The changed immunolocalization of START-domain-containing 6 (StarD6) during the development of testes in rat perinatal hypothyroidism

Thyroid hormones have an essential role in maintaining the normal developmental structure of testes during the neonatal stage. START-domain-containing 6 (StarD6) is exclusively expressed in germ cells during spermatogenesis; however, its biological role in rat perinatal hypothyroidism is not clear. After hypothyroidism was induced by daily administration of 0.05% 6-propyl-2-thiouracil (PTU), the pattern of StarD6 immunolocalization was examined from gestation day 15 to postnatal day 49. In normal rats, the labelling of StarD6 was confined to the germ cells from the third-week postpartum. In contrast, its immunoreactivity in hypothyroidal rats was not detected until the fourth-week postpartum. The immunolocalization pattern of StarD6 differed from that of normal adult rats during the seventh-week postpartum. StarD6 was clearly detected in the Leydig cells of the perinatal hypothyroid rats from the fifth-week postpartum. Therefore, StarD6 may play a pivotal role, not only in the spermatogenesis of normal rats, but also in the steroidogenesis of Leydig cells under perinatal hypothyroidism.     

Immunolocalization of steroidogenic acute regulatory protein-related lipid transfer (START) domain-containing proteins in the developing cerebellum of normal and hypothyroid rats

Cholesterol transport proteins are a prerequisite for neurosteroidogenesis. Steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain-containing proteins, such as StAR and START domain-containing 6 (StarD6), are known to be distributed in the brain. Since perinatal hypothyroidism affects cerebellar development, we examined postnatal changes in StAR and StarD6 immunolocalization in the developing cerebellum of control and hypothyroid rats. Pregnant Sprague-Dawley rats were given 0.05% 6-propyl-2-thiouracil (PTU) or water from gestation day 11 until postnatal day (P) 28, and were then killed together with age-matched control rats. As shown by calbindin D-28k immunostaining, the developing cerebellar cytoarchitecture and Purkinje cells were affected by PTU-induced hypothyroidism as compared to control rats. The immunolocalization of StAR and StarD6 generally followed the maturation pattern of Purkinje cells from the vermis to the cerebellar hemisphere. StAR immunostaining first appeared in the Purkinje cells of the vermis at P7 in both control and hypothyroid rats. In control rats, a few StarD6 immunoreactive cells were seen at birth and a nuclear localization of StarD6 in Purkinje cells was obvious at P14. PTU-induced hypothyroidism delayed the appearance of StarD6 immunopositive cells until P7. Moreover, the nuclear localization of StarD6 in PTU-treated rats was not obvious at P14. An adult-like distribution of StAR and StarD6 was achieved by P21 in control and hypothyroid rats. These results suggest that StarD6 may affect the development of Purkinje cells during the first and second postnatal weeks, a known period of thyroid hormone action.     

老年学习记忆减退大鼠脑线粒体DNA缺失

目的和方法:测定老年大鼠脑mtDNA缺失型(以下简称缺失型),缺失mtDNA比例,探讨mtDNA缺失与老年性学习记忆减退的关系,为研究其分子机制提供基础资料。用Morris水迷宫将老年大鼠(24个月)筛选为老年学习记忆正常和学习记忆减退两组。用稀释PCR法测定大鼠大脑皮质、海马和小脑缺失型mtDNA比例。结果:青老年鼠大脑皮质、海马和小脑均存在mtDNA缺失,片段为4834bp;青年鼠的缺失比例约为0.00018%。老年记忆正常鼠上述3个脑区缺失型mtDNA缺失比例分别是青年鼠的6倍、6倍和2倍;老年记忆减退大鼠上述脑区的缺失型mtDNA比例分别是老年记忆正常鼠的2倍、1.8倍和3倍。结论:衰老时脑组织缺失型mtDNA增多,而老年学习记忆减退鼠较老年学习记忆正常鼠mtDNA缺失进一步成倍增加,表明相关脑区的mtDNA缺失在老年学习记忆减退的细胞分子机制中发挥重要作用。     

大白鼠脑与松果体β肾上腺素能受体的老年性变化

目的 研究老年大鼠大脑皮质、海马、小脑皮质和松果体β-肾上腺素能受体的变化.方法 选用老年(20个月)和青年(3个月)SD大白鼠,以放射配基受体结合分析法测定大鼠大脑皮质、海马、小脑皮质和松果体β受体密度、比较2个年龄组上述脑区β受体含量变化及松果体β受体日夜周期变化.结果 与青年组比较,老年组大脑皮质、海马β受体含量明显降低(P<0.01),小脑皮质β受体含量变化不明显(P>0.05),松果体β受体含量在青年组白天明显高于夜间,而老年组变化不明显.结论 衰老时大鼠大脑皮质和海马β受体含量减少,松果体β受体含量的日夜周期改变消失.     

