Effect of immunization with neurospecific proteins and tubulin on learning in the rat

The paper deals with the effect of anticerebral antibodies on rats learning in T-shaped maze with food reinforcement. The rats of August line were immunized by neurospecific proteins 10-40-4 from the human and rat's brain, 14-3-2 and cerebral tubuline. Rats immunized by bovine serum albumine (BSA) served as a control of the influence of antibodies to noncerebral protein. Rats injected with Freund adjuvant and saline, served as control of Freund adjuvant action. At the time of antibodies formation (from the 7-th day after the last injection) the rats were trained in a T-shaped maze during 4 days. The acquisition of conditioned reactions was inhibited in rats immunized with neurospecific proteins 10-40-4 from the rats brain, 14-3-2 and cerebral tubuline. Antibodies to neurospecific protein 10-40-4 from the rats brain produced the greatest effect on the process of learning. They elicited a significant decrease of conditioning and an increase of the number of errors. Antibodies to the neurospecific protein 10-40-4 from the human brain and to the BSA, i.e. to proteins which are not present in the nervous tissue of the rats, did not affect the learning. The obtained results indirectly confirm the permeability for the antibodies of the blood-brain barrier in immunized animals.     

A blood-CSF barrier function controls embryonic CSF protein composition and homeostasis during early CNS development

In vertebrates, early brain development takes place at the expanded anterior end of the neural tube, which is filled with embryonic cerebrospinal fluid (E-CSF). Most of the proteins contained within the E-CSF, which play crucial roles in CNS development, are transferred from the blood serum. Two important questions are how E-CSF is manufactured and how its homeostasis is controlled. In this respect, the timing of the blood-CSF barrier formation is controversial. Recently, the concept of a functional dynamic barrier has been introduced. This type of barrier is different from that found in adults and is adapted to the specific requirements and environment of the developing nervous system. In this study, we injected a number of proteins into the outflow of the heart and into the cephalic cavities and examined their transport rate between these two embryo compartments. The results indicated that a functional blood-CSF barrier dynamically controls E-CSF protein composition and homeostasis in chick embryos before the formation of functional choroid plexuses. We also showed that proteins are transferred through transcellular routes in a specific area of the brain stem, close to the ventral mesencephalic and prosencephalic neuroectoderm, lateral to the ventral midline, in particular blood vessels. This study contributes to our understanding of the mechanisms involved in CNS development, as this blood-CSF interface regulates the composition of E-CSF by regulating its specific composition.     

Astrocyte–Endotheliocyte Axis in the Regulation of the Blood–Brain Barrier

Neurochemical Research - The evolution of blood–brain barrier paralleled centralisation of the nervous system: emergence of neuronal masses required control over composition of the...     

Dissemination pathways for poliovirus cells to animals models

It is considered there are two main pathways for poliovirus dissemination towards the central nervous system in humans. One is the pathway through the blood brain barrier. The orally ingested virus invades into the blood circulation, and then the virus permeates into the central nervous system through the blood brain barrier. The other is the neural pathway. In this pathway, the intramuscularly-inoculated virus is transported through the axons from the synapse to the cell body in the central nervous system. We have developed the oral infection system using the mouse models. Moreover, we proposed the possibility that PV is transcytosed through the brain capillary epithelia in a specific manner. As for the neural pathway, we have proved that PV is endocytosed into CD155 containing vesicles and the vesicles are retrogradely transported in the axon of rat primary motor neuron. We have also shown that the cytoplasmic dynein takes part in the transport.     

