Biogenic Aldehydes in Brain: Characteristics of a Reaction Between Rat Brain Tissue and Indole-3-Acetaldehyde

When indole-3-acetaldehyde was incubated with rat brain tissue, an aldehyde dehydrogenase-independent irreversible disappearance of the aldehyde was found. This was accompanied by an increase in absorbance at 240-400 nm, with a peak at 310 nm. The results suggested that this change in absorbance was caused by a membrane-bound nonenzymatic reaction between indole-3-acetaldehyde and phospholipids. A similar reaction occurred between indole-3-acetaldehyde and pure preparations of phosphatidylethanolamine and phosphatidylserine, but not phosphatidylcholine. Indole-3-acetaldehyde levels also decreased slightly when incubated with albumin but absorbance at 310 nm was unaltered. It is suggested that nonenzymatic reactions between indole-3-acetaldehyde (or other biogenic aldehydes) and membrane components might occur in vivo, and could be involved in the effects of drugs such as ethanol and barbiturates.     

Cyclic GMP Formation and Inositol Phosphate Accumulation Do Not Share Common Origins in Rat Brain Slices

Cyclic GMP formation and inositol phospholipid hydrolysis were studied in rat brain slices to determine if the two processes have common origins. Muscarinic cholinergic stimulation enhanced [3H]inositol phosphate ([ 3H]IP) accumulation from slices prelabelled with [3H]inositol but did not affect cyclic GMP formation in the cortex, striatum, or cerebellum. An elevated level of extracellular K+ stimulated accumulation of both cyclic GMP and [3H]IP in cortex slices. The former, but not the latter, was reduced by lipoxygenase and phospholipase A2 inhibition. Calcium channel activation enhanced and blockade reduced K+-stimulated [3H]IP formation without affecting the cyclic GMP level, and there were differences in the Ca2+ requirements for the two responses. Thus, there is no support for the concept that guanylate cyclase activation inevitably accompanies inositol phospholipid breakdown, and the evidence presented demonstrates that K+ stimulation promotes cyclic GMP and [3H]IP accumulation by different transducing pathways.     

Ethanolamine Base-Exchange Reaction in Rat Brain Microsomal Subfractions

Crude microsomal fractions have been subfractionated by differential ultracentrifugation into subfractions A, B, and C, corresponding to light smooth, heavy smooth, and rough microsomal membranes, respectively. The purity and the vesiculation of the membranes were checked biochemically. Subfraction C showed the highest ethanolamine base-exchange activity, both on phospholipid and protein bases. The other two subfractions had roughly similar activities. The kinetic behavior of the enzyme activity, although anomalous, was similar in the three subfractions. Treatment of the vesicles with Pronase or with mercury-dextran produced inactivation of the ethanolamine base-exchange reaction in the three subfractions. These findings suggest that the active site of base-exchange activity would be localized on the external leaflet of the vesicles. Treatment of the membranes with trinitrobenzenesulfonic acid (TNBS) has shown that the newly synthesized phosphatidylethanolamine (PE) belongs to a pool easily reacting with the probe, independent of the subfraction investigated. On the other hand, the distribution of the bulk membrane PE reacting with TNBS differs in the three subfractions examined. It is concluded that the newly synthesized PE and probably the active site of the enzyme are on the external leaflet of the membrane in all subfractions and that the ethanolamine base-exchange reaction has similar properties in all subfractions.     

Source of Methyl Groups in Brain and Nerve Tissue in the Rat

Previous studies that demonstrated that mouse brain accumulated significantly more radioactivity from subcutaneously administered 5-methyltetrahydrofolate labelled in the methyl group compared to the label in the folate moiety are open to two interpretations. The methyl group could have been transferred to another compound (probably methionine) prior to its transport into the brain. Alternatively, if plasma 5-methyltetrahydrofolate per se is significantly involved in the provision of methyl groups to brain and nerve tissue it would be expected that the folate moiety would be returned to the plasma to complete the cycle and thus would appear not to have been taken up. In this article, using competition experiments that exploit the differences in the mechanism of transport of methionine and 5-methyltetrahydrofolate into brain and nerve, evidence is presented that in the rat the methyl group of 5-methyltetrahydrofolate is transported after its conversion to methionine.     

Multiple Forms of Choline Acetyltransferase from Rat Brain

WE have found that several different forms of choline acetyl-transferase (ChAc) from rat brain can be separated by isoelectric focusing. Such heterogeneity of ChAc is of particular interest in the context of its ultrastructural localization. Subcellular fractionation^1–4 and histochemistry⁵ have shown that the enzyme in rat, in conditions of low ionic strength and pH, adhered to several different membranous structures.     