Low glutathione levels in brain regions of aged rats

Glutathione (GSH) was measured in 6 regions of brain and liver of young adult, middle-aged and aged rats. GSH levels were significantly lower in cortex, cerebellum, striatum, thalamus and hippocampus of aged rats, while no changes were observed in liver as compared to young adult rats. On the other hand, lipid peroxidation as measured by thiobarbituric acid-reactive products increased significantly in all the regions of brain examined and in the liver of aged rats. Since GSH plays an important role as a cellular protectant against oxygen radical-mediated injury, decreased levels of GSH in aged rat brain are indicative of the vulnerability of the aged cerebral tissue to oxidative injury.     

In vivo molecular imaging of the GABA/benzodiazepine receptor complex in the aged rat brain

The GABA-ergic system, known to regulate neural tissue genesis during cortical development, has been postulated to play a role in cerebral aging processes. Using in vivo molecular imaging and voxel-wise quantification, we aimed to assess the effects of aging on the benzodiazepine (BDZ) recognition site of the GABA(A) receptor. To visualize BDZ site availability, [(11)C]-flumazenil microPET acquisitions were conducted in young and old rats. The data were analyzed and region of interest analyses were applied to validate the voxel-wise approach. We observed decreased [(11)C]-flumazenil binding in the aged rat brains in comparison with the young control group. More specifically, clusters of reduced radioligand uptake were detected in the bilateral hippocampus, cerebellum, midbrain, and bilateral frontal and parieto-occipital cortex. Our results support the pertinence of voxel-wise quantification in the analysis of microPET data. Moreover, these findings indicate that the aging process involves declines in neural BDZ recognition site availability, proposed to reflect alterations in GABA(A) receptor subunit polypeptide expression.     

Reduced serine racemase expression contributes to age-related deficits in hippocampal cognitive function

To gain insight into the contribution of d-serine to impaired cognitive aging, we compared the metabolic pathway and content of the amino acid as well as d-serine-dependent synaptic transmission and plasticity in the hippocampus of young and old rats of the Wistar and Lou/C/Jall strains. Wistar rats display cognitive impairments with aging that are not found in the latter strain, which is therefore considered a model of healthy aging. Both mRNA and protein levels of serine racemase, the d-serine synthesizing enzyme, were decreased in the hippocampus but not in the cerebral cortex or cerebellum of aged Wistar rats, whereas the expression of d-amino acid oxidase, which degrades the amino acid, was not affected. Consequently, hippocampal levels of endogenous d-serine were significantly lower. In contrast, serine racemase expression and d-serine levels were not altered in the hippocampus of aged Lou/C/Jall rats. Ex vivo electrophysiological recordings in hippocampal slices showed a marked reduction in N-methyl-d-aspartate-receptor (NMDA-R)-mediated synaptic potentials and theta-burst-induced long-term potentiation (LTP) in the CA1 area of aged Wistar rats, which were restored by exogenous d-serine. In contrast, NMDA-R activation, LTP induction and responses to d-serine were not altered in aged Lou/C/Jall rats.These results further strengthen the notion that the serine racemase-dependent pathway is a prime target of hippocampus-dependent cognitive deficits with aging. Understanding the processes that specifically affect serine racemase during aging could thus provide key insights into the treatment of memory deficits in the elderly.     

大鼠海马微血管构筑的定量分析及衰老变化

目的:观察并定量分析大鼠海马的微血管构筑及衰老变化。方法:采用单宁酸-氯化铁媒染微血管的方法分别观察青龄、老龄大鼠海马与额叶皮质的微血管构筑,并采用MiVnt图像分析系统对海马与额叶皮质的微血管密度(MVD)及微血管面积密度(MVA)进行定量分析。结果:老龄大鼠海马、皮质的微血管数量显著减少,分布杂乱、扭曲缠结。定量分析显示老龄大鼠海马、皮质的MVD值和MVA值均明显低于青龄大鼠,差异具有统计学意义。结论:老龄大鼠海马及额叶皮质的MVD值、MVA值均明显低于青龄大鼠,这是老年血管性痴呆发生机制的主要形态学依据。     

