Zonal centrifuge profiles of rat brain homogenates: Instability in sucrose,stability in iso-osmotic Ficoll-sucrose

By substituting iso-osmotic Ficoll-sucrose for hyperosmotic sucrose between the densities of 1.043 and 1.088 in sucrose density gradients in the B-XV rotor of an Anderson-NIH-AEC zonal centrifuge system, it was possible to stabilize the zonal centrifuge absorbancy profiles of adult rat brain homogenates. The reason for the instability in ordinary sucrose gradients was found to be the interaction of myelin with other brain structures in hyperosmotic sucrose. No such interaction occurred in isoosmotic sucrose (0.32 M) with or without Ficoll. In Ficoll-sucrose, myelin was separated at three reproducible densities of 1.054, 1.060, and 1.066 gm/ml. No myelin appeared at a density if 1.094 gm/ml, which represented the main collection point in ordinary sucrose. Synaptosomes were separated at peak densities of 1.072 and 1.152 gm/ml. Mitochondria were obtained at a density of 1.176 gm/ml. Areas under zonal centrifuge absorbancy profiles of rat brain homogenates were found to be constant regardless of the values of ω²t that were reached.     

Glycogen phosphorylase activity and immunoreactivity during pre- and postnatal development of rat brain

Catalytic activity and immunoreactivity of glycogen phosphorylase were studied in pre- and postnatal rat brain. The catalytic activity was assayed in brain homogenates; immunoreactivity was investigated by immunoblot analysis using a monoclonal anti-bovine brain glycogen phosphorylase antibody. The cellular localization and intensity of immunoreactivity were analysed on paraffin-embedded sections utilizing the same monoclonal antibody. The catalytic activity increased 10-fold from embryonic day 16 to adult; immunoreactivity became detectable on embryonic day 16 and increased in intensity as the enzyme activity rose to adult values. The first cellular elements to be stained immunohistochemically were ependymal cells lining the ventricles, ependymal cells of the choroid plexus, meningeal cells and a selected population of neurons in the brain stem. The immunoreactivity of plexus cells and meningeal cells was reduced or absent in the adult rat brain. The earliest appearance of glycogen phosphorylase immunoreactivity in astroglial cells was seen at postnatal day 9 in the hippocampus. The staining pattern of the adult brain was reached at day 22 post partum. The developmental changes in glycogen deposition and in glycogen phophorylase activity and immunoreactivity may indicate a variable physiological role of glycogen metabolism for different cell types in the pre- and postnatal periods.Dedicated to Professor Helmut Leonhardt on the occasion of his 75th birthday     

Properties and Ontogenic Development of Membrane-Bound Histidine Decarboxylase from Rat Brain

Abstract: Histidine decarboxylase (HD) activity was determined in high-speed fractions (100,000 g for 60 min) obtained from whole rat brain homogenates. Twenty-eight percent of the HD activity was associated with membranes, and the remaining was soluble. Several properties of the soluble and membrane-bound HD were compared. No significant differences in the values of K _m for histidine and pyridoxal 5'-phosphate were observed. The solubilization of membrane-bound HD with Triton X-100 resulted in an increase of 60% over the nonsolubilized activity with no changes in the K _m for substrate and cofactor. The proportion of free pyridoxal 5'-phosphate-independent activity was identical in both fractions. The soluble and membrane-bound forms of the enzyme differ slightly in their pH-activity profiles, although both enzymes showed an optimum pH near 6.5. The HD activities present in soluble and membrane fractions were determined at different postnatal ages. The soluble activity increased until day 90, whereas the membrane-bound activity became stabilized from day 20.     

A centrifugation study of rat-liver mitochondria, lysosomes and peroxisomes during the perinatal period.

