经验改变大鼠听皮层神经元的特征频率

应用常规电生理学技术,以神经元的特征频率和频率调谐曲线为指标,研究大鼠听皮层神经元特征频率的可塑性. 结果表明,在给予的条件刺激频率和神经元特征频率相差1.0 kHz范围内,条件刺激可诱导50%以上神经元特征频率发生完全偏移,并可分为向频率调谐曲线的低频端偏移、高频端偏移,或两侧均可偏移三种类型. 其中,神经元的特征频率高、Q₁₀-dB值大和频率调谐曲线对称指数大于零的神经元,其特征频率偏向频率调谐曲线高频端的概率更高. 结果提示,经验可改变大鼠听皮层神经元的特征频率,为深入研究中枢神经元功能活动可塑性的机制提供了重要实验资料.     

Effect of prenatal treatment with methylazoxymethanol on carbachol-, norepinephrine- and glutamate-stimulated phosphoinositide metabolism in the neonatal,young, and adult offspring

Carbachol-, norepinephrine- and glutamate-stimulated phosphoinositide metabolism was investigated in the neonatal, young and adult cerebral cortex slices of rats prenatally treated with methylazoxymethanol (MAM) on gestational day 15 (GD15) or GD19. In rat offspring treated on GD15 there was a significant reduction in the accumulation of [³H]inositol phosphates induced by carbachol and a significant increase in the accumulation of [³H]inositol phosphates induced by norepinephrine on day 7, whereas no changes were observed at the other ages. No significant changes, on the other hand, were observed for glutamate-stimulated phosphoinositide metabolism in GD15 treated rats and for carbachol-, norepinephrine- and glutamate-stimulated phosphoinositide metabolism in animals treated on GD19 at any of the different ages evaluated. These results indicate that treatment with MAM on GD15, which results in a marked microencephaly, causes a marked alteration of muscarinic and ₁-adrenergic receptor-stimulated phosphoinositide metabolism during brain development and that these alterations undergo adaptive changes in the adult brain.     

Increased insulin-like growth factor-I gene expression precedes left ventricular cardiomyocyte hypertrophy in a rapidly-hypertrophying rat model system

Chronic pressure overload leads to an increase in the size, i.e. hypertrophy, of cardiomyocytes in the heart. However, the molecular mechanisms underlying this hypertrophy are not understood. Insulin-like growth factor-I (IGF-I) synthesized locally in the heart is known to be associated with the hypertrophic process. So far, however, cardiac IGF-I gene expression in the widely used rat model system has only been shown to be increased when the hypertrophy induced by pressure-overload was already established. Therefore, the question of whether IGF-I serves as an initiating or early-enhancing factor for the cardiac hypertrophy remains unanswered. Here, cardiac hypertension and hypertrophy were rapidly induced in the rat by complete constriction of the abdominal aorta between the origins of the renal arteries. Carotid arterial systolic blood pressure remained unchanged in sham rats but increased rapidly in the pressure-overloaded constricted rats with a sustained hypertension established by 3 days. Hypertrophy of left ventricular (LV) cardiomyocytes in constricted rats also occurred by 3 days. However, this hypertrophy was preceded by increases in LV IGF-I mRNA and protein which occurred within 1 day. These results support the hypothesis that cardiac-synthesized IGF-I is an initiating or early-enhancing factor for hypertrophy of LV cardiomyocytes.     

Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures

Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×10⁹ cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions.     

Activation of Cl− Currents in Cultured Rat Retinal Pigment Epithelial Cells by Intracellular Applications of Inositol-1,4,5-triphosphate: Differences Between Rats with Retinal Dystrophy (RCS) and Normal Rats