老年大鼠空间记忆减退与cNOS基因表达变化

目的：从基因表达角度研究原生型NOS（cNOS,包括神经元型nNOS和内应型eNOS）与老年记忆减退发生的关系。方法：用Morris水迷宫将老年大鼠筛选为记忆正常和记忆减退两组,以老年记忆减退大鼠作为老年记忆减退模型;用半定量反转录一聚合酶链反应（RT－PCR）方法测定nNOS和eNOS的mRNA含量。结果：（1）海马组织中老年记忆正常组和老年记忆减退组较青年组nNOS和eNOSmRNA含量均下降,nNOS以记忆减退组下降更为显著;（2）小脑组织中老年记忆减退组nNOS和eNOSmRNA含量的下降与老年记忆正常组和青年组均有显著差异。（3）额叶组织nNOSmRNA含量三组之间均无显著差异;eNOSmRNA含量老年记忆正常组和老年记忆减退组技青年组均下降,以记忆减退组下降更为显著。结论：老年记忆减退的发生可能与有关脑区cNOS基因表达下降有关。     

Possible involvement of proteasome inhibition in aging: implications for oxidative stress

Oxidative stress may contribute to the cellular alterations, which occur as the result of aging, and the nervous system is particularly vulnerable to aging associated oxidative injury. The multicatalytic proteasome (MCP) is responsible for the majority of protein degradation and is sensitive to oxidative stress. To determine if MCP activity is altered during aging, studies were conducted in multiple tissues from aged Fisher 344 rats. Analysis of heart, lung, kidney, and liver revealed decreased MCP activity in 12, 24, and 28 month old rats, compared with 3 week or 3 month old animals. The spinal cord, hippocampus, and cerebral cortex demonstrated age dependent decreases in MCP activity, but at no timepoint was MCP activity decreased in either the brain stem or cerebellum. Oxidative injury and the lipid oxidation product 4-hydroxynonenal caused decreased MCP activity in neural PC6 cells, while application of MCP inhibitors was sufficient to induce cell death in neural PC6 cells. Together, these data indicate a role for MCP inhibition in cellular dysfunction associated with aging and oxidative injury.     

Regulation of phorbol ester binding and protein kinase C activity in aged rat brain

Protein kinase C (PKC) function was analyzed in aged male Sprague-Dawley rat brain using two different approaches: the binding of [3H]-phorbol-12,13-dibutyrate and the in vitro phosphorylation of histone H1. In cortex the binding was decreased while in cerebellum no age-related modifications were observed. In hippocampus the binding capacity was increased in old animals and the affinity decreased. The kinase activity in both soluble and particulate fractions was decreased in cortex, increased in hippocampus and unmodified in cerebellum. The area selective, age-dependent modifications in neuronal PKC may sustain short- and long-term regional changes of neuronal excitability.     

Noggin在大鼠中枢神经系统发育过程中的表达

目的 研究Noggin基因在大鼠中枢神经系统(CNS)发育过程中的表达。方法 地高辛标记的cRNA探针原位杂交组织化学技术。结果 ISHH结果显示，在胚胎期(E16)大鼠，noggin mRNA阳性细胞主要位于大脑皮质、海马、丘脑与下丘脑的部分核团。新生期(P1-P2)大鼠，noggin在大脑皮质与海马的表达均降低，而在丘脑与延脑的表达增强；生后1周(P1W)noggin在脑内的表达明显降低，生后2周noggin在脑内的表达开始升高，在大脑皮质与海马升高尤为明显。生后1个月，noggin表达继续升高，在额叶皮质、顶叶皮质、扣带皮质、梨状皮质及海马的齿状回可检测到强阳性信号；而在丘脑的侧核、网状核、腹内侧核与腹外侧核可见中等强度的阳性信号。此外，在下丘脑的室旁核和视上核亦可见密集深染的阳性神经元。生后3个月，noggin阳性细胞在脑内的表达开始降低；生后18个月，noggin表达降至最低，仅见散在的阳性神经元。此外，在不同发育期大鼠的脊髓亦未观察到noggin mRNA阳性细胞。结论 提示noggin基因参与大鼠生后CNS的发育。     

Comparison of hippocampal synaptosome proteins in young-adult and aged rats

The hippocampus is important in learning and memory functions but its ability to aid in these functions declines during aging. In this study, we examined hippocampal proteins whose expressions changed in the aging process. A comparison of synaptosome proteins of hippocampus prepared from young-adult (9-week-old) rats with those from aged (30-month-old) rats by two-dimensional fluorescence difference gel electrophoresis revealed 24 spots that were expressed differently among about 1000 spots detected in both young-adult and aged rat samples. Nineteen of these 24 spots were identified by peptide mass fingerprinting. These proteins included chaperone proteins and proteins related to the cytoskeleton, neurotransmission, signal transduction and energy supply. The cytoskeleton-related proteins included actin and T-complex 1, which is thought to play a role in actin folding. Actin was up-regulated but T-complex 1 was down-regulated in aged rat synapses. These results suggest that age-dependent changes of actin filament formation are related to neuronal dysfunction associated with aging.     