We have investigated the intracellular distribution of several enzymes on homogenates of late foetal, early postnatal and adult rat livers. Homogenates were subjected to differential centrifugations in 0.25 M sucrose and four fractions were isolated which corresponded to the N (nuclear) ML (total mitochondrial) P (microsomal) and S (soluble) fractions of de Duve et al. (1955). In general the age of the animal did not significantly affect the distribution pattern. Reference enzymes of mitochondria, lysosomes and peroxisomes were mainly recovered in the total mitochondrial fraction (ML). Glucose-6-phosphatase and esterase, both located in the endoplasmic reticulum, were chiefly associated with the microsomal fraction P together with galactosyltransferase (a reference enzyme of the Golgi apparatus). 5'-Nucleotidase, (a plasma membrane enzyme) exhibits a bimodal distribution and is mainly recovered in the N and the P fractions. Such results indicate that the membrane composition of the fractions isolated by the fractionation scheme was used, does not appreciably differ for the late foetal, early postnatal and adult rat livers. An analytical fractionation of the mitochondrial (ML) fraction of livers at different stages of development was performed by isopycnic centrifugation in sucrose gradients and in glycogen gradients using sucrose solutions of various concentrations as the solvents. The distribution of mitochondria, lysosomes and peroxisomes were assessed by establishing the distribution of their reference enzymes. Some physical characteristics of the particles were deduced from the manner in which the distributions were influenced by the sucrose concentration of the centrifugation medium. The distribution of liver mitochondrial enzymes one day prenatal differs strikingly from that of enzymes one day postnatal; foetal mitochondria seem characterized by a high osmotic space and a high hydrated matrix density; neonatal mitochondria seem devoid of an osmotic space and the density of their hydrated matrix is markedly lower than that of the foetal mitochondria. As ascertained by the distribution of mitochondrial enzymes in a sucrose 2H2O gradient, the high density of a foetal mitochondria matrix does not mainly originate from a lower amount of hydration water. The behavior of lysosomal enzymes in media with increasing concentrations of sucrose suggests that lysosomes originating from late foetal rat liver are endowed with a very small osmotic space. As for the peroxisomes, our results do not display significant behavior differences in centrifugations that would indicate physicochemical changes of these particles during the perinatal period.     

Abnormal Expression of the Myelin-Associated Glycoprotein in the Central Nervous System of Dysmyelinating Mutant Mice

Total cytoplasmic brain RNA was isolated at two different ages from three neurological mutant mice (qk/qk, jp/Y, and shi/shi) and their apparently normal littermates. This RNA was translated in vitro in a rabbit reticulocyte lysate system. Myelin-associated glycoprotein (MAG)-related polypeptides were immunoprecipitated from equal amounts of total translation products derived from mRNA of mutant animals, normal littermates, or control animals. The developmentally regulated synthesis of MAG polypeptides was compared among the mutants and normal animals. mRNA from qk/qk brains synthesized an overabundance of p67MAG (five- to sevenfold) which may be compensation for a decreased synthesis of p72MAG. mRNA from jp/Y brains synthesized less than 10% of normal amounts of both MAG polypeptides. The quantity of MAG synthesized by 15-day shi/shi brain mRNA was slightly decreased compared with normal brain mRNA but the quantity of MAG synthesized by adult shi/shi brain mRNA was normal. No apparent differences were detected in the sizes of the MAG polypeptides synthesized by any of the mutants studied. The data suggest that the genetic defect in qk/qk mutants directly or indirectly affects the coordinated developmental regulation of MAG polypeptide synthesis leading to an overabundance of the MAG polypeptide that is normally found in older animals. The jp/Y mutation appears to affect general myelin protein synthesis. Finally, shi/shi mutants may have a delayed synthesis of MAG. The data are discussed in the light of recent observations concerning the synthesis of myelin proteins and their proposed role in myelin assembly.     

Intracellular distribution of creatine kinase isoenzymes in the brains and hearts of rats at different stages of postnatal development]

The total activity and range of the creatine kinase (CK) isozymes have been studied in the homogenate and subcellular fractions (nuclei, mitochondria, cytoplasm) of the rat brain and heart during postnatal ontogenesis. The total activity of CK in the brain and heart of newborn rats was found to be 4 and 2 times less, resp., than in those of adults. The age patterns were established in the activity of cytoplasmic (CK-1, CK-2 and CK-3) and mitochondrial (CK-4) isozymes. During the whole postnatal development the rat brain contains only one cytoplasmic isozyme, CK-1. In the heart of newborn rats, as compared with adults, the content of CK-1 and CK-2 is much higher and that of CK-3 lower. On the 12-15th day of life the range of the CK isozymes approaches that characteristic of adult animals. The activity of CK-4 was found in the brain on the 5-7th day of life and in the heart on 12-15th day. In the range of the CK isozymes in the adult brain the content of mitochondrial CK amounts to 19.3% and in the heart to 16.5%. The data obtained complement the literary ones suggesting the low level of energy-forming processes in the brain and heart cells at the early stages of the rat postnatal development.     

