Chronic social isolation induces NF-κB activation and upregulation of iNOS protein expression in rat prefrontal cortex

Exposure of an organism to stress, results in oxidative stress and increased nitric oxide (NO) production in the brain. The role of the processes caused by chronic stress in the prefrontal cortex has not been fully investigated. Considering that chronic stress increases NO production by the enzyme nitric oxide synthase (NOS), we examined the cytosolic neuronal (nNOS) or inducible (iNOS) protein levels in the prefrontal cortex of rats exposed to 21 d of chronic social isolation stress, an animal model of depression, alone or in combination with 2 h of acute immobilization or cold (4 °C) stress (combined stress). Antioxidative status via cytosolic CuZnSOD and mitochondrial MnSOD activity, cytosolic redox status via reduced glutathione (GSH) concentration were determined. Furthermore, cytosolic inducible heat shock protein 70 (Hsp70i), cytosolic/nuclear distributions of NF-κB and serum corticosterone (CORT) were also investigated to elucidate the possible mechanism involved in the cellular NOS pathway. Our results showed that both acute stressors led to increases of CORT and nNOS protein while iNOS protein expression was unaffected. In contrast to the acute stress, chronic social isolation compromised hypothalamic–pituitary-adrenal axis functioning such that the normal stress response was impaired following subsequent acute stressors. Downregulated redox GSH status as well as decreased activity of CuZnSOD and MnSOD suggests the existence of oxidative stress which remained as such following combined stressors. Changes in redox-status associated with decreased Hsp70i protein expression enabled NF-κB translocation into the nucleus, causing increased cytosolic nNOS and iNOS protein expression. Results suggest that NOS signaling pathway plays a differential role between acute and chronic stress whereby state of oxidative/nitrosative stress after chronic social isolation is caused, at least in part, by NF-κB activation and increased iNOS protein expression.     

Repeated 6-Hz Corneal Stimulation Progressively Increases FosB/ΔFosB Levels in the Lateral Amygdala and Induces Seizure Generalization to the Hippocampus

Exposure to repetitive seizures is known to promote convulsions which depend on specific patterns of network activity. We aimed at evaluating the changes in seizure phenotype and neuronal network activation caused by a modified 6-Hz corneal stimulation model of psychomotor seizures. Mice received up to 4 sessions of 6-Hz corneal stimulation with fixed current amplitude of 32 mA and inter-stimulation interval of 72 h. Video-electroencephalography showed that evoked seizures were characterized by a motor component and a non-motor component. Seizures always appeared in frontal cortex, but only at the fourth stimulation they involved the hippocampus, suggesting the establishment of an epileptogenic process. Duration of seizure non-motor component progressively decreased after the second session, whereas convulsive seizures remained unchanged. In addition, a more severe seizure phenotype, consisting of tonic-clonic generalized convulsions, was predominant after the second session. Immunohistochemistry and double immunofluorescence experiments revealed a significant increase in neuronal activity occurring in the lateral amygdala after the fourth session, most likely due to activity of principal cells. These findings indicate a predominant role of amygdala in promoting progressively more severe convulsions as well as the late recruitment of the hippocampus in the seizure spread. We propose that the repeated 6-Hz corneal stimulation model may be used to investigate some mechanisms of epileptogenesis and to test putative antiepileptogenic drugs.     

Barrels, stripes, and fingerprints in the brain—Implications for theories of cortical organization

In the last decade improvements in the histological processing of cortical tissue in conjunction with the investigation of additional mammalian species in comparative brain studies has expanded the information available to guide theories of cortical organization. Here I review some of these recent findings in the somatosensory system with an emphasis on modules related to specializations of the peripheral sensory surface. The diversity of modular representations, or cortical “isomorphs” suggest that information from the sensory sheet guides many of the features of cortical maps and suggest that cortex is not constrained to form circular units in the form of a traditional cortical column.     

Quantitative mapping of cocaine-induced ΔFosB expression in the striatum of male and female rats

ΔFosB plays a critical role in drug-induced long-term changes in the brain. In the current study, we evaluated locomotor activity in male and female rats treated with saline or cocaine for 2 weeks and quantitatively mapped ΔFosB expression in the dorsal striatum and nucleus accumbens of each animal by using an anti-FosB antibody that recognizes ΔFosB isoforms preferentially. Behavioral analysis showed that while there was little difference between males and females that sensitized to cocaine, nonsensitizing rats showed a large sex difference. Nonsensitizing males showed low behavioral activation in response to cocaine on the first day of treatment, and their activity remained low. In contrast, nonsensitizing females showed high activation on the first day of treatment and their activity remained high. Western blot and immunohistochemical analyses indicated that basal levels of ΔFosB were higher in the nucleus accumbens than the dorsal striatum, but that the effect of cocaine on ΔFosB was greater in the dorsal striatum. Immunostaining showed that the effect of cocaine in both the dorsal striatum and nucleus accumbens was primarily to increase the intensity of ΔFosB immunoreactivity in individual neurons, rather than to increase the number of cells that express ΔFosB. Detailed mapping of ΔFosB-labeled nuclei showed that basal ΔFosB levels were highest in the medial portion of the dorsal striatum and dorsomedial accumbens, particularly adjacent to the lateral ventricle, whereas the cocaine-induced increase in ΔFosB was most pronounced in the lateral dorsal striatum, where basal ΔFosB expression was lowest. Sex differences in ΔFosB expression were small and independent of cocaine treatment. We discuss implications of the sex difference in locomotor activation and regionally-specific ΔFosB induction by cocaine.     

Enzyme activities of monoamine oxidase,cathechol-o-methyltransferase and γ-aminoubutyric acid transaminase in primary astroglial cultures and adult rat brain from different brain regions

The activities of monoamine oxidase (MAO), cathechol-O-methyltransferase (COMT) and -aminobutyric acid transaminase (GABA-T) were measured in primary cultures from newborn rat cultivated from 6 different brain regions. These primary cultures contained mostly astroglial cells, evaluated by the presence of the glial fibrillary acidic protein (GFAp, -albumin) and the S-100 protein. The enzyme activities in the corresponding brain areas from adult rat were also quantified. MAO activities were on the same level in 14-day old cultures and in adult rat brain homogenates, with significantly lower values in brain stem as compared to the other brain regions examined. COMT activities were on a higher level in the cultures than in adult rat brain homogenates. Astroglial cells from hippocampus were found to have the highest and those from brain stem the lowest COMT-activities. GABA-T activities were lower in the cultures than in adult rat homogenates. No significant differences were seen in the various astroglial cultures. Accumulation of [³H]dopamine and [³H]-aminobutyric acid (GABA) visualized by autoradiography showed only a slight uptake of dopamine in comparison with the uptake of GABA. It is concluded that astroglial cells in culture have enzymatic properties similar to those of astroglial cells in different brain regions of adult rat brain. Studies are in progress to evaluate if the regional heterogeneity observed among cultivated astroglial cells is affected by in vivo differentiation until cultivation and/or time in culture.     

Chemical ionization mass spectrometric measurement of α-methyldopa and s-dopa metabolites in rat brain regions

In order to study combined s-dopa and s-α-methyldopa metabolism in brain, we have developed a chemical ionization mass spectrometric method for simultaneously measuring nanomole quantities of dopamine, α-methyldopamine, norepinephrine, and α-methylnorepinephrine from tissue samples dissected from rat brain. Deuterium-labeled analogs of each of the four catecholamines were synthesized and used as internal standards for quantification. Following addition of the internal standards to the tissue sections, the catecholamines were extracted and separated from amino acids by ion-exchange chromatography. Derivatization with HCl·ethanol and pentafluoropropionic anhydride produced a family of eight derivatives with unique parent masses which were applied without gas chromatographic separation to the direct inlet probe of the mass spectrometer. The mass region containing the derivatives was repetitively scanned, and the individual amines were quantified by comparing their peak heights with the deuterium-labeled internal standards. To validate the solid probe method in vivo, animals were treated with varying combinations of s-dopa and s-α-methyldopa, and their brains were analyzed for amine concentrations. Results from rat hypothalamus, brainstem, and corpus striatum are presented.     

Amyloid β-peptide directly induces spontaneous calcium transients,delayed intercellular calcium waves and gliosis in rat cortical astrocytes

The contribution of astrocytes to the pathophysiology of AD (Alzheimer''s disease) and the molecular and signalling mechanisms that potentially underlie them are still very poorly understood. However, there is mounting evidence that calcium dysregulation in astrocytes may be playing a key role. Intercellular calcium waves in astrocyte networks in vitro can be mechanically induced after Aβ (amyloid β-peptide) treatment, and spontaneously forming intercellular calcium waves have recently been shown in vivo in an APP (amyloid precursor protein)/PS1 (presenilin 1) Alzheimer''s transgenic mouse model. However, spontaneous intercellular calcium transients and waves have not been observed in vitro in isolated astrocyte cultures in response to direct Aβ stimulation in the absence of potentially confounding signalling from other cell types. Here, we show that Aβ alone at relatively low concentrations is directly able to induce intracellular calcium transients and spontaneous intercellular calcium waves in isolated astrocytes in purified cultures, raising the possibility of a potential direct effect of Aβ exposure on astrocytes in vivo in the Alzheimer''s brain. Waves did not occur immediately after Aβ treatment, but were delayed by many minutes before spontaneously forming, suggesting that intracellular signalling mechanisms required sufficient time to activate before intercellular effects at the network level become evident. Furthermore, the dynamics of intercellular calcium waves were heterogeneous, with distinct radial or longitudinal propagation orientations. Lastly, we also show that changes in the expression levels of the intermediate filament proteins GFAP (glial fibrillary acidic protein) and S100B are affected by Aβ-induced calcium changes differently, with GFAP being more dependent on calcium levels than S100B.     

TNF-α induces upregulation of EGFR expression and signaling in human colonic myofibroblasts

The myofibroblast has recently been identified as an important mediator of tumor necrosis factor-α (TNF-α)-associated colitis and cancer, but the mechanism(s) involved remains incompletely understood. Recent evidence suggests that TNF-α is a central regulator of multiple inflammatory signaling cascades. One important target of TNF-α may be the signaling pathway downstream of the epidermal growth factor receptor (EGFR), which has been associated with many human cancers. Here, we show that long-term exposure of 18Co cells, a model of human colonic myofibroblasts, with TNF-α led to a striking increase in cell surface EGFR expression, an effect that was completely inhibited by cycloheximide. Subsequent EGFR binding by EGF and heparin binding (HB)-EGF was associated with enhanced EGFR tyrosine kinase activity, prolonged ERK activation, and a significant increase in cyclooxygenase-2 (COX-2) expression compared with 18Co cells treated with EGF and HB-EGF alone. TNF-α also increased EGFR expression and signaling in primary myofibroblasts isolated from human colon tissue. TNF-α-induced upregulation of EGFR may be a plausible mechanism to explain the exaggerated cellular responsiveness that characterizes inflammatory bowel disease and that may contribute to a microenvironment that predisposes to colitis-associated cancer through enhanced COX-2 expression.     

β-adrenergic receptor regulation,through cyclic AMP,of nerve growth factor expression in rat cortical and cerebellar astrocytes

1. Type 1 astrocytes prepared from 3-day rat cortex and cerebellum express the 1.3-kb nerve growth factor (NGF) mRNA and synthesize and release beta-NGF. 2. Isoproterenol (IP), a beta-adrenergic agonist, stimulates NGF mRNA content in cortical astrocytes; this increase is blocked by the beta-adrenergic antagonist propranolol but not the alpha-antagonist phenoxybenzamine. The EC50 for the effect of IP is 5 nM. 3. IP increases astrocyte cyclic AMP as does forskolin, which directly activates adenylate cyclase and also increases NGF mRNA content. Cerebellar astrocytes contain about one-third as much NGF mRNA, which can also be increased by forskolin and cyclic AMP. 4. These results suggest that CNS astrocytes can serve as a source of NGF and that the NGF gene is one of the class of cyclic AMP regulated genes.     

Decreased glutamine synthetase,increased citrulline–nitric oxide cycle activities,and oxidative stress in different regions of brain in epilepsy rat model

To understand their role in epilepsy, the nitric oxide synthetase (NOS), argininosuccinate synthetase (AS), argininosuccinate lyase (AL), glutamine synthetase (GS), and arginase activities, along with the concentration of nitrate/nitrite (NOx), thiobarbituric acid reactive substances (TBARS), and total antioxidant status (TAS), were estimated in different regions of brain in rats subjected to experimental epilepsy induced by subcutaneous administration of kainic acid (KA). The short-term (acute) group animals were killed after 2 h and the long term (chronic) group animals were killed after 5 days of single injection of KA (15 mg/kg body weight). After decapitation of rats, the brain regions were separated and in their homogenates, the concentration of NOx, TBARS and TAS and the activities of NOS, AS, AL, arginase and glutamine synthetase were assayed by colorimetric methods. The results of the study demonstrated the increased activity of NOS and formation of NO in acute and chronic groups epilepsy. The activities of AS and AL were increased and indicate the effective recycling of citrulline to arginine. The activity of glutamine synthetase was decreased in acute and chronic groups of epilepsy compared to control group and indicate the modulation of its activity by NO in epilepsy. The activity of arginase was not changed in acute group; however it was decreased in chronic group and may favor increased production of NO in this condition. The concentration TBARS were increased and TAS decreased in acute and chronic groups of epilepsy and supports the oxidative stress in epilepsy.     

15-Deoxy-Δ12,14-prostaglandin J2 induces Cox-2 expression in human osteosarcoma cells through MAPK and EGFR activation involving reactive oxygen species

Prostaglandins (PGs), important modulators in bone biology, may also contribute to tumor formation and progression in human osteosarcoma. 15-Deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)), a metabolite of PGD(2) and PPARγ-ligand, exerts a panel of biological activities via receptor-dependent and -independent mechanisms. As inducible cyclooxygenase-2 (Cox-2) is a candidate inflammatory marker in human osteosarcoma and a rate-limiting enzyme in PG biosynthesis, this study aimed at investigating intracellular redox status and signaling cascades leading to Cox-2 induction in human MG-63 osteosarcoma cells. 15d-PGJ(2) induced the accumulation of reactive oxygen species (ROS) that in turn may lead to upregulation of Cox-2 via two different routes in a PPARγ-independent manner. First, phosphorylation of p38 MAPK directly enhances Cox-2 expression by promoting mRNA stability. Second, 15d-PGJ(2) induces activation of epidermal growth factor receptors and downstream activation of Cox-2 via phosphorylation of p42/44 MAPK. Glutathione precursor molecules reversed enhanced ROS levels and Cox-2 expression. Functional activity of Cox-2 expression was tested by measurement of PGE(2) and PGF(2α). The synthetic compound 9,10-dihydro-15d-PGJ(2) lacking the α,β-unsaturated carbonyl group in the cyclopentenone ring did not exhibit the cellular responses observed with 15d-PGJ(2). We conclude that the electrophilic carbon atom of 15d-PGJ(2) is responsible for alterations in intracellular redox status and Cox-2 expression.