Endothelial vesicles as a transport system in the blood-brain barrier. Morphometric study during development

The vesicles and vacuoles of the endothelia, morphological expression of endocytosis and transendothelial transport, are quite absent in the mature neural endothelia. In order to study the temporal sequence of the vesicle and vacuole modifications during the blood brain barrier (bbb) setting up, the extent of these structures was morphometrically analyzed on electron micrographs of neural microvessels in the optic tectum of 8, 14, and 17 day chick embryos, fixed after an intracardial injection of the permeability marker horseradish peroxidase. During the development, endocytosis and transendothelial transport change, since a statistically significant reduction of both vesicles and vacuoles was recorded at the 17th incubation day. The temporal coincidence between decrease of endocytosis-transport processes and appearance of astrocytic endfeet close to the vessel wall, suggests that the glial cells might control, besides the tight junction formation, the expression of other properties of the bbb-provided endothelia.     

In vitro insertion of the myelin proteolipid apoprotein into oligodendrocyte plasma membranes

Uncoated vesicles (UCV) loaded with the myelin proteolipid apoprotein covalently tagged with fluorescein (PLP_F) were found to interact with isolated oligodendrocytes from bovine brain at 4°C as well as at 37°C. After 1.5 hours of incubation, the labeled protein was localized in the cell membranes. After 2.5 hours the fluorescence intensity associated with the oligodendrocytes decreased and completely disappeared at t=3.5 hours. Addition of KCl or EDTA in the incubation medium significantly hindered the interaction with cells. In contrast, the elimination of membrane proteins from UCV did not perturb cell labeling. A specific role of PLP was suggested since UCV loaded with a soluble protein (BSA_F) led to a weak cell labeling.Abbreviations IAF 5-iodacetamidofluorescein - BSA bovine serum albumin - BSA BSA labelled with IAF - PLP proteolipid apoprotein - PLP_F aqueous form of PLP tagged with IAF - CV coated vesicles - UCV uncoated vesicles - UCV^*PLP_F UCV loaded with PLP_F - MV model vesicles This work was suported by Cnrs and INSERM.     

Brain induces the expression of an early cell surface marker for blood-brain barrier-specific endothelium.

Capillaries derived from the perineural vascular plexus invade brain tissue early in embryonic development. Considerably later they differentiate into blood-brain barrier (BBB)-forming blood vessels. In the chick, the BBB as defined by impermeability for the protein horseradish peroxidase develops around embryonic day 13. We have previously found that brain endothelial cells start to express a number of proteins at around the same time, suggesting that these proteins play a role in BBB function. Here we describe a 74 kd protein defined by the monoclonal antibody HT7 that is expressed on the surface of chick embryonic blood cells and brain endothelial but on no other endothelial cells. This protein is not detectable on early embryonic brain endothelium, but is expressed by these cells on embryonic day 10. It is absent in choroid plexus endothelial cells which represent permeable fenestrated endothelial cells. The antigen is expressed on choroid plexus epithelium which is the site of the blood-cerebrospinal fluid barrier. Since it is also found in basolateral membranes of kidney tubules, it may be involved in specific carrier mechanisms. Embryonic mouse brain tissue transplanted on the chick chorio-allantoic membrane induces the expression of this antigen on endothelial cells derived from the chorio-allantois. Brain tissue can therefore induce in endothelial cells in vivo the expression of a molecule characteristic of brain endothelium.     

Detection and isolation of glycoproteins and nuclear proteins in the bovine brain

Using ammonium sulphate precipitation, ion exchange chromatography and preparative isoelectrofocusing, 9 organ-specific glycoproteins and 16 specific nuclear proteins were isolated from bovine brain nervous tissue in a homogeneous state. The isolation of proteins was controlled by a solid phase immunoenzymatic analysis. The molecular weight, subunit composition and isoelectric points of the proteins were determined and their ability to interact with immobilized calf thymus DNA and concanavalin A was demonstrated. It was assumed that the multiplicity of specific proteins of brain tissue is a molecular basis which provides for the functional specificity of the nervous tissue at large.     