Postnatal changes of cathepsin D activity in rat liver and brain

Total and specific activity of cathepsin D (EC. 3.4.23.5) were measured in rat liver and brain from 1 to 98 days of age. The activity of cathepsin D in the liver of adult and newborn rats was the same while in the rat brain it was higher in adult than in newborn rats. In the liver maximum specific activity of cathepsin D occurred on the 10th postnatal day and minimum on the fourth day of age. In the brain maximum specific activity of the enzyme occurred on the 14th postnatal day. Total activity of cathepsin D increased after birth in rat liver and brain. These results are discussed in relation to the functional role of cathepsin D in the rat liver and the brain.     

FETAL DEVELOPMENT: THE EFFECTS OF MATURATION ON IN VITRO PROTEIN SYNTHESIS BY MOUSE BRAIN TISSUE

—The elucidation of the translational regulatory events which function during the critical fetal and neonatal period is an important prerequisite to our understanding of normal, as well as abnormal, brain growth and differentiation. Brain cell suspensions and cell-free homogenates were employed to study the protein synthetic activity during the maturation of fetal- neural tissue. The results clearly demonstrated that while neural tissue from 1-day postnatal mice was 10 times more active in protein synthesis than brain tissue from adult mice, the former was many fold less active in translational events than fetal neural tissue from 13-day post-zygotic mice. Fetal polypeptide synthetic activity was found to decrease from the 13th day to the 19th day post-zygotic. This decrement in the translational activity was not due to amino acid availability or pools, or to differences, quantitatively or qualitatively, in polysome concentrations. The enhanced rate of protein synthetic activity measured with neural tissue from 13-day post-zygotic mice was shown to be due to an increase in rate of protein synthesis and not to an enhanced rate of protein degradation.     

METABOLISM OF HIGHLY BASIC PROTEINS OF RAT BRAIN DURING POSTNATAL DEVELOPMENT

Abstract— (1) The encephalitogenic basic protein obtained from adult rat brain by treatment with 0·03 N-HCl was demonstrable in the brain on the 10th day after birth. It showed a marked increase in quantity during the phase of active myelination.
(2) The proteins extracted under similar conditions from 5-day old rat brain contained several highly basic proteins other than the encephalitogenic basic protein. These basic proteins, which were electrophoretically similar to highly basic proteins extracted similarly from adult rat liver, are histones.
(3) For metabolic studies the entire group of highly basic proteins in the acid extract was obtained after one-step adsorption of other proteins on DEAE-cellulose equilibrated at pH 9·8
(4) After injection of [¹⁴C]lysine the fractions containing highly basic proteins, water soluble non-basic proteins and other tissue proteins of the brain showed higher relative specific radioactivities during the period 1–10 days after birth than during later stages of postnatal development. The fraction containing proteolipid protein, another myelin protein, showed a low relative specific radioactivity throughout the whole period of postnatal development. The relative specific radioactivity of proteolipid protein was somewhat higher in young than in adult rat brain.     

Glucocorticoid receptors in developing rat brain and liver

Dexamethasone receptors were measured by conventional equilibrium steroid binding studies in rat liver and brain cytosol, during late prenatal and postnatal development, Receptor binding could be detected in both cytosol preparations as early as the 17th day of gestation. Receptor levels in the cytosol from intact animals reached adult values by the 1st day after birth in both tissues. Using adrenalectomized animals an increase which reached adult values was observed during the first postnatal week for liver and the second postnatal week for brain. At physiological concentrations of endogenous glucocorticoids depletion of receptor from the cytosol of intact animals was minimal at postnatal day 1 and reached adult levels by day 7. chromatographie analysis in DEAE-Sephadex A50 minicolumns of unactivated and activated receptor constituents revealed the same pattern as that of adult animals. Glucocorticoid receptor complex from developing liver and brain was shown to be capable of binding to isolated adult liver nuclei after in vitro activation. However full capacity, for nuclear binding was observed in vivo, after injection of inducing doses of [³H]dexamethasone: By the end of the first week after birth adult nuclear binding capacity was observed in experiments in vivo while values peaked during the second week, in both tissues studied.     

