of rat basal forebrain neurons based on patch-clamp analysis of low-voltage activated (LVA) calcium and tetrodotoxin-resistant (TTX-R) sodium currents combined with single-cell RT-PCR analysis. Neurons were identified by mRNA expression of choline acetyltransferase (ChAT+, cholinergic) and glutamate decarboxylase (GAD67, GABAergic). Four cell types were encountered: ChAT+, GAD+, ChAT+/GAD+ and ChAT-/GAD- cells. Both ChAT+ and ChAT+/GAD+ cells (71/75) displayed LVA currents and most (34/39) expressed mRNA for LVA Ca(2+) channel subunits. Ca(v)3.2 was detected in 31/34 cholinergic neurons and Ca(v)3.1 was expressed in 6/34 cells. Three cells expressed both subunits. No single neurons showed Ca(v)3.3 mRNA expression, although BF tissue expression was observed. In young rats (2-4 mo), ChAT+/GAD+ cells displayed larger LVA current densities compared to ChAT+ neurons, while these latter neurons displayed an age-related increase in current densities. Most (29/38) noncholinergic neurons (GAD+ and ChAT-/GAD-) possessed fast TTX-R sodium currents resembling those mediated by Na(+) channel subunit Na(v)1.5. This subunit was expressed predominately in noncholinergic neurons. No cholinergic cells (0/75) displayed fast TTX-R currents. The TTX-R currents were faster and larger in GAD+ neurons compared to ChAT-/GAD- neurons. The properties of ChAT+/GAD+ neurons resemble those of ChAT+ neurons, rather than of GAD+ neurons. These results suggest novel features of subtypes of cholinergic and noncholinergic neurons within the BF that may provide new insights for understanding normal BF function.

To determine neuronal and glial responses to copper (Cu) elevation in the CNS, human neuroblastoma and astrocytoma cells were used to compare their responses to Cu in terms of reactive oxygen species (ROS) generation and expression of enzymes responsible for anti-oxidation. Astrocytoma cells, not neuroblastoma cells, were responsive to Cu and Cu elevation was associated with ROS generation. Intracellular Cu levels as determined by inductively coupled plasma-mass spectrometry (ICP-MS), and expression levels of copper-transporting ATPase (ATP7A) and human copper transporter 1 (hCtr1) as detected by quantitative reverse transcription-polymerase chain reaction (RT-PCR), were comparable in both cell lines. Differences in Cu-induced ROS between two cell lines paralleled superoxide dismutase (SOD)-catalase expression as detected by Western blot analysis. Copper,zinc-SOD (Cu,Zn-SOD) and catalase protein levels were upregulated by Cu in neuroblastoma cells while Cu,Zn-SOD was down-regulated by Cu and catalase level was not changed in astrocytoma cells. Manganese-SOD (Mn-SOD) was not responsive to Cu in either cell line. Furthermore, 78-kDa glucose-regulated protein aggregation and upregulation were observed in Cu-treated astrocytoma cells, but not neuroblastoma cells. These data suggest that neurons use the SOD-catalase system to scavenge Cu-induced ROS while glia rely on the endoplasmic reticulum stress response to compensate for the reduction of ROS scavenging capacity.

Members of the MRG family of G-protein coupled receptors (GPCRs) are expressed predominately in small diameter sensory neurons of the dorsal root ganglia (DRG) suggesting a possible role in nociception. However, the large expansion of this gene family in rodents, combined with the lack of strict rodent orthologs for many of the human MRG genes, limits the usefulness of rodent models to evaluate human MRG involvement in nociception. Furthermore, the high degree of similarity between related rodent Mrg genes suggests that pharmacological approaches to define the function of individual receptors will prove difficult. The creation of an animal model to examine human MRG function will, therefore, require the identification of human MRG orthologs in a non-rodent species. Here we report the identification of MRGD, MRGE, and several MRGX orthologs in the crab-eating macaque, Macaca fascicularis. Similar to their human counterparts, all isolated macaque genes were expressed in dorsal root ganglia neurons. In the case of macaque MrgX2 and MrgD, expression was co-localized with the known nociceptive neuronal markers, IB4, VR1, and SP. Although expression in DRG neurons was the prominent feature of this family, we also found that MrgE was expressed in numerous brain regions of macaque, mouse, and human.

