Developmental Expression and Functional Characterization of the 4C5 Antigen in the Postnatal Cerebellar Cortex

Abstract: The monoclonal antibody 4C5 recognizes a neuron-specific surface antigen (4C5 antigen) in the CNS and PNS of the rat. In the present study we investigated the expression of 4C5 antigen in the developing cerebellum of the rat and the functional role of this molecule during cerebellar morphogenesis. Immunoblotting and immunohistochemistry in sections of cerebellar cortex revealed an age-dependent decrease in the expression of the 4C5 antigen. In cerebellar primary cell cultures, 4C5 immunoreactivity was detected both on granule and on Purkinje neurons. Granule cell migration was inhibited in cerebellar explants derived from 8-day-old rats and cultured for 2 days in the presence of antibodies against the 4C5 antigen. Electron microscope immunocytochemistry revealed that in 8-day-old rat cerebellum, 4C5 immunoreactivity was localized on the cell bodies of granule neurons in the external and internal granular layers and on parallel fibers in the developing molecular layer as well as at contact sites between these cellular elements. It was not detected on Bergmann glia. These results suggest strongly that the 4C5 antigen is involved in granule cell migration during cerebellar development, possibly via neuron-neuron interactions.     

Circadian Oscillations of Molecular Clock Components in the Cerebellar Cortex of the Rat

The central circadian clock of the mammalian brain resides in the suprachiasmatic nucleus (SCN) of the hypothalamus. At the molecular level, the circadian clockwork of the SCN constitutes a self-sustained autoregulatory feedback mechanism reflected by the rhythmic expression of clock genes. However, recent studies have shown the presence of extrahypothalamic oscillators in other areas of the brain including the cerebellum. In the present study, the authors unravel the cerebellar molecular clock by analyzing clock gene expression in the cerebellum of the rat by use of radiochemical in situ hybridization and quantitative real-time polymerase chain reaction. The authors here show that all core clock genes, i.e., Per1, Per2, Per3, Cry1, Cry2, Clock, Arntl, and Nr1d1, as well as the clock-controlled gene Dbp, are expressed in the granular and Purkinje cell layers of the cerebellar cortex. Among these genes, Per1, Per2, Per3, Cry1, Arntl, Nr1d1, and Dbp were found to exhibit circadian rhythms in a sequential temporal manner similar to that of the SCN, but with several hours of delay. The results of lesion studies indicate that the molecular oscillatory profiles of Per1, Per2, and Cry1 in the cerebellum are controlled, though possibly indirectly, by the central clock of the SCN. These data support the presence of a circadian oscillator in the cortex of the rat cerebellum. (Author correspondence: mrath@sund.ku.dk)     

γ-Aminobutyric Acid Affects the Developmental Expression of Neuron-Associated Proteins in Cerebellar Granule Cell Cultures

The effect of gamma-aminobutyric acid (GABA) on the expression of the neuron-associated D2 and neuron-specific enolase (NSE) was studied during development in culture of cerebellar granule cells. It was found that the presence of GABA during culture development increased the overall protein content. D2 content was also increased but not above the general increase in protein whereas NSE increased above the general level of protein. The presence of GABA in the growth medium also appeared to accelerate the changes in molecular forms of D2 and NSE seen during neuronal development. This suggests that GABA promotes or accelerates the general maturation of neurons, as these two neuron-associated proteins otherwise differ from each other with respect to their subcellular localization and their physiological and biochemical properties.     

Age and Sex Dependent Alteration in Presenilin Expression in Mouse Cerebral Cortex

(1) Presenilin (PS) expression is regulated by several cellular and extracellular factors which change with age and sex. Both age and sex are key risk factors for Alzheimer’s disease (AD), which is linked to mutations in PS genes. (2) We have analyzed the effect of age and sex on PS expression by northern hybridization and western blot analysis using the cerebral cortex of adult (24 ± 2 weeks) and old (65 ± 5 weeks) mice. (3) Our results demonstrate that PS1 was downregulated and PS 2 was upregulated in old mice of both sexes. The level of PS 1 was relatively higher and that of PS 2 was lower in female than male mice of same age group. Taken together, these findings show age and sex dependent alteration in PS expression, which in turn may influence the signal transduction pathways and consequently brain functions.     