Microglial cells of the rat brain in postnatal period (comparative immunocytochemical analysis)

In the course of the brain’s development, distribution of microglial cells was studied in rats using immunocytochemical detection. To identify the microglial cells, antibodies to lipocortin 1 (LC1) and phosphotyrosine (PT) were used. On postnatal day 1, LC1-positive microglial cells of an ameboid shape were distributed mainly in the subventricular zone; their mean density was 31±8 cells/mm² (counted across the total area of frontal sections). On postnatal day 7, microglial cells of an intermediate type were located throughout the whole brain; their density was 54±15 cells/mm². On the 15th day, LC1-positive cells were of a ramified shape, and their density reached 104±20 cells/mm² (the microglial cell density in the mature normal brain was 103±3 cells/mm²). On postnatal day 7, PT-positive cells were similar in their morphology to LC1-positive cells of an intermediate type, while their mean density was 32 cells/mm². In the mature brain, the density of PT-positive microglia was 53±5 cells/mm²; the shape of the cells in the white and gray matter of the brain was, on the whole, similar to that of LC1-positive microglia. Therefore, LC1 is a specific marker for different types of microglial cells in the developing brain. Our data about 3D distribution and morphological peculiarities of microglial cells at different stages of postnatal development are consistent with the hypothesis on the neuroectodermal origin of microglia.     

Postnatal thyroxine modifies effects of early androgen on lordosis

The sensitivity of female rats to the organizational effects of postnatal androgen was examined after néonatal manipulations known to affect the rate of brain development: thyroxine (T₄) administration and handling at birth. Testosterone propionate (TP) was injected subcutaneously in oil on postnatal day 6 to littermates that (i) had been undisturbed at birth; (ii) had received saline injections (S) and associated handling (H) on the day of birth (postnatal day 1) and the following day; and (iii) had received T₄ in saline (1 μg/g body wt) and H on postnatal days 1 and 2. Estrous cycles at 45 and 90 days of age, ovulation at Day 100 and sexual receptivity (lordosis score) at Days 115 and 125 were used to evaluate changes in TP effects. The majority of animals treated with 100 μg TP on postnatal day 6 exhibited persistent estrus (PE) at 45 and 90 days of age as expected. Neither T₄ nor S pretreatment on postnatal days 1 and 2 changed the incidence of PE. A reduction in ovarian weights and incidence of ovulation at 100 days of age supported cycle data in that only one out of 25 androgenized rats showed ovulation, compared with 16 of 22 controls. At approximately 115 days of age 2 μg of estradiol benzoate were administered for 3 days and 0.5 mg progesterone given on the 4th day 4 hr prior to placing females with sexually vigorous males. T₄-TP females exhibited higher (< .01) median lordosis scores than their S-TP littermates. The latter results were replicated in a second test conducted 10 days later (p = .002). In addition, the second test indicated that the S-TP group had lower (p = .005) lordosis scores than littermates given only TP neonatally. The results of these studies demonstrate that pretreatment with T₄ and handling, which are known, respectively, to hasten and retard the chronology of brain maturation, can exert differential effects on behavioral manifestations of postnatal TP without modifying androgen-induced sterility.     

Membrane-bound neuraminidase in the developing mouse brain

The specific activity of membrane-bound neuraminidase towards exogenously added gangliosides in the developing mouse brain was investigated. While whole brain was examined in fetuses, studies were carried out comparatively in cortex and cerebellum of postnatal stages. Considerable differences were found among brain areas. There was a rapid increase in forebrain neuraminidase activity before birth. This high level of activity was maintained throughout the first postnatal week, followed by a slow leveling-off to adult values on the 30th day. In the cerebellum a rapid increase in neuraminidase activity occurred between the 10th and 16th postnatal day. After having reached the maximum, enzyme activity declined rapidly, with adult values being reached on the 21st day. Neuraminidase activity in the adult cerebellum exceeds that in the cortex by 65.9 %. The results are discussed in reference to developmental changes in ganglioside metabolism and the possible involvement of neuraminidase in its regulation is pointed out.     