Muscarinic receptor-mediated GTP-Eu binding in the hippocampus and prefrontal cortex is correlated with spatial memory impairment in aged rats

The present study examined muscarinic receptor/G-protein coupling in the hippocampus and the prefrontal cortex of young and aged Long-Evans rats characterized for spatial learning ability in the Morris water maze. In a highly sensitive time-resolved fluorometry GTP-Eu binding assay, muscarinic-mediated GTP-Eu binding was severely blunted in hippocampus (-32%) and prefrontal cortex (-34%) as a consequence of aging. Furthermore, the magnitude of decreased muscarinic-mediated GTP-Eu binding was significantly correlated with the severity of spatial learning impairment in hippocampus and prefrontal cortex of aged rats and was specifically decreased in the subset of aged rats that were spatial learning impaired when compared to the aged unimpaired and the young rats. Western blot data indicated a preservation of the membrane-bound M1 receptor and the Galphaq/11 protein in both brain regions. These data demonstrate that muscarinic signaling is severely impaired as a consequence of normal aging in a manner that is closely associated with age-related cognitive decline.     

Quantitative autoradiographic analysis of 125I-pindolol binding in Fischer 344 rat brain: changes in beta-adrenergic receptor density with aging

Age-related changes in beta-adrenergic receptor density in Fischer 344 rat brain were examined using in vitro 125I-pindolol (IPIN) binding and quantitative autoradiographic analysis. Localized protein concentrations were determined using a new quantitative histological technique, and these were used to normalize the densities of receptors. Saturation binding studies in brain sections revealed 40-50% decreases in beta-adrenergic receptor density in the thalamus of 23-25-month-old and the cerebellum and brainstem of both 18-19-month-old and 23-25-month-old compared to 4-6-month-old rats. The loss of cerebellar beta-adrenergic receptors may be correlated with reports of deficits in sensitivity to beta-adrenergic-mediated transmission in the cerebellum of aged rats. No changes in specific IPIN binding with age were observed in rat cortex or hippocampus. In all areas examined no age-related differences were observed in receptor affinity. No changes in protein concentration were found in any of the areas examined in the different aged animals. These results demonstrate a region-specific loss of beta-adrenergic receptors with age in the brain of Fischer 344 rats.     

Changes of mouse brain gangliosides during aging from young adult until senescence

The brain gangliosides from young adult to senescent mice (BDF1 and C57BL/6) were studied. The total ganglioside concentrations of the whole brains were almost constant from young adulthood until the beginning of senescence, but decreased constant from young adulthood until the beginning of senescence, but decreased during senescence to about 80% of the constant level observed at the period before the beginning of senescence. In spite of the constancy of the ganglioside concentrations at the period before the beginning of senescence, the composition gradually changed, with an increase of GM1 and decreases of GD1b, GT1b and GQ1b. During the senescence, all of the gangliosides decreased in their concentrations, but GD1a, GT1b and GQ1b decreased to a markedly greater extent (GT1b greater than GD1a greater than GQ1b). The regional gangliosides in olfactory bulb, cerebrum cortex, cerebrum white matter, hippocampus, hypothalamus, cerebellum and medulla oblongata were compared between 3- and 30-month-old mice of both strains. Significant changes in ganglioside concentrations were observed in both strains in the cerebrum and the hippocampus. In the cerebrum white matter, cerebellum and medulla oblongata, GM1 and GM4 contents significantly increased in the senescent mice.     

Acute exposure to sarin increases blood brain barrier permeability and induces neuropathological changes in the rat brain: dose-response relationships

We hypothesize that a single exposure to an LD(50) dose of sarin induces widespread early neuropathological changes in the adult brain. In this study, we evaluated the early changes in the adult brain after a single exposure to different doses of sarin. Adult male rats were exposed to sarin by a single intramuscular injection at doses of 1, 0.5, 0.1 and 0.01 x LD(50). Twenty-four hours after the treatment, both sarin-treated and vehicle-treated (controls) animals were analyzed for: (i) plasma butyrylcholinesterase (BChE) activity; (ii) brain acetylcholinesterase (AChE) activity, (iii) m2 muscarinic acetylcholine receptor (m2 mAChR) ligand binding; (iv) blood brain barrier (BBB) permeability using [H(3)]hexamethonium iodide uptake assay and immunostaining for endothelial barrier antigen (EBA); and (v) histopathological changes in the brain using H&E staining, and microtubule-associated protein (MAP-2) and glial fibrillary acidic protein immunostaining. In animals treated with 1 x LD(50) sarin, the significant changes include a decreased plasma BChE, a decreased AChE in the cerebrum, brainstem, midbrain and the cerebellum, a decreased m2 mAChR ligand binding in the cerebrum, an increased BBB permeability in the cerebrum, brainstem, midbrain and the cerebellum associated with a decreased EBA expression, a diffuse neuronal cell death and a decreased MAP-2 expression in the cerebral cortex and the hippocampus, and degeneration of Purkinje neurons in the cerebellum. Animals treated with 0.5 x LD(50) sarin however exhibited only a few alterations, which include decreased plasma BChE, an increased BBB permeability in the midbrain and the brain stem but without a decrease in EBA expression, and degeneration of Purkinje neurons in the cerebellum. In contrast, animals treated with 0.1 and 0.01 x LD(50) did not exhibit any of the above changes. However, m2 mAChR ligand binding in the brainstem was increased after exposure to all doses of the sarin.Collectively, the above results indicate that, the early brain damage after acute exposure to sarin is clearly dose-dependent, and that exposure to 1 x LD(50) sarin induces detrimental changes in many regions of the adult rat brain as early as 24 hours after the exposure. The early neuropathological changes observed after a single dose of 1 x LD(50) sarin could lead to a profound long-term neurodegenerative changes in many regions of the brain, and resulting behavioral abnormalities.     

Comparative distribution of nicotinic receptor subtypes during development, adulthood and aging: an autoradiographic study in the rat brain

The distribution in the rat brain of high affinity nicotinic heteromeric acetylcholine receptors and of low affinity nicotinic, alpha7-containing, homomeric receptors was studied using in vitro light microscopic autoradiography. As ligands, we used [3H]epibatidine, or [125I]epibatidine, and [125I]alpha-bungarotoxin, respectively. In adult animals, the two types of binding sites were widely distributed in many different brain structures, including the brainstem, cerebellum, mesencephalic structures, limbic system and cortex, but their anatomical distribution differed markedly. Only in rare instances could a co-localization be observed, for example in the superficial layer of the superior colliculus. In developing animals, both types of labeling were strongly expressed during embryonic and postnatal phases. Their distributions were qualitatively similar to those observed in adult animals, with a few noticeable exceptions in the cerebral cortex, hippocampus and brain stem. In aging animals, neither the distribution nor the density of nicotinic binding sites was significantly altered. Our conclusions are the following. (a) There is little overlap in the distribution of heteromeric and alpha7-containing homomeric nicotinic receptors in the rat brain. (b) The abundance of neuronal nicotinic receptors during embryonic and postnatal development suggests that they may play a role in the establishment of neuronal connectivity. (c) The expression of neuronal nicotinic receptors is unaltered in middle aged animals, suggesting that in the rat these receptors do not play any major role in aging process.     

Elevation of cytoskeletal protein breakdown in aged Wistar rat brain

Previous studies indicated there is an overall increase of proteolysis in aging rat brains. We monitored the potential degradation of cytoskeletal proteins in neuronal tissue taken from cerebral cortex and cerebellum of young (3 month) and aging (17, 21 and 23.5 month) Wistar rats. We found significant age-dependent proteolysis of cytoskeletal proteins (alphaII-spectrin and microtubule-associated protein MAP-2A/B) in the cerebral cortex and the cerebellum. The pattern of alphaII-spectrin breakdown shows a marked increase in 150- and 145-kDa fragments (SBDP150 and SBDP145, respectively), but we did not detect the caspase-3-mediated 120-kDa fragment (SBDP120) in aged rat brains, suggesting the involvement of the calpain proteases. The pattern of MAP-2A/B breakdown in aged rat brains mirrors that produced by in vitro calpain digestion of 3-month control rat brain MAP-2A/B. In aged rat brains, there is no significant increase in pro-caspase-3 processing; rather, there is a moderate reduction in pro-caspase-3 protein and caspase-3 hydrolytic activity in the cortex. These results point to selective susceptibility of cytoskeletal proteins to calpain-mediated degradation, but not caspase-3 in aging rat brains.