Developmental Changes in Distribution of Acidic Fibroblast Growth Factor in Rat Brain Evaluated by a Sensitive Two-Site Enzyme Immunoassay

We developed a sensitive two-site enzyme immunoassay (EIA) system for acidic fibroblast growth factor (aFGF), using a polyclonal antibody raised in rats. This assay is based on the sandwiching of the antigen between anti-aFGF antibody immunoglobulin G (IgG) coated on plates and biotinylated anti-aFGF antibody IgG; the detection of biotinylated IgG was performed by enzyme reaction of streptavidin-conjugated beta-D-galactosidase (beta-D-galactoside hydrolase; EC 3.2.1.23). Our system was specific for aFGF, because basic fibroblast growth factor, which shares a 55% homology of amino acid sequence with aFGF, hardly cross-reacted at all. The sensitivity of this system (0.2 ng/ml) enabled us to quantify endogenous immunoreactive aFGF in the CNS. Using this two-site EIA system, we examined the levels of aFGF in various regions of rat brain and their developmental changes. At the early stage of neonatal development, i.e., 2 days after birth, all brain regions registered low aFGF levels (less than 10 ng/g tissue). However, at the young adult stage (21- to 49-day-old animals), an extremely high level of aFGF (75-90 ng/g tissue) was found in the ponsmedulla; relatively high levels (30-40 ng/g tissue) were found in the diencephalon and mesencephalon; and comparatively low aFGF levels (5-15 ng/g tissue) were found in various other brain regions such as the frontal cortex, piriform cortex, hippocampus, olfactory bulb, cerebellum, and striatum. This marked change in the regional distribution of aFGF in the rat brain during postnatal development from 2 to 21 days after birth suggests that this factor plays a significant role in the brain during this period.     

衰老大鼠的某些脑区组织中游离氨基酸水平的改变

使用D 半乳糖建立衰老大鼠模型组与同龄、同饲的正常对照组大鼠的某些脑区游离氨基酸 (FAA)水平的比较发现 :( 1 )衰老模型组的海马、纹状体以及皮层等脑区中谷氨酸 (Glu)、天门冬氨酸 (Asp)水平明显降低 ;( 2 )γ 氨基丁酸 (GABA)水平在衰老模型组大鼠的海马 ,纹状体以及小脑等脑区中明显升高 ;( 3)衰老模型组的皮层、小脑、海马、纹状体等脑区的牛磺酸 (Tau)水平明显下降。以此探讨动物衰老与脑区游离氨基酸水平的关系     

Clozapine but not haloperidol suppresses the changes in the levels of neuropeptides in MK-801-treated rat brain regions

Noncompetitive NMDA receptor antagonist (+)MK-801 is known to induce neurotoxicity and schizophrenia-like symptomatology where atypical neuroleptic clozapine is effective in contrast to typical neuroleptic, haloperidol. Although neuropeptides are implicated in memory and cognition, their roles in schizophrenia are not well understood. In the present study, we therefore examined the possible roles of neuropeptides, cholecystokinin (CCK) and somatostatin (SS) in the posterior cingulate/retrosplenial cortices (PC/RSC), frontal cortex, and hippocampus of a MK-801-induced schizophrenia-like model rat brain. This study further investigated the pretreated effect of atypical versus typical neuroleptics on the peptidergic system. SS mRNA and peptide levels significantly decreased in the PC/RSC and hippocampus but not in the frontal cortex 3 days after 0.5 mg/kg MK-801 treatment whereas CCK mRNA and peptide levels significantly decreased in all of the brain regions examined. Pretreatment with clozapine but not haloperidol completely recovered the changes in both mRNA and peptide levels of SS and CCK in those brain regions. These data suggest that peptidergic system in the brain presumably plays an important role in the control of negative schizophrenia.     

Differential effects of chronic treatment with haloperidol and clozapine on the level of preprosomatostatin mRNA in the striatum,nucleus accumbens,and frontal cortex of the rat