Methyl mercury toxicity in the chick embryo

The toxicity of methyl mercury (mHg) in the developing chick embryo was investigated. The relationship of dose, time of administration (i.e., days 4-9 of development), and body levels of mercury was examined. The LD50 for mHg injected into the yolk sac on day 5 of incubation was 40-50 micrograms. Embryos dying within 24 hours showed increased total body mHg levels when compared to survivors (219 +/- 67 vs. 105 +/- 41 micrograms/gm, mean +/- SD). Absorption was dose-related, with a good correlation between mortality and body, blood, and brain levels. Daily analysis of body mHg levels after injection on day 5 showed continued mHg accumulation (0.88 +/- 0.35 micrograms/embryo/day). However, the rate of embryo growth exceeded the rate of mHg absorption, resulting in a progressive decrease in mHg in concentration in tissues (from 94.5 +/- 34.2 micrograms/gm on day 6 to 45.3 +/- 13.4 on day 9). Administration after day 5 resulted in a significant reduction in levels of mHg in the brain on day 18 (from 11.4 +/- 2.1 micrograms/gm when given on day 5 to 8.4 +/- 2.3 when given on day 9) and in mortality (from 64% to 33%). Because blood mHg levels remained unchanged, the increased brain levels and higher mortality early in embryogenesis may reflect facilitated transfer of mHg across a poorly developed blood-brain barrier. Later in development, the reduced mortality and lower brain mHg levels correspond to the formation of specialized interendothelial junctions and a more effective blood-brain barrier.     

Isolation and some physico-chemical properties of a new neurospecific protein 10-40-4 from human brain

A method for isolation of a neurospecific protein 10-40-4 from human brain has been elaborated. This procedure includes immunoaffinity chromatography of a Sepharose 4B-IgG fraction of rabbit antisera against the protein fraction containing the antigen. The isolated protein cannot be detected in protein extracts of various organs and human blood serum by immunochemical methods. This indicates that the protein is specific for nervous tissue. The values of molecular weight (74 000) and pI (4.7) of the isolated protein suggest that the protein does not contain the carbohydrate component and reveals limited tissue specificity. The properties of protein 10-40-4 differ from those of the well-known neurospecific proteins, such as S-100, enolase 14-3-2 and glial fibrillar acid protein GFA.     

Clathrin-coated vesicles in nervous tissue are involved primarily in synaptic vesicle recycling

The recycling of synaptic vesicles in nerve terminals is thought to involve clathrin-coated vesicles. However, the properties of nerve terminal coated vesicles have not been characterized. Starting from a preparation of purified nerve terminals obtained from rat brain, we isolated clathrin-coated vesicles by a series of differential and density gradient centrifugation steps. The enrichment of coated vesicles during fractionation was monitored by EM. The final fraction consisted of greater than 90% of coated vesicles, with only negligible contamination by synaptic vesicles. Control experiments revealed that the contribution by coated vesicles derived from the axo-dendritic region or from nonneuronal cells is minimal. The membrane composition of nerve terminal-derived coated vesicles was very similar to that of synaptic vesicles, containing the membrane proteins synaptophysin, synaptotagmin, p29, synaptobrevin and the 116-kD subunit of the vacuolar proton pump, in similar stoichiometric ratios. The small GTP-binding protein rab3A was absent, probably reflecting its dissociation from synaptic vesicles during endocytosis. Immunogold EM revealed that virtually all coated vesicles carried synaptic vesicle proteins, demonstrating that the contribution by coated vesicles derived from other membrane traffic pathways is negligible. Coated vesicles isolated from the whole brain exhibited a similar composition, most of them carrying synaptic vesicle proteins. This indicates that in nervous tissue, coated vesicles function predominantly in the synaptic vesicle pathway. Nerve terminal-derived coated vesicles contained AP-2 adaptor complexes, which is in agreement with their plasmalemmal origin. Furthermore, the neuron-specific coat proteins AP 180 and auxilin, as well as the alpha a1 and alpha c1-adaptins, were enriched in this fraction, suggesting a function for these coat proteins in synaptic vesicle recycling.     