猫小脑皮质Glu/GABA表达的老年性变化

衰老导致小脑的生理功能下降,但其神经机制仍然不清楚。为此,利用免疫组织化学方法标记猫小脑皮质内谷氨酸(Glutamate,Glu)和γ-氨基丁酸(γ-Aminobutiric acid,GABA)免疫反应阳性(Glu-IR和GABA-IR)结构,探讨青年猫和老年猫小脑皮质Glu/GABA表达的老年性变化及其可能影响。并利用Image-Pro Express图像分析软件对小脑皮质各层Glu和GABA免疫反应阳性细胞密度及其灰度值进行测量。结果显示:与青年猫相比,老年猫小脑皮质内的Glu免疫反应阳性浦肯野细胞密度、颗粒层Glu免疫反应阳性细胞密度及其两者的免疫阳性反应灰度值均显著下降(P<0.01)(免疫反应强度与平均灰度值成反比);老年猫分子层、浦肯野细胞层GABA免疫反应阳性神经元密度及其免疫反应强度均显著下降(P<0.01);颗粒层GABA免疫反应阳性神经元密度无显著变化(P>0.05),但神经元免疫反应强度显著减弱(P<0.01)。研究结果提示,衰老过程中猫小脑皮质出现神经元Glu的表达增强、GABA的表达减少等,可能是小脑神经元丢失和精确调控能力下降等的重要原因之一。     

Ascorbic Acid Attenuates Lead-Induced Alterations in the Synapses in the Developing Rat Cerebellum

Biological Trace Element Research - We evaluated the effect of lead (Pb) and ascorbic acid treatment of pregnant female rats on cerebellar development in pups. Pb was administered in drinking water...     

Developmental Expression of HNK-1-Reactive Antigens in the Rat Cerebellum and Localization of Sulfoglucuronyl Glycolipids in Molecular Layer and Deep Cerebellar Nuclei

Monoclonal antibody HNK-1-reactive carbohydrate epitope is expressed on proteins, proteoglycans, and sulfoglucuronyl glycolipids (SGGLs). The developmental expression of these HNK-1-reactive antigens was studied in rat cerebellum. The expression of sulfoglucuronyl lacto-N-neotetraosylceramide (SGGL-1) was biphasic with an initial maximum at postnatal day one (PD 1), followed by a second rise in the level at PD 20. The level of sulfoglucuronyl lacto-N-norhexaosyl ceramide (SGGL-2) in cerebellum was low until PD 15 and then increased to a plateau at PD 20. The levels of SGGLs increased during postnatal development of the cerebellum, contrary to their diminishing expression in the cerebral cortex. The expression of HNK-1-reactive glycoproteins decreased with development of the rat cerebellum from PD 1. Several HNK-1-reactive glycoproteins with apparent molecular masses between 150 and 325 kDa were visualized between PD 1 and PD 10. However, beyond PD 10, only two HNK-1-reactive bands at 160 and 180 kDa remained. The latter appeared to be neural cell adhesion molecule, N-CAM-180. A diffuse HNK-1-reactive band seen at the top of polyacrylamide electrophoretic gels was due mostly to proteoglycans. This band increased in its reactivity to HNK-1 between PD 15 and PD 25 and then decreased in the adult cerebellum. The lipid antigens were shown by two complementary methodologies to be localized primarily in the molecular layer and deep cerebellar nuclei as opposed to the granular layer and white matter. A fixation procedure which eliminates HNK-1-reactive epitope on glycoproteins and proteoglycans, but does not affect glycolipids, allowed selective immunoreactivity in the molecular layer and deep cerebellar nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental Expression of HNK-1-Reactive Antigens in Rat Cerebral Cortex and Molecular Heterogeneity of Sulfoglucuronylneolactotetraosylceramide in CNS Versus PNS

Monoclonal antibody HNK-1 reacts with a carbohydrate epitope present in proteins, proteoglycans, and sulfoglucuronylglycolipids (SGGLs). On high-performance TLC plates, SGGLs of the CNS from several species migrated consistently slower than those from the PNS, a result indicating possible differences in the structures. The structural characteristics of the major SGGL, sulfoglucuronylneolactotetraosylceramide (SGGL-1), from CNS was compared with those of SGGL-1 from PNS. Although the composition, sequence, and linkages of the carbohydrate moiety of the SGGL-1 species were identical, SGGL-1 from CNS contained mainly short-chain fatty acids, 16:0, 18:0, and 18:1, amounting to 85% of the total fatty acids, whereas SGGL-1 from PNS contained large proportions (59%) of long-chain fatty acids (greater than 18:0). These differences in the fatty acid composition accounted for the different migration pattern observed. The developmental expression of SGGLs and HNK-1-reactive proteins was studied in rat cerebral cortex between embryonic day (ED) 15 to adulthood. SGGLs in the rat cortex were maximally expressed around ED 19 and almost completely disappeared by postnatal day (PD) 20. This expression was contrary to their increasing expression in the cerebellum and sciatic nerve with postnatal development. Six to eight protein bands with a molecular mass of greater than 160 kDa were HNK-1 reactive in the rat cerebral cortex at different ages. The major HNK-1 reactivity to the 160-kDa protein band seen in ED 19 to PD 10 cortex decreased and completely disappeared from the adult cortex, whereas several other proteins remained HNK-1 reactive even in the adult. Western blot analyses of the neural cell adhesion molecules (N-CAMs) during development of the rat cortex with a polyclonal anti-N-CAM antibody showed that the major HNK-1-reactive protein bands were not N-CAMs. Between PD 1 and 10, 190-200-kDa N-CAM was the major N-CAM, and between PD 15 to adulthood, 180-kDa N-CAM was the only N-CAM present in the rat cortex.     