1. The goal of this work was to determine the effects of typical and atypical neuroleptics on the level of preprosomatostatin messenger RNA (mRNA) in regions of the rat brain innervated by dopaminergic neurons. 2. Quantitative in situ hybridization histochemistry was used to measure the levels of mRNA encoding preprosomatostatin in neurons of the striatum, the nucleus accumbens, and the medial and lateral agranular areas of the frontal cortex in adult rats treated with either haloperidol or clozapine. 3. In untreated animals, the density of neurons containing preprosomatostatin mRNA was higher in the nucleus accumbens than in the striatum and frontal cortex. The intensity of labeling per neuron, however, was higher in the striatum than in the two other areas examined, suggesting that the expression of preprosomatostatin mRNA is differentially regulated in these brain regions. Chronic administration of haloperidol (1 mg/kg for 28 days) induced a significant decrease in the labeling for preprosomatostatin mRNA in neurons of the nucleus accumbens, frontal cortex, and medial but not lateral striatum. Treatment with clozapine (20 mg/kg for 28 days) increased the levels of preprosomatostatin mRNA in the nucleus accumbens but not in the striatum or the frontal cortex. 4. These results support a role for dopamine in the regulation of central somatostatinergic neurons. The differences in the effects of haloperidol, a neuroleptic which induces extrapyramidal side effects, and clozapine, which does not, suggest that somatostatinergic neurons may play an important role in the regulation of motor behavior.     

Regional Distribution of the GABAA/Benzodiazepine Receptor (α Subunit) mRNA in Rat Brain

A human cDNA clone containing the 5' coding region of the GABAA/benzodiazepine receptor alpha subunit was used to quantify and visualize receptor mRNA in various regions of the rat brain. Using a [32P]CTP-labelled antisense RNA probe (860 bases) prepared from the alpha subunit cDNA, multiple mRNA species were detected in Northern blots using total and poly A rat brain RNA. In all brain regions, mRNAs of 4.4 and 4.8 kb were observed, and an additional mRNA of 3.0 kb was detected in the cerebellum and hippocampus. The level of GABAA/benzodiazepine receptor mRNA was highest in the cerebellum followed by the thalamus = frontal cortex = hippocampus = parietal cortex = hypothalamus much greater than pons = striatum = medulla. In situ hybridization revealed high levels of alpha subunit mRNA in cerebellar gray matter, olfactory bulb, thalamus, hippocampus/dentate gyrus, and the arcuate nucleus of the hypothalamus. These data suggest the presence of multiple GABAA/benzodiazepine receptor alpha subunit mRNAs in rat brain and demonstrate the feasibility of studying the expression of genes encoding the GABAA/benzodiazepine receptor after pharmacological and/or environmental manipulation.     

Assay of 2-Hydroxyputrescine in Various Regions of Rat Brain by Gas Chromatography-Mass Spectrometry

2-Hydroxyputrescine in seven regions of single rat brains was measured with a sensitive, specific assay by gas chromatography-mass spectrometry. The regions were the cerebral cortex, cerebellum, medulla oblongata, hypothalamus, striatum, hippocampus, and midbrain. The level of 2-hydroxyputrescine was very high in the cerebral cortex and cerebellum, high in the medulla oblongata, hypothalamus, and hippocampus, and low in the striatum and midbrain. The level of 2-hydroxyputrescine in the cerebellum was significantly higher than in the striatum and midbrain.     

RNA polymerase I and II activities in different brain regions of young, adult, and old rats

The activities of RNA polymerase I and II were assayed in nuclei isolated from different regions (cerebral cortex, cerebellum, hypothalamus, hippocampus, corpus striatum and pituitary) of brains from young (10 days), adult (6 months), and old (2 years) rats. The RNA polymerases I and II activities generally increased during maturation, i.e., from 10 days to 6 months of postnatal age and then showed a decrease from 6 months to 2 years of age in all the regions except in cerebral cortex where the RNA polymerase II activity was highest at 10 days but showed a gradual decrease through the lifespan up to 2 years.     

α-Linolenic Acid Dietary Deficiency Alters Age-Related Changes of Dopaminergic and Serotoninergic Neurotransmission in the Rat Frontal Cortex

Abstract: The effects of α-linolenic acid diet deficiency on rat dopaminergic and serotoninergic neurotransmission systems were investigated in the frontal cortex, striatum, and cerebellum of male rats 2, 6, 12, and 24 months of age. The diet deficiency induced a severe decrease in the 22:6n-3 fatty acid levels in all regions and a compensatory increase in n-6 fatty acid levels. A recovery in the levels of 22:6n-3 was observed in deficient rats between 2 and 12 months of age; however, this recovery was lower in frontal cortex than in striatum and cerebellum. In the striatum and the cerebellum, dopaminergic and serotoninergic receptor densities and endogenous dopamine and serotonin levels were affected by aging regardless of the diet. In contrast, a 40–75% lower level of endogenous dopamine in the frontal cortex occurred in deficient rats according to age. The deficiency also induced an 18–46% increase in serotonin 5-HT₂ receptor density in the frontal cortex during aging, without variation in endogenous serotonin level, and a 10% reduction in density of dopaminergic D₂ receptors. Monoamine oxidase-A and -B activities showed specific age-related variations but regardless of the diet. Our results suggest that a chronically α-linolenic-deficient diet specifically affects the monoaminergic systems in the frontal cortex.     