Biogenic Aldehydes in Brain: On Their Preparation and Reactions with Rat Brain Tissue

When 1 mM serotonin, dopamine, or norepinephrine was incubated with a monoamine oxidase preparation (mitochondrial membranes) in the presence of 4 mM sodium bisulfite, 85-95% of the amines were oxidized to the corresponding aldehydes. In the absence of bisulfite, the recoveries were only approximately 30%, and dark colored products were formed during the incubations. The aldehydes derived from tyramine, octopamine, methoxytyramine, and normetanephrine were also prepared by the use of this method. The bisulfite-aldehyde compounds were stable during storage at -20 degrees C. Bisulfite-free aldehyde solutions were made by diethylether extraction. When the aldehydes derived from dopamine or serotonin were incubated with rat brain homogenates, they were found to disappear in an aldehyde dehydrogenase- and aldehyde reductase-independent manner. The disappearance of the latter aldehyde was more pronounced, and the results indicated that this aldehyde may react with both proteins and phospholipids.     

Affinity States of Rat Brain Opioid Receptors in Different Tissue Preparations

The binding of [3H]Tyr-D-Ala-Gly-(N-Me)Phe-Gly-ol ([3H]DAGO) and [3H]Tyr-D-Thr-Gly-Phe-Leu-Thr ([3H]DTLET), selective agonists for mu- and delta-opioid binding sites, respectively, has been investigated using different rat brain tissue preparations and buffer systems. The results were compared with the binding of the ligands to crude membrane fractions in Tris-HCl, the most commonly used preparation for binding studies. In both rat brain membranes and intact cells, Krebs-HEPES induced a decrease in the affinities of [3H]DAGO and [3H]DTLET, but little modification was observed when 20-microns tissue slices were used, whatever the brain area studied. The dissociation rate of [3H]DTLET was clearly dependent on the tissue preparation used, because the koff value of this ligand in Krebs-HEPES was 2.5-fold higher in membrane fractions than that measured in intact cells. The kinetic dissociation constant of [3H]DTLET in membrane fractions in Krebs-HEPES was 6.5-fold greater than that measured in Tris-HCl. In intact cells, the koff value for [3H]DTLET was lower than that found in membrane fractions in Krebs-HEPES and similar to that observed in membrane preparations in Tris-HCl supplemented with 30 mM NaCl. These data suggest (a) that the koff constant of [3H]DTLET was regulated by the ionic environment of the delta-opioid receptor, which is clearly dependent on the preservation of cellular structure, and (b) that opioid receptors could exist under different states that are regulated, in part, by the intracellular Na+ concentration.     

SNAP-25在大鼠蛛网膜下腔出血后脑组织中的表达研究

目的:观察蛛网膜下腔出血(SAH)后突触小体相关蛋白(SNAP-25)在大鼠脑组织中的表达变化.方法:42只成年健康雄性SD大鼠,随机分为正常组、对照组、SAH后1、3、5、7、14天共7组,经大鼠自体股动脉(尾动脉)非肝素化动脉血注入视交叉池致蛛网膜下腔出血模型.蛛网膜下腔出血后1、3、5、7、14天后处死大鼠,取颞叶皮层、海马、小脑做标本,Western blot及免疫组化测定SNAP-25,采用SPSS16.0软件分析.结果:与正常组相比,对照组无明显差异(P＞0.05);SAH组中SAH后1天的SNAP-25蛋白水平降低明显(P＜0.05);SAH后3天蛋白水平达到最低值(P＜0.01);SAH后5、7天的SNAP-2蛋白水平虽降低但呈逐渐上升趋势(P＜0.05);SAH后14天的SNAP-2蛋白水平与正常组相比无明显差异(P＞0.05).结论:SNAP-2在SAH后72小时明显下降,后逐渐上升,至14天时基本恢复正常.     

Changes in Rat Brain Tissue Microstructure and Stiffness during the Development of Experimental Obstructive Hydrocephalus

Understanding neural injury in hydrocephalus and how the brain changes during the course of the disease in-vivo remain unclear. This study describes brain deformation, microstructural and mechanical properties changes during obstructive hydrocephalus development in a rat model using multimodal magnetic resonance (MR) imaging. Hydrocephalus was induced in eight Sprague-Dawley rats (4 weeks old) by injecting a kaolin suspension into the cisterna magna. Six sham-injected rats were used as controls. MR imaging (9.4T, Bruker) was performed 1 day before, and at 3, 7 and 16 days post injection. T2-weighted MR images were collected to quantify brain deformation. MR elastography was used to measure brain stiffness, and diffusion tensor imaging (DTI) was conducted to observe brain tissue microstructure. Results showed that the enlargement of the ventricular system was associated with a decrease in the cortical gray matter thickness and caudate-putamen cross-sectional area (P < 0.001, for both), an alteration of the corpus callosum and periventricular white matter microstructure (CC+PVWM) and rearrangement of the cortical gray matter microstructure (P < 0.001, for both), while compression without gross microstructural alteration was evident in the caudate-putamen and ventral internal capsule (P < 0.001, for both). During hydrocephalus development, increased space between the white matter tracts was observed in the CC+PVWM (P < 0.001), while a decrease in space was observed for the ventral internal capsule (P < 0.001). For the cortical gray matter, an increase in extracellular tissue water was significantly associated with a decrease in tissue stiffness (P = 0.001). To conclude, this study characterizes the temporal changes in tissue microstructure, water content and stiffness in different brain regions and their association with ventricular enlargement. In summary, whilst diffusion changes were larger and statistically significant for majority of the brain regions studied, the changes in mechanical properties were modest. Moreover, the effect of ventricular enlargement is not limited to the CC+PVWM and ventral internal capsule, the extent of microstructural changes vary between brain regions, and there is regional and temporal variation in brain tissue stiffness during hydrocephalus development.     