When exposed to the cold, the body temperature of the ruin lizard (Podarcis sicula), an ectothermic vertebrate, comes into equilibrium with that low environmental temperature. During this time, the behavioral output of the circadian clock, locomotor activity, disappears. We tested the activity of the circadian clockwork at low temperature (6 degrees C) by following the expression of one of its essential components, the Period2 (Per2) gene. Here we show that lizard Per2 (lPer2) expression, which is rhythmic and paralleling the behavioral rhythm of locomotor activity at higher temperature (29 degrees C), becomes constantly high at low temperature. When lizards are re-exposed to high temperature, rhythmic lPer2 expression is re-established after 2 days of adaptation and coincides with onset of locomotor activity. The alteration of the lPer2 expression pattern at low temperature indicates that the activity of the molecular feedback loop is modified under these conditions.

This study determined the expression levels of tyrosine hydroxylase (TH), arginine vasopressin (AVP), and prepro-neuropeptide Y (NPY) mRNA in the brain following acute and chronic air-puff stress. Acute stress increased TH gene expression in the locus coeruleus of Wistar-Kyoto (WKY) rats (+80%), but only modestly in spontaneously hypertensive rats (SHR; +19%). Hypothalamic AVP mRNA in SHR was persistently elevated in the supraoptic nucleus (+24% acute; +19% chronic), but reduced in the magnocellular region of the paraventricular nucleus (-15% acute; -20% chronic). In the arcuate nucleus, NPY mRNA expression increased by 60% and 81% in response to acute and chronic air-puff stress, respectively, in SHR. These data suggest differential adaptation to air-puff stress between WKY and SHR.

Food restriction is associated with a number of endocrine disturbances. We validated the experimental conditions for several house-keeping genes and determined the effects of 12 day 50% food restriction on hypothalamic and pituitary transcription of genes involved in different neuroendocrine systems, using real-time quantitative polymerase chain reaction (PCR). A total of 7 nuclear receptors and 12 neuropeptides and peptide hormones were investigated in the dorsal and ventral hypothalamus and the pituitary gland in rats. In the hypothalamus, food restriction reduced mRNA levels of estrogen receptor alpha (ERalpha), progesterone receptor, glucocorticoid receptor, thyroid hormone receptor alpha and beta, pro-opiomelanocortin (POMC), growth hormone-releasing factor (GHRF), corticotropin-releasing factor (CRF), thyrotropin-releasing factor (TRF), somatostatin, and increased that of neuropeptide Y (NPY). In the pituitary, the treatment reduced growth hormone (GH), luteinizing hormone beta (LHbeta) and thyrotropin beta, but increased ERalpha mRNA levels. The study provides a map of how food restriction affects the regulation of a number of transcripts involved in neuroendocrine control.

Glia maturation factor beta (GMFB) was identified as a growth and differentiation factor acting on neurons as well as glia. We investigated the expression of GMFB during 56 days after cryogenic brain injury, using immunohistochemistry, reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting and enzyme immunoassay. Immunohistochemical analysis demonstrated that the GFAP-positive astrocytes around the lesion expressed GMFB protein, peaking 14 days after injury. Weak astrocytic expression of GMFB-immunoreactivity was seen in sham-operated animal brains. Cryogenic injury (CI) induced GMFB mRNA in the lesioned side after 7 days with a maximum at 14 days. Western blotting revealed the induction of GMFB protein starting 1 day after injury, and continuing until 14 days after injury. In the enzyme immunoassay, GMFB protein concentration peaked 14 days after injury in extracts from the injured side of the brain, whereas in serum it peaked 1 day after injury. These data indicate that the expression of GMFB increased in the astrocytes around the lesioned area after cortical cryogenic brain injury. These findings may provide new insight into GMFB function in pathological conditions following brain injury.

Postsynaptic density-95 (PSD-95) is an evolutionarily conserved synaptic adaptor protein that is known to bind many proteins including the NMDA receptor. This observation has implicated it in many NMDA receptor-dependent processes including spatial learning and synaptic plasticity. We have cloned and characterised the murine PSD-95 gene. In addition, we have identified two previously uncharacterised splice variants of the major murine PSD-95 transcript (PSD-95alpha): PSD-95alpha-2b results from an extension of exon 2 and PSD-95alpha-Delta18 from the temporal exclusion of exon 18. The presence of PSD-95alpha-2b sequences in other PSD-95 family members implicates this peptide stretch as functionally significant. Another potential transcript (PSD-95gamma) was also identified based on examination of EST databases. Immunoprecipitation assays demonstrate that proteins corresponding in size to PSD-95alpha-Delta18 and PSD-95gamma interact with the NMDA receptor, suggesting an important biological role for these isoforms. Finally, we have performed bioinformatics analyses of the PSD-95 mRNA untranslated regions, identifying multiple translational control elements that suggest protein production could be regulated post-transcriptionally. The variety of mRNA isoforms and regulatory elements identified provides for a high degree of diversity in the structure and function of PSD-95 proteins.