Decreased levels of free d-aspartic acid in the forebrain of serine racemase (Srr) knock-out mice

d-Serine, an endogenous co-agonist of the N-methyl-d-aspartate (NMDA) receptor is synthesized from l-serine by serine racemase (SRR). A previous study of Srr knockout (Srr-KO) mice showed that levels of d-serine in forebrain regions, such as frontal cortex, hippocampus, and striatum, but not cerebellum, of mutant mice are significantly lower than those of wild-type (WT) mice, suggesting that SRR is responsible for d-serine production in the forebrain. In this study, we attempted to determine whether SRR affects the level of other amino acids in brain tissue. We found that tissue levels of d-aspartic acid in the forebrains (frontal cortex, hippocampus and striatum) of Srr-KO mice were significantly lower than in WT mice, whereas levels of d-aspartic acid in the cerebellum were not altered. Levels of d-alanine, l-alanine, l-aspartic acid, taurine, asparagine, arginine, threonine, γ-amino butyric acid (GABA) and methionine, remained the same in frontal cortex, hippocampus, striatum and cerebellum of WT and mutant mice. Furthermore, no differences in d-aspartate oxidase (DDO) activity were detected in the forebrains of WT and Srr-KO mice. These results suggest that SRR and/or d-serine may be involved in the production of d-aspartic acid in mouse forebrains, although further detailed studies will be necessary to confirm this finding.     

Characterization of NMDA Receptor Subunit-Specific Antibodies: Distribution of NR2A and NR2B Receptor Subunits in Rat Brain and Ontogenic Profile in the Cerebellum

Abstract: Selective antisera for NMDA receptor subunits NR2A and NR2B have been developed. Each antiserum identifies a single band on an immunoblot at ∼175 kDa that appears to be the appropriate subunit of the NMDA receptor. Using these antisera the relative densities of the subunits in eight areas of adult rat brain have been determined. The NR2A subunit was found to be at its highest level in hippocampus and cerebral cortex, to be at intermediate levels in striatum, olfactory tubercle, midbrain, olfactory bulb, and cerebellum, and to be at lowest levels in the pons-medulla. The NR2B subunit was found to be expressed at its highest levels in the olfactory tubercle, hippocampus, olfactory bulb, and cerebral cortex. Intermediate levels were expressed in striatum and midbrain, and low levels were detected in the pons-medulla. No signal for NR2B was found in the cerebellum. These regional distributions were compared with that for [³H]MK-801 binding sites. It was found that although the distribution of the NR2A subunit corresponds well with radioligand binding, the distribution of the NR2B subunit does not. The ontogenic profiles of NR2A and NR2B subunits in the rat cerebellum were also determined. Just following birth [postnatal day (P) 2] NR2A subunits are undetectable, whereas NR2B subunits are expressed at amounts easily measurable. Beginning at about P12 the levels of NR2A rise rapidly to reach adult levels by P22. At the same time (P12), levels of NR2B protein begin to decline rapidly to reach undetectable levels by 22 days after birth. The results suggest that NMDA receptors are likely to be composed of different subunits in different parts of the brain and that even in the same tissue the receptors are likely to show different properties at various times during development due to alterations in the subunit composition of the receptor.     

Corticosterone administration and lipid metabolism in brain regions during development.

Effect of corticosterone on lipid contents of different brain regions and the effect of age on the sensitivity of these regions to corticosterone have been studied. Corticosterone administration (40 mg/kg body wt, sc) to 17-day-old rat for 3 days led to significant decrease in phospholipid content of cerebellum and increase in cholesterol contents of hippocampus and striatum. However, there was no effect on cerebral cortex and brain stem lipids. This alteration in lipids was associated with decrease in [U-14C] glucose incorporation into cholesterol and phospholipids, decrease in plasma beta-hydroxy butyrate levels and increase in beta-hydroxy butyrate dehydrogenase activity in hippocampus and striatum, thereby suggesting that suppression of glucose utilization by corticosterone was compensated by higher utilization of ketone bodies for lipid synthesis in these regions. The sensitivity to corticosterone appears to be age-specific as, at 20-day, cerebellum, hippocampus and striatum were susceptible, at 10-day only hippocampus and at 40- and 90-day none of these regions responded to the treatment.     