Role of Ascorbic Acid in Scavenging Free Radicals and Lead Toxicity from Biosystems

Free radicals are reactive species that are responsible for damaging normal cells and creating diseases in humans. Antioxidants from natural resources or as supplements can scavenge these radicals. A MedLine search indicates that vitamin C is the most investigated antioxidant responsible for the elimination of free radicals. Its chelating property for the removal of neurotoxic lead, which creates oxidative stress in the human biosystem, was investigated and results indicate its great potential as a lead-detoxifying agent.     

An analysis of the cross-reactivity of autoantibodies to GAD65 and GAD67 in diabetes

Background

Autoantibodies to GAD65 (anti-GAD65) are present in the sera of 70–80% of patients with type 1 diabetes (T1D), but antibodies to the structurally similar 67 kDa isoform GAD67 are rare. Antibodies to GAD67 may represent a cross-reactive population of anti-GAD65, but this has not been formally tested.

Methodology/Principal Findings

In this study we examined the frequency, levels and affinity of anti-GAD67 in diabetes sera that contained anti-GAD65, and compared the specificity of GAD65 and GAD67 reactivity. Anti-GAD65 and anti-GAD67 were measured by radioimmunoprecipitation (RIP) using ¹²⁵I labeled recombinant GAD65 and GAD67. For each antibody population, the specificity of the binding was measured by incubation with 100-fold excess of unlabeled GAD in homologous and heterologous inhibition assays, and the affinity of binding with GAD65 and GAD67 was measured in selected sera. Sera were also tested for reactivity to GAD65 and GAD67 by immunoblotting. Of the 85 sera that contained antibodies to GAD65, 28 contained anti–GAD67 measured by RIP. Inhibition with unlabeled GAD65 substantially or completely reduced antibody reactivity with both ¹²⁵I GAD65 and with ¹²⁵I GAD67. In contrast, unlabeled GAD67 reduced autoantibody reactivity with ¹²⁵I GAD67 but not with ¹²⁵I GAD65. Both populations of antibodies were of high affinity (>10¹⁰ l/mol).

Conclusions

Our findings show that autoantibodies to GAD67 represent a minor population of anti-GAD65 that are reactive with a cross-reactive epitope found also on GAD67. Experimental results confirm that GAD65 is the major autoantigen in T1D, and that GAD67 per se has very low immunogenicity. We discuss our findings in light of the known similarities between the structures of the GAD isoforms, in particular the location of a minor cross-reactive epitope that could be induced by epitope spreading.     

多巴胺受体1在皖西白鹅小脑皮质发育中的表达

采用普通染色及免疫组化SABC染色法研究皖西白鹅小脑皮质的发育和多巴胺受体1(DRD1)阳性细胞在其发育中的表达.结果表明,小脑皮质在胚龄13 d(E13)由外向内分为外颗粒层(EGL)、浦肯野细胞层(PCL)和内颗粒层(IGL),E19由外向内分为EGL、分子层(ML)、PCL和IGL.随发育天数的增加,EGL的厚度和细胞层次呈先升后降的变化趋势,细胞密度逐渐下降;ML厚度逐渐增大,在E24到E28时增值最大;浦肯野细胞(PC)在E13、E19、E24和E28时随胚龄增大逐渐增大,在E28后趋于稳定,细胞密度随着发育天数的增加逐渐下降,在小脑皮质发育中还发现有一部分PC呈多层排列,且细胞层次逐渐变少;IGL厚度呈先升后降的变化趋势,细胞密度呈上升趋势.外颗粒层和内颗粒层在E13、E19、E24和E28时有DRD1阳性细胞表达,分子层在E24、E28、日龄7 d(P7)和15d(P15)有阳性细胞表达,PC在所检测的6个时段均有阳性表达.研究表明,小脑皮质的发育主要与细胞增殖、迁移和凋亡有关,外颗粒层的逐渐消失是以细胞迁移和凋亡为主,多层PC逐渐退化成单层是与细胞凋亡和正常突触联系的建立有关;DRD1在皖西白鹅小脑皮质发育中对外颗粒层细胞和PC起着重要作用.     