Neurobehavioral Abnormalities in the HIV-1 Transgenic Rat Do Not Correspond to Neuronal Hypometabolism on 18F-FDG-PET

Motor and behavioral abnormalities are common presentations among individuals with HIV-1 associated neurocognitive disorders (HAND). We investigated whether longitudinal motor and behavioral performance in the HIV-1 transgenic rat (Tg), a commonly used neuro-HIV model, corresponded to in vivo neuronal death/dysfunction, by using rotarod and open field testing in parallel to [¹⁸F] 2-fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET). We demonstrated that age-matched non-Tg wild type (WT) rats outperformed the HIV-1 Tg rats at most time points on rotarod testing. Habituation to rotarod occurred at 8 weeks of age (fifth weekly testing session) in the WT rats but it never occurred in the Tg rats, suggesting deficits in motor learning. Similarly, in open field testing, WT rats outperformed the Tg rats at most time points, suggesting defective exploratory/motor behavior and increased emotionality in the Tg rat. Despite the neurobehavioral abnormalities, there were no concomitant deficits in ¹⁸F-FDG uptake in Tg rats on PET compared to age-matched WT rats and no significant longitudinal loss of FDG uptake in either group. The negative PET findings were confirmed using ¹⁴C- Deoxy-D-glucose autoradiography in 32 week-old Tg and WT rats. We believe that the neuropathology in the HIV-1 Tg rat is more likely a consequence of neuronal dysfunction rather than overt neurodegeneration/neuronal cell death, similar to what is seen in HIV-positive patients in the post-ART era.     

Multiple Approaches to Analyse the Data for Rat Brain Acetylcholinesterase Inhibition by Cyclophosphamide

Acetylcholinesterase (AChE) is an externally oriented membrane-bound enzyme and its main physiological role is termination of chemical transmission at cholinergic synapses and secretory organs by rapid hydrolysis of the neurotransmitter acetylcholine (ACh). Nevertheless, it is well known that cyclophosphamide (CP; nitrogen mustard derivative) is an eminent anticancer drug. The present work addresses multiple approaches to analyze an identical data for rat brain AChE inhibition by CP. These different angels of analysis based on two classical (Lineweaver–Burk as well as Dixon) plots, their secondary replots, a new graphical approach and general built-in equations of GOSA. Thus various kinetic constants (K _I, K _s, K _m, k _sl, V _mao, K _i, k _sli, S _lo, K _maxi, S _K0.5, k _cat and k _sp) were estimated and mode of inhibition discussed in the current study.     

改良的大鼠脑组织总RNA抽提方法

目的:建立一种简单方便、结果稳定的抽提大鼠脑组织总RNA的方法。方法:在传统一步法抽提动物组织总RNA的基础上,通过增加抽提步骤使总RNA与细胞DNA、蛋白质、细胞残片等干扰总RNA质量的杂质有效分离。结果:建立了可以快速、稳定地获得高纯度、未被降解的大鼠脑组织总RNA的方法。结论:采用改良的方法抽提到的总RNA比用传统的一步法得到的总RNA的完整性和均一性好,可直接应用于分子生物学操作。     

Rapidly Raise Blood Sugar Will Aggravate Brain Damage After Severe Hypoglycemia in Rats