Increase in brain 125I-cholecystokinin (CCK) receptor binding following chronic haloperidol treatment, intracisternal 6-hydroxydopamine or ventral tegmental lesions

Specific 125I-CCK receptor binding was significantly increased in brain tissue taken from guinea pig or mouse following chronic (2-3 week) daily administration of haloperidol (2-3 mg/kg/day). Scatchard analysis indicated the increase in CCK binding was due to an increased receptor number (B max) with no change in affinity (Kd). In guinea pigs, the increased CCK binding was observed in the mesolimbic regions and frontal cortex, but not in striatum, hippocampus nor posterior cortex. In mice, however, the increases occurred in both pooled cerebral cortical-hippocampal tissue, and in the remainder of the brain. Enhanced CCK receptor binding was also observed in membranes prepared from whole brain of mice one month following intracisternal injection of 6-hydroxydopamine. Additionally, an increase in CCK binding was observed in mesolimbic regions and frontal cortex, but not striatum or hippocampus, of guinea pigs 3 weeks after an unilateral radiofrequency lesions of the ipsilateral ventral tegmentum. The present studies demonstrate that three different procedures which reduce dopaminergic function in the brain enhance CCK receptor binding. The data provide further support for a functional interrelationship between dopaminergic systems and CCK in some brain regions and raise the possibility that CCK may play a role in the antipsychotic action of neuroleptics.     

Expression of mRNAs Encoding Subunits of the NMDA Receptor in Developing Rat Brain

Abstract: Developmental changes in the levels of N -methyl- d -aspartate (NMDA) receptor subunit mRNAs were identified in rat brain using solution hybridization/RNase protection assays. Pronounced increases in the levels of mRNAs encoding NR1 and NR2A were seen in the cerebral cortex, hippocampus, and cerebellum between postnatal days 7 and 20. In cortex and hippocampus, the expression of NR2B mRNA was high in neonatal rats and remained relatively constant over time. In contrast, in cerebellum, the level of NR2B mRNA was highest at postnatal day 1 and declined to undetectable levels by postnatal day 28. NR2C mRNA was not detectable in cerebellum before postnatal day 11, after which it increased to reach adult levels by postnatal day 28. In cortex, the expression of NR2A and NR2B mRNAs corresponds to the previously described developmental profile of NMDA receptor subtypes having low and high affinities for ifenprodil, i.e., a delayed expression of NR2A correlating with the late expression of low-affinity ifenprodil sites. In cortex and hippocampus, the predominant splice variants of NR1 were those without the 5' insert and with or without both 3' inserts. In cerebellum, however, the major NR1 variants were those containing the 5' insert and lacking both 3' inserts. The results show that the expression of NR1 splice variants and NR2 subunits is differentially regulated in various brain regions during development. Changes in subunit expression are likely to underlie some of the changes in the functional and pharmacological properties of NMDA receptors that occur during development.     

The effects of acute ethanol exposure and ageing on rat brain glutathione metabolism

Abstract

Binge alcohol consumption in adolescents is increasing, and it has been proposed that immature brain deals poorly with oxidative stress. The aim of our work was to study the effect of an acute dose of ethanol on glutathione (GSH) metabolism in frontal cortex, hippocampus and striatum of juvenile and adult rats. We have observed no change in levels of glutathione produced by acute alcohol in the three brain areas studied of juvenile and adult rats. Only in the frontal cortex the ratio of GSH/GSSG was increased in the ethanol-treated adult rats. GSH levels in the hippocampus and striatum were significantly higher in adult animals compared to young ones. Higher glutathione peroxidase (GPx) activity in adult rats was observed in frontal cortex and in striatum. Our data show an increased GSH concentration and GPx activity in different cerebral regions of the adult rat, compared to the young ones, suggesting that age-related variations of total antioxidant defences in brain may predispose young brain structures to ethanol-induced, oxidative stress-mediated tissue damage.     

The effects of acute ethanol exposure and ageing on rat brain glutathione metabolism

Binge alcohol consumption in adolescents is increasing, and it has been proposed that immature brain deals poorly with oxidative stress. The aim of our work was to study the effect of an acute dose of ethanol on glutathione (GSH) metabolism in frontal cortex, hippocampus and striatum of juvenile and adult rats. We have observed no change in levels of glutathione produced by acute alcohol in the three brain areas studied of juvenile and adult rats. Only in the frontal cortex the ratio of GSH/GSSG was increased in the ethanol-treated adult rats. GSH levels in the hippocampus and striatum were significantly higher in adult animals compared to young ones. Higher glutathione peroxidase (GPx) activity in adult rats was observed in frontal cortex and in striatum. Our data show an increased GSH concentration and GPx activity in different cerebral regions of the adult rat, compared to the young ones, suggesting that age-related variations of total antioxidant defences in brain may predispose young brain structures to ethanol-induced, oxidative stress-mediated tissue damage.