MORPHOLOGICAL AND BIOCHEMICAL CHANGES IN RAT SYNAPTOSOME FRACTIONS DURING NEONATAL DEVELOPMENT

A biochemical and quantitative morphologic study of presynaptic endings during postnatal development was carried out in subcellular fractions from cerebral cortex of 1, 4, 8, 12, and 18 day old and adult rats. Crude mitochondrial fractions were subfractionated in Ficoll gradients and all resulting fractions were examined in the electron microscope. Presynaptic terminals and other intact processes were counted. Protein content and enzyme activities were assayed in the fractions and in total brain homogenate. In the first and fourth day of life, most of the presynaptic terminals were found in two "light" fractions, between supernatant and 7.5% Ficoll, where they accounted, respectively, for 6 and 22% of all the processes. Progressively with age, more presynaptic terminals were found in the traditional "synaptosomal" fractions between 7.5 and 13% Ficoll. In that region of the gradient, 40, 54, 75, and 89% of the processes were presynaptic endings at 8, 12, and 18 postnatal days and in the adult animal, respectively. A similar shift from the lighter to the heavier fractions was observed in the distribution of choline acetyltransferase and acetylcholinesterase between days 8 and 12. The rate of increase of the specific activity of these two enzymes paralleled that of the percentage of the presynaptic endings after day 8. This study indicates that subcellular fractions can be used to study formation and maturation of synapses during postnatal development.     

Subclass of astroglia in mouse cerebellum recognized by monoclonal antibody

A monoclonal antibody was detected that distinguishes astrocyte subclasses in mouse cerebellum. This antibody, designated anti-M1, is the product of a hybridoma that arose from the fusion of NS1 myeloma cells and splenocytes derived from a rat immunized with crude membranes from early postnatal mouse cerebella. The distribution and regulation of M1 antigen expression in vivo were examined by indirect immunofluorescence on frozen thin sections of mouse brain. M1 expression shows differing age dependencies within subpopulations of astroglia. M1 is first detectable around postnatal day 7 in white matter astrocytes and persists in this cell type throughout adulthood. By postnatal day 10, M1 is additionally detected in Bergmann glial fibers and in granule layer astrocytes. M1 expression in these latter astrocytic cell types is transient and cannot be detected after the fourth postnatal week. Cerebella of adult neurological mutant weaver mice show abnormal persistence of M1 antigen expression in Bergmann glial fibers. In monolayer cultures of early postnatal cerebella, M1 antigen is detected in a subpopulation of the glial fibrillary acidic protein positive astrocytes. M1 antigen can be detected only in fixed cultured cells which allow intracellular penetration of the antibody. The developmental regulation of M1 expression and the abnormal expression of M1 in weaver mutant cerebella suggest that M1 may be a useful marker for astroglial maturation and differentiation.     

Glycosylation of the sodium channel β4 subunit is developmentally regulated and involves in neuritic degeneration

Aberrant protein glycosylation plays major roles in neurodegenerative diseases, including Parkinson's disease (PD). Glycoproteomics showed that the glycosylation of sodium channel β4 was significantly increased in human brain tissue. β4-specific antibodies reacted in immunoblot assays with the 35- and 38-kDa bands from the membrane fractions isolated from neonatal PD transgenic mice but only with the 35-kDa band of the neonatal wild-type mice. The size of the 38-kDa immunoreactive protein is in close agreement with previously reported, suggesting heavy glycosylation of this protein in adult wild-type and neonatal PD transgenic brain tissues. However, the neonatal wild-type mice membrane fractions only contained the 35-kDa immunoreactive protein, and the additional 38-kDa band was not shown until postnatal day 7. Enzymatic deglycosylation of the membrane preparations only converted the 38-kDa band into a faster migrating protein, which was consistent with heavy glycosylation of this protein. The glycosylated state of β4 was developmentally regulated and was altered in disease state. Neurite outgrowth assay demonstrated that overexpression of deglycosylated mutant β4-MUT accelerated neurite extension and increased the number of filopodia-like protrusions, when compared with β4-WT and the vector. These results suggest that extensive glycosylation of β4 subunit play roles in morphological changes, and the altered glycosylation may be involved in the pathogenesis of PD.     

移植视网膜NOS阳性神经元的发育

目的 观察不同年龄组段大鼠正常视网膜及移植视网膜内NOS阳性神经元的发育情况及其定位分布。方法 实验分正常视网膜发育组和移植视网膜发育组,应用还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）组织化学方法显示。结果 1、NOS阳性神经元最早出现于生后第五天（P5）,P18时阳性神经元数目达到最高峰,2、移植视网膜具有正常视网膜的各层结构和相似的生长规律,NOS阳性神经元在生后第4天移植视网膜（TP4）中出现,TP12数量达到高峰值,TP22后降至正常成年鼠水平。结论 根据NOS阳性神经元的定位,分布,推测其为无长突细胞,移位无长突细胞及节细胞。     

Comparative study of appearance of water-soluble antigens and brain esterase fractions in ontogenesis of two strains of rats

By means of immunoelectrophoresis of rat brain, 12 water-soluble antigens were detected, five of which were found to be specific to the brain. Histochemical reactions have identified two antigens that are not specific to the brain, lactate dehydrogenase and esterase. By means of enzymoelectrophoresis, 14 esterase fractions were determined. An immunoautoradiographic study of the synthesis of some antigens specific to the brain was carried out. It was found that rats responding to sound by epileptic seizures develop more slowly than normal rats. In particular, in rats resistant to auditory stimulus the antigenic spectrum typical of the brain of the adult rat (12 antigens) is formed by day 14 of postnatal life, while in rats of the sensitive strain this pattern takes 17 days to completely form. The last brain-specific antigen for rats susceptible to audiogenic epilepsy appears on day 17, while this antigen is present in normal rats on day 14. Fast-moving esterase fractions are detected earlier in ontogenesis in rats resistant to sound than in sensitive rats.     

Ontogenic pattern of dopamine, acetylcholine, and acetylcholinesterase in the brains of normal and hypothyroid rats.

The influence of neonatal thyroidectomy (Tx) on developmental changes in dopamine (DA), acetylcholine (ACh), and acetylcholinesterase (AChE) was studied in the whole brain of rats. In control animals, brain levels of ACh gradually increased and attained adult values at the 70th day. In contrast, AChE activity showed a rapid increase between the 7th and 30th days. Levels of DA were low during the early postnatal life but markedly increased to reach adult values of 1.47 mug/g at the 30th day, after which no further enhancement was noted. Neonatal Tx interfered with the normal growth of the animals, decreased brain weights, and markedly influenced the developmental pattern of both DA and ACh in the brain. The concentration of DA in 30-day-old hypothyroid rats was 46% of the control values. In contrast, brain ACh levels in Tx rats were consistently above those seen in controls, being significantly higher, by 49 and 64%, at 15 and 30 days, respectively. Activity of AChE in brains of hypothyroid animals was not significantly different from that in controls. Treatment of Tx rats with thyroid hormone virtually restored the levels of DA and ACh to values in control animals.     

Uridine kinase activities in developing, adult and neoplastic rat tissues.

Uridine kinase activities were found chiefly in the soluble fractions of rat tissues. In normal adults the activities ranged from 13 munits/g in skeletal muscle to 178 munits/g in colon. Enzyme activities in several rat neoplasms were significantly higher (e.g. in a fibrosarcoma, mammary carcinoma, renal carcinoma, pancreatic carcinoma and lymphocytic lymphoma, but not in a fast-growing Morris hepatoma). The activities were not related to tumour growth rates or sizes. In normal foetal liver, lung, brain, heart and kidney, uridine kinase concentrations equalled or exceeded those in the adult homologous tissue, but maximal activities in liver were reached 3--5 days post partum. In suckling rats the intestinal activity decreased substantially immediately after birth and normally did not rise again until late in the third postnatal week. Premature upsurges could be evoked by an injection of cortisol or by starvation of the pups overnight. Pancreatic activity was absent from 1-day-old rats, and only about 5% of the adult activity was reached by day 20; adult activities were attained rapidly after weaning. In pancreas, precocious formation or uridine kinase was elicited by overnight starvation of 2-week-old rats.