Hypoglycemia can cause rapid and severe brain damage. We studied the impact of hypoglycemic brain damage in the insulin-induced hypoglycemic rats. Thirty male rats were divided into normal blood sugar control group (group A), the blank group (group B), and the experimental group which was further divided into four groups according to the level of blood glucose reperfusion i.e., blood glucose ≤3 mmol/L (Group C), ≤6 mmol/L (Group D), ≤9 mmol/L (Group E), and >9 mmol/L (Group F). Each groups had five rats. TUNEL and FJB staining were used to observe the apoptosis and necrosis in the rat hippocampus CA1 and DG regions and transmission electron microscopy for ultra-structures. We observed that neuronal apoptosis and necrosis of group A and B were not obvious. The apoptotic and necrotic neuron cell densities in the hippocampus CA1 and DG regions were moderately detected in group C, D, and E, while we found it maximum in group F. No significant difference was found in apoptotic and necrotic neuron cell density in the hippocampus CA1 and DG regions in group A and B. Apoptotic and necrotic cell density was significantly increased in all experimental groups as compared to the control group. Moreover, the apoptotic and necrotic cell density was significantly higher in group F than other experimental groups (group C, D, and E). However, apoptosis and necrosis in hippocampus CA1 and DG regions was not differed significantly among groups C, D, and E. All results were well supported by transmission electron microscopy. In conclusion, under the condition of the same blood glucose level, the degree of brain damage related to the blood glucose level with hypoglycemia and rapid blood glucose increased after hypoglycemia could cause more significant brain damage.     

Multiple Forms of Choline-O-Acetyltransferase in Mouse and Rat Brain: Solubilization and Characterization

Three forms of acetyl coenzyme A: choline-O-acetyltransferase (EC 2.3.1.6, ChAT) have been isolated from mouse and rat forebrain synaptosomes with a 100 mM sodium phosphate (NaP) buffer of pH 7.4, a high-salt solution (500 mM NaCl), and a 2% Triton DN-65 solution, respectively. The Triton-solubilized form of ChAT differed from the other two forms in its capacity to acetylate homocholine, its pH profile, and its sensitivity to denaturation. NaCl-solubilized ChAT could be distinguished from the other two forms with respect to pH profile, sensitivity to inhibition by 4-(1-naphthylvinyl) pyridine (in the presence of Triton), and apparent Km value for choline acetylation. The caudate and putamen of rat brain contained the highest amount of ChAT activity, based on tissue wet weight, and the cerebellum contained the least of the brain regions examined; only the cerebellum had more membrane-bound than soluble ChAT. Septal lesion reduced ChAT activity in the NaP- and Triton-solubilized fractions prepared from hippocampus by 68% and 64%, respectively, whereas it reduced the activity of the NaCl-solubilized fraction by only 21%. These results suggest that three different forms of ChAT may exist in both mouse and rat brain.     

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.     

Determination of 1-Methylimidazole-4-Acetic Acid in Brain Tissue from Rat and Mouse

The concentration of the histamine metabolite 1-methylimidazole-4-acetic acid was determined in brain tissue from rat and mouse with a gas chromatographic-mass spectrometric method. Mouse brain contained 1.7-3.2 nmol/g, depending on the strain. The concentration in cerebrum from Sprague-Dawley rats was 1.2 nmol/g, whereas cerebellum contained 0.24 nmol/g. The concentration of tele-methylhistamine in mouse brain was 1.4-2.2 nmol/g. The concentration of 1-methylimidazole-4-acetic acid in rat brain after death did not change significantly during 2 h at room temperature.     

Heparin-Binding Proteins in the Rat Brain

Using a histochemical technique, we found that in rat embryos heparin-binding sites are localized within ventricular regions of the neural tube. The highest intensity of the heparin-binding activity was observed in the membranes of migrating nerve cells. Heparin-binding membrane-associated proteins were isolated and purified from the brains of newborn rats; molecular masses of two such proteins were measured (19 and 28 kdalton). The level of affinity for binding of heparan sulfate to the purified proteins was characterized by equilibrium constants of 1.7 · 10^-3 and 6.7 · 10^-3. Binding of heparan sulfate to the above proteins was more intensive at low ion force and pH values within the 3.0 to 4.0 range and about 6.0.     

The Dopamine Metabolite 3-Methoxytyramine Is Not a Suitable Indicator of Dopamine Release in the Rat Brain

It has been postulated that changes in the concentration of 3-methoxytyramine (3-MT) in the brain might reflect changes in the release of 3,4-dihydroxyphenylethylamine (DA, dopamine) and, therefore, might be used as an index of dopaminergic activity in the brain. 3-MT is known to accumulate rapidly after death. Killing by microwave irradiation (MWR) is considered to be the method of choice to obtain "undisturbed" 3-MT concentrations. We measured striatal 3-MT concentrations even lower than those following MWR when the brains were excised and frozen in dry ice very rapidly (typical time between decapitation and freezing of the brain 22 s). There was a linear increase in striatal 3-MT concentration when the time between decapitation and freezing was varied between 13 and 300 s. Extrapolation to time zero indicated negligible amounts of 3-MT at the time of decapitation. In addition, it was observed that DA, 3,4-dihydroxyphenylacetic acid, and homovanillic acid decompose during the cooling phase after heating the brain by microwave. It is concluded that MWR induces artifactual changes in the postmortem levels of DA and metabolites. Consequently 3-MT cannot be considered to be a reliable indicator of DA release in the rat brain.