"青春期"大鼠前额皮质内BDNF与trkB的表达

目的:探讨"青春期"大鼠前额皮质内BDNF与trkB的表达。方法:出生后35天大鼠作为"青春期"大鼠,出生后15天、75天大鼠分别作为幼年期与成年期对照,每组大鼠各6只,用ABC免疫组织化学方法与图像分析相结合技术检测前额皮质内BDNF与trkB免疫反应的强度和免疫阳性产物平均光密度值的变化。结果:各时间点大鼠前额皮质内均可见BDNF与trkB免疫阳性产物。35天组BDNF与trkB免疫反应最强,阳性产物平均光密度值最高,与其它两组相比差异有显著性(p<0.05)。结论:"青春期"大鼠前额皮质内BDNF与trkB高表达,提示在此时期内前额皮质对BDNF的需求最多。     

Glycosidases and Cerebellar Ontogenesis in the Rat

Abstract: Five glysosidases (α- and β-D-mannosidase, β-D-galactosidase, β- N -acetyl-glucosaminidase, and α-L-fucosidase) were studied during the postnatal development of the rat cerebellum. Each glycosidase has a particular developmental pattern. Transient decreases in the specific activities of β-mannosidase, β-D-galactosidase, and β-D- N -acetyl-glucosaminidase could be correlated with the phase of massive cell multiplication. A possible more specific role for α-D-mannosidase is discussed.     

The Similar Expression Pattern of MHC Class I Molecules in Human and Mouse Cerebellar Cortex

The major histocompatibility complex (MHC) class I molecules are considered to be important in the immune system. However, the results reported in the past decade indicate that they also play important roles in the central nervous system. Here we examined the expression of MHC I and β2-microglobulin (β2m) in human and mouse cerebellar cortex. The results show that MHC I molecules are expressed both in human and mouse cerebellar cortex during brain development. The expression of H-2K^b/D^b is gradually increased with the development of mouse cerebellar cortex, but finally decreased to a very low level. Similarly, the expression of HLA-B/C genes is increased in developing human cerebellar cortex, but decreased after birth. The spatial and temporal expression of β2m overlaps mostly with that of HLA-B/C molecules, and they are co-expressed in Purkinje cells. Our findings provide a fundamental basis to reveal the functions of neuronal MHC class I molecules in the development of human cerebellum.     

Developmental and Regional Changes of mRNA for a Cerebellar Protein (Spot 35) in the Rat Brain

Spot 35 protein is a cerebellar Ca-binding protein. Because Southern blot analysis showed evidence for the nucleotide sequence of spot 35 protein cDNA in the rat genome, we applied cDNA to quantitate the mRNA of rat spot 35 protein. The size of this mRNA was about 1,900 nucleotides in length, and that of mRNA from bovine cerebellum was larger. We also examined the developmental changes and regional distribution of spot 35 protein mRNA in rat brains by dot-blot analysis using cDNA as a probe. During postnatal days 1-20, a rapid increase of mRNA levels was observed. Further, the level of mRNA for spot 35 protein was found in the cerebellum and was negligible in the cerebral cortex, striatum, and hippocampus.     

Effects of Lead and/or Cadmium on the Oxidative Damage of Rat Kidney Cortex Mitochondria

Lead acetate (300 mg/L) and/or cadmium chloride (50 mg/L) were administered as drinking water to Sprague–Dawley rats for 8 weeks to investigate the possible combined effects of these metals on the damage in renal cortex mitochondria. Increased malonaldehyde levels due to exposure to these metals were detected by colorimetric method, which demonstrated the lipid peroxidation in the renal cortex. Ultrastructural observations and real-time quantitative PCR analyses were performed on kidney cortex pieces to identify the mitochondrial damage and quantify the relative expression levels of cytochrome oxidase subunits (COX-I/II/III), respectively. The most striking ultrastructural modifications involved distortion of mitochondrial cristae and an unusual arrangement, which were more evident when the mixture was ingested. There were significant differences in the expression levels of COX-I, II, and III between the control group and the exposed groups, whereas those in the (lead+cadmium) group were all significantly lower than that in the lead or cadmium group. In conclusion, there was an obvious synergistic oxidative damage effect of lead combined with cadmium on rat kidney cortex mitochondria, which increased defects in mitochondrial oxidative metabolism.     

Reciprocal Regulation of Ornithine Decarboxylase and Choline Kinase Activities by Their Respective Reaction Products in the Developing Rat Cerebellar Cortex

Changes in the activity of choline kinase were measured in the cerebellum during development. Early transient increase was found in the enzyme activity just prior to and during birth. This period of increase did not coincide with the periods of transient elevation in ornithine decarboxylase and choline acetyltransferase previously observed in the developing cerebellum. The effects of the naturally occurring polyamines (putrescine, spermidine, and spermine) on choline kinase and choline acetyltransferase activities, and of phosphorylcholine (the product of the reaction catalyzed by choline kinase) on ornithine decarboxylase and choline acetyltransferase activities, were also examined. Choline acetyltransferase activity was not influenced by either polyamines or phosphorylcholine. However, choline kinase activity from 7-day-old, but not from adult, cerebellum was increased 25% in the presence of 4 mM spermine. In contrast, low spermidine concentrations (less than 2 mM) inhibited choline kinase activity selectively in 7-day-old cerebellum. Ornithine decarboxylase activity from 7-day-old cerebellum was inhibited in a concentration-dependent manner by phosphorylcholine. The present data together with other previous reports suggest that: (a) polyamines may play a role in choline utilization during development via their regulation of choline kinase activity, on the one hand, and of acetylcholinesterase activity on the other; and (b) during development, a reciprocal regulation of choline kinase and ornithine decarboxylase activities by their respective reaction products may exist, whereby choline kinase activity is regulated in a complex manner by polyamines and, in turn, ornithine decarboxylase is inhibited by phosphorylcholine.     

Olfactory Enrichment Influences Adult Neurogenesis Modulating GAD67 and Plasticity-Related Molecules Expression in Newborn Cells of the Olfactory Bulb

The olfactory bulb (OB) is a highly plastic region of the adult mammalian brain characterized by continuous integration of inhibitory interneurons of the granule (GC) and periglomerular cell (PGC) types. Adult-generated OB interneurons are selected to survive in an experience-dependent way but the mechanisms that mediate the effects of experience on OB neurogenesis are unknown. Here we focus on the new-generated PGC population which is composed by multiple subtypes. Using paradigms of olfactory enrichment and/or deprivation combined to BrdU injections and quantitative confocal immunohistochemical analyses, we studied the effects of olfactory experience on adult-generated PGCs at different survival time and compared PGC to GC modulation. We show that olfactory enrichment similarly influences PGCs and GCs, increasing survival of newborn cells and transiently modulating GAD67 and plasticity-related molecules expression. However, PGC maturation appears to be delayed compared to GCs, reflecting a different temporal dynamic of adult generated olfactory interneuron integration. Moreover, olfactory enrichment or deprivation do not selectively modulate the survival of specific PGC phenotypes, supporting the idea that the integration rate of distinct PGC subtypes is independent from olfactory experience.     

Aging Diminishes Serotonin-Stimulated Arachdonic Acid Uptake and Cholinergic Receptor-Activated Arachidonic Acid Release in Rat Brain Cortex Membrane

Abstract: Synaptoneurosomal and synaptosomal fractions from the brain cortex of adult (4-month-old) and aged (27-month-old) rats were used for studies on the uptake and subsequent release of [¹⁴C]arachidonic acid ([¹⁴C]AA) from brain lipids. The incorporation of AA and the pattern of its uptake into lipids of the aged brain cortex synapto-neurosomes and synaptosomes were not significantly different when compared with those in the adult brain cortex fractions. Serotonin (5-HT), at 10 μM to 1 μM in the presence of pargyline and the agonist of the 5-HT_1A receptor, buspirone, stimulated AA uptake into membrane lipids, mainly into phosphatidylinositol, by about 40% exclusively in adult brain synaptoneurosomes. Aging significantly diminished the effect of 5-HT on AA uptake. Synaptoneurosomal and synaptosomal fractions prelabeled with [¹⁴C]AA were used subsequently for investigation of voltage-dependent, muscarinic and 5-HT receptor-mediated AA release. Aging diminished markedly carbachol-stimulated Ca²⁺-dependent AA liberation from membrane lipids of synaptoneurosomes and synaptosomes. Moreover, aging decreased voltage-dependent and 5-HT₂ receptor-mediated AA release. These results show that aging affects receptor-dependent AA uptake and pre-and postsynaptic receptor-mediated AA release. These modulations of AA incorporation and release in aged brain may be of patho-physiological significance, in view of the importance of these processes for signal transmission in the brain. The changes of receptor-dependent processes of deacylation and reacylation may be responsible for alteration in the function of neuronal cells and may affect learning and memory ability and brain plasticity during aging.