Using the whole-cell configuration of the patch-clamp technique, we studied the conditions necessary for the activation of Cl⁻-currents in retinal pigment epithelial (RPE) cells from rats with retinal dystrophy (RCS) and nondystrophic control rats. In RPE cells from both rat strains, intracellular application of 10 μm inositol-1,4,5-triphosphate (IP3) via the patch pipette led to a sustained activation of voltage-dependent Cl⁻ currents, blockable by 1 mm 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). IP3 activated Cl⁻ currents in the presence of a high concentration of the calcium chelator BAPTA (10 mm) in the pipette solution, but failed to do so when extracellular calcium was removed. Intracellular application of 10⁻⁵ m Ca²⁺ via the patch pipette also led to a transient activation of Cl⁻ currents. When the cells were preincubated in a bath solution containing thapsigargin (1 μm) for 5 min before breaking into the whole-cell configuration, IP3 failed to activate voltage-dependent currents. Thus, IP3 led to release of Ca²⁺ from cytosolic calcium stores. This in turn activated an influx of extracellular calcium into the submembranal space by a mechanism as yet unknown, leading to an activation of calcium-dependent chloride currents. In RPE cells from RCS rats, which show an increased membrane conductance for calcium compared to normal rats, we observed an accelerated speed of Cl⁻-current activation induced by IP3 which could be reduced by nifedipine (1 μm). Thus, the increased membrane conductance to calcium in RPE cells from RCS rats changes the response of the cell to the second messenger IP3. Received: 17 July 1995/Revised: 31 January 1996     

Interstitial noradrenaline concentration of rat hearts as influenced by cellular catecholamine uptake mechanisms

Marked concentration differences of noradrenaline (NA) between the vascular and the interstitial compartment were detected by sampling interstitial transudate from isolated perfused rat hearts. The ratios of vascular/interstitial concentration amounted to 7.4 to 1.3 depending on the concentration of NA administered (3 × 10^–9 to 10^–6 M). These concentration differences were abolished by inhibitors of uptake₁ desipramine (DMI) I and uptake, (O-methyl-isoprenaline (OMI)). Neuronal uptake, was characterized by a K_m of 0.22 mol/l and a V_max of 370 pmol × min^–1 × gWWT^–1, extraneuronal uptake₂ by a K_UPTAKE of = 0.313 min^–4.The apparent permeability surface area (P×S)-product calculated from uptake rate and transcapillary concentration difference was significantly decreased by administrating 100 mol/l (NA) in presence of DMI. A presumed endothelial uptake mechanism contributing to catecholamine translocation was investigated in endothelial cells in culture. These cells showed a specific noradrenaline uptake with a K_m of 4.35 mol/l and a V_max of about 75 pmol × min^–1 x gWWT^–1. Any inhibiton by inhibitors of both of the two noradrenaline uptakes was lacking. The uptake rate of this mechanism is insufficient to contribute to the diffusive conductivity of the capillary wall (P × S-product). We conclude from our investigations on interstitial concentrations of catecholamines and transcapillary concentration differences, that the capillary wall, owing to its metabolic and diffusional characteristics, influences the exchange of catecholamines to a substantial and physiologically relevant extent.     

大鼠基底前脑NGF-R及CHAT免疫反应阳性神经元的分布:免疫双标法

本文用免疫组化双标法观察了神经生长因子受体(NGF-R)及胆碱乙酰转移酶(ChAT)免疫反应阳性神经元在成鼠基底前脑内的分布,结果发现嗅结节、隔内侧核、斜角带核、腹侧苍白球及基底大细胞核均有NGF-R及ChAT免疫反应阳性神经元.免疫组化双标染色发现,大部分免疫反应阳性神经元的NGF-R与ChAT共存,部分神经元呈单纯NGF-R或ChAT阳性,但这种NGF-R和ChAT的共存情况在不同区域不完全相同.在隔内侧核和斜角带核,大多数的NGF-R阳性神经元和ChAT阳性神经元共存,但在腹侧仓白球和基底大细胞核,两者共存的神经元较前两区为少.此外ChAT阳性神经元在尾壳核中分布较均匀,而NGF-R阳性神经元较少见.研究结果表明,大多数胆碱能神经元有NGF-R,提示NGF对胆碱能神经元的保护和激活作用,部分可能是通过直接与NGF受体的结合而发生作用.     

β-Adrenergic Receptors Mediate a Stress-Induced Decrease in IGF-II mRNA in the Rat Cerebellum

1. Exposure to a combined forced swimming–confinement stress resulted in a decrease in insulin-like growth factor II (IGF-II) mRNA levels in the whole brain (without the cerebellum) and in the isolated brain areas of the cerebral cortex, the hippocampus, and the cerebellum.2. In an effort to elucidate the neurotransmitter systems involved in this stress-induced decrease, animals were injected prior to exposure to the stress, with either propranolol, diazepam, or MK-801.3. Administration of diazepam or MK-801 did not affect the stress-induced decrease in IGF-II mRNA in any of the three brain areas examined.4. Administration of propranolol prior to the exposure to the stress inhibited the stress-induced decrease in IGF-II mRNA in the cerebellum. Propranolol had no such effect in the cerebral cortex or the hippocampus.5. Our results suggest that in the cerebellum, the stress-induced decrease in IGF-II mRNA is mediated by ₂-adrenergic receptors.     

S-adenosyl-l-methionine inhibits phosphoinositide metabolism in the rat brain synaptosomal suspensions

S-adenosyl-l-methionine (AdoMet) has been reported to affect events linked to noradrenergic neurotransmission. In the present work, we studied the effect of AdoMet on norepinephrine (NE)-stimulated inositol phosphate production in³H-inositol-labelled crude synaptosomal suspensions of rat brain. AdoMet (50–1000 M) decreased both the synthesis of labelled polyphosphoinositide (30–50%) and the release of inositol mono- and bisphosphate (40–50%). The AdoMet effect was not dependent on NE concentration (10–1000 M), suggesting that the inhibition of inositol phosphate release was not the result of a modification of the norepinephrine binding to its receptor sites. S-adenosyl-L-homocysteine (AdoHcy) (1 mM) an inhibitor of methyltransferase activities, partially inhibited (70%) the AdoMet (0.1 mM) effect, indicating that the methylation processes cannot explain all the effects observed. We conclude that, in addition to previously reported effects of AdoMet on NE transport, AdoMet may reduce NE-linked intracellular signalling.     

Inhibitory effect of regucalcin on protein phosphatase activity in the nuclei of rat kidney cortex

The role of regucalcin, which is a regulatory protein of calcium signaling, in the regulation of protein phosphatase activity in the nuclei of rat kidney cortex was investigated. Protein phosphatase activity towards phosphotyrosine, phosphoserine, and phosphothreonine was found in the nuclei. The enzyme activity towards three phosphoamino acids was significantly increased by the addition of calcium chloride (10-50 microM) in the enzyme reaction mixture. This increase was significantly inhibited by trifluoperazine (25 or 50 microM), an antagonist of calmodulin. The presence of regucalcin (50 or 100 nM) in the enzyme reaction mixture caused a significant decrease in protein phosphatase activity towards three phosphoamino acids. This effect was also seen in the presence of calcium (25 microM) and/or calmodulin (5 microg/ml). Protein phosphatase activity towards three phosphoamino acids was significantly increased in the presence of anti-regucalcin monoclonal antibody (25 or 50 ng/ml) in the enzyme reaction mixture. This effect was completely blocked by the addition of regucalcin (100 nM). The effect of antibody (25 ng/ml) in increasing protein phosphatase activity towards phosphotyrosine was significantly inhibited by vanadate (10(-4) M). Also, the antibody's effect towards phosphoserine and phosphothreonine was significantly inhibited by cyclosporin A (10(-5) M). Endogenous regucalcin was found in the nuclei of rat kidney cortex using Western blot analysis. Nuclear regucalcin level was significantly reduced by the administration of saline (0.9% NaCl) for seven days in rats. Protein phosphatase activity towards three phosphoamino acids was significantly decreased by saline administration. The effect of anti-regucalcin monoclonal antibody (25 ng/ml) in increasing protein phosphatase activity towards three phosphoamino acids was weakened in the renal cortex nuclei of saline-administrated rats. The present study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein phosphatase activity in the nuclei of rat kidney cortex cells.