Nerve growth factor (NGF) is required for the survival of neurons. We have addressed the effect of platelet-activating factor (PAF), one of the mediators of ischemic injury of the brain, on NGF expression in astrocytes. Normal human astrocytes in culture were stimulated with PAF, and levels of NGF mRNA and protein were analyzed by reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative PCR and enzyme-linked immunosorbent assay (ELISA). PAF increased the expressions of NGF mRNA and protein in astrocytes in time- and concentration-dependent manners. After 48-h stimulation, 10 nmol/L PAF increased the levels of NGF protein in astrocyte-conditioned medium by 1.4-fold. The PAF-induced stimulation of NGF expression was further enhanced (2.1-fold of the control) in the cells under hypoxic culture condition. BN52021 (Ginkgolide B), an antagonist for PAF binding sites, suppressed the effect of PAF. We conclude that PAF enhances NGF gene expression in human astrocytes, and the PAF-induced increase in the expression of NGF under hypoxia may benefit the protection of the nervous tissue by promoting neuronal survival.

The regulatory mechanisms leading to IL-20 expression during infection have not been elucidated. In the present study, we found that bacterial lipopolysaccharide (LPS) induced IL-20 expression in the primary cultured glial cells and RAW264.7 macrophage cell line. Pretreatment with protein synthesis inhibitor puromycin or cycloheximide failed to inhibit the expression of IL-20, suggesting that the expression was not dependent on de novo protein synthesis. Myeloid differentiation factor 88 (MyD88) is an important adaptor molecule for Toll-like receptor signaling. We observed complete inhibition of LPS-induced expression of IL-20 in the primary cultured glial cells prepared from MyD88-deficient mice. Furthermore, a p38 MAP kinase inhibitor, SB203580, inhibited LPS-induced expression of IL-20 mRNA. LPS-induced p38 MAP kinase phosphorylation was delayed in MyD88-deficient glial cells. Therefore, it is suggested that LPS induces IL-20 expression through MyD88-p38-dependent mechanisms. As dexamethasone inhibited LPS-induced IL-20 expression, the expression of IL-20 is regulated by a negative feedback loop mediated through glucocorticoids. Therefore, it is suggested that IL-20 may play a crucial role in inflammatory conditions in the brain.

Netrins are a family of secreted proteins that function as tropic cues directing axon growth and cell migration during neural development. Beta-netrin or netrin-4 (NTN4), the fourth member of this family, was previously reported by Koch (M. Koch, J.R. Murrell, D.D. Hunter, P.F. Olson, W. Jin, D.R. Keene, W.J. Brunken, R.E. Burgeson. A novel member of the netrin family, beta-netrin, shares homology with the beta chain of laminin: identification, expression, and functional characterization. J. Cell Biol. 151 (2000) 221-234) and Yin (Y. Yin, J.R. Sanes, J.H. Miner, Identification and expression of mouse netrin-4. Mech. Dev. 96 (2000) 115-119), respectively. However, whether there is another isoform of netrin-4 and the expression patterns have not been fully determined. Here we report the cloning of a novel isoform of human netrin-4 using rapid amplification of complementary DNA (cDNA) ends (RACE) technique and analysis on the netrin-4 mRNA expression in adult rat brain using in situ hybridization. The new isoform lacks the signal peptide region and might represent a truncated form at the Laminin-N terminal domain of human netrin-4. Examination of the expression pattern of netrin-4 mRNA in adult rat brain revealed that the netrin-4 mRNA was widely expressed in many regions of the brain. High levels of netrin-4 mRNA was found in the pyramidal cell layer of the cerebral cortex, prepiriform cortex, amygdaloid nuclei, pyramidal layer of hippocampus, Purkinje's cells, medial cerebellar nucleus and interposed cerebellar nucleus, medial nucleus of the trapezoid body and mitral cell layer of the olfactory bulb. Moderate signals were present in frontal cortex, several thalamic and hypothalamic nuclei. The widespread expression of netrin-4 in adult rat brain indicates that netrin-4 is not only important for axon guidance during embryonic development, but also critical to neuronal plasticity in adult stage.