扬子鳄胚胎中脑视叶的组织发生

观察了16例不同时间扬子鳄胚胎中脑视叶的组织发生过程。胚胎孵育第6d,三个脑泡明显;孵育第9～10d,中脑泡可分细胞层和边缘层或纤维层,中脑水管未形成;孵育第18d,视叶隆起于中脑背侧,中脑水管形成,视叶分三层;孵育第24d,视叶分5层;孵育第34d,视叶分化为6层;孵育第51d,视叶分化为8层,与初生扬子鳄中脑视叶分层相同。     

Relative stability of alpha-melanotropin and related analogues to rat brain homogenates

alpha-Melanotropin (alpha-MSH) retains less than 1% of its original activity after a 60 min incubation with 10% rat brain homogenate. [Nle4,D-Phe7]-alpha-MSH is nonbiodegradable in rat serum (240 min incubation) and still maintains 10% of its original activity in 10% rat brain homogenate (240 min incubation). The related fragment analogue, Ac-[Nle4,D-Phe7]-alpha-MSH4-10-NH2, retains 50% of its activity after a 240 min incubation in rat brain homogenate, whereas Ac-[Nle4,D-Phe7]-alpha-MSH4-11-NH2 is totally resistant to inactivation by rat brain homogenate. Both [Nle4,D-Phe7]-fragments are resistant to degradation by rat serum, but [Nle4]-alpha-MSH, Ac-[Nle4]-alpha-MSH4-10-NH2 and Ac-[Nle4]-alpha-MSH4-11-NH2 are rapidly inactivated under both conditions. The cyclic melanotropin, [Cys4,Cys10]-alpha-MSH, is inactivated in rat brain homogenate as is the shorter Ac-[Cys4,Cys10]-alpha-MSH4-10-NH2 analogue, but neither cyclic melanotropin is inactivated upon incubation in serum from rats. Ac-[Cys4,D-Phe7,Cys10]-alpha-MSH4-10-NH2 is resistant to inactivation by either rat serum or a brain homogenate. Some of these melanotropin analogues may provide useful probes for the localization and characterization of putative melanotropin receptors in both the central nervous system and peripheral tissues.     

Effect of cardiac arrest on brain weight and the permeability of the blood-brain and blood-spinal cord barrier to albumin and tumor necrosis factor-alpha

Time-dependent changes in brain and spinal cord were studied in mice in a cardiac arrest model. A transient decrease in body weight and a prolonged decrease in brain weight occurred after arrest whereas spinal cord weight was unchanged. The permeability of the blood-brain barrier (BBB) to I131-albumin and I131 tumor necrosis factor-alpha (TNF) showed maximal, non-significant increases on day 5 after cardiac arrest, but the permeability of the blood-spinal cord barrier (BSCB) to both materials was unchanged with time. We conclude that selective weight loss occurs in the brain after cardiac arrest with the integrity of the BBB and BSCB remaining intact to serum proteins and minimal alteration in the blood to CNS transport of TNF.     

Aldehyde dehydrogenase activity in the barrier brain structures

The histochemical method was used to study the aldehyde dehydrogenase (EC 1.2.1.3.; ALDH) activity in capillaries and glial structures of different regions in the rat central nervous system (CNS). The occurrence of three metabolic barriers for aldehydes on systemic level in the CNS has been shown. They are: the barrier between blood and the nervous tissue (represented by capillary endothelium and surrounding astrocytes ALDH), that between blood and cerebrospinal fluid (ALDH in ependymocytes of vascular plexus), and that between cerebrospinal fluid and nervous tissue (ALDH of ependymocytes covering brain cavities). On the single microregions level a similar barrier is between interstitial fluid and neurons (ALDH of satellite oligodendrocytes).     

Diabetes-induced biochemical changes in central and peripheral catecholaminergic systems

A great variety of alterations have been described in the nervous system of diabetic animals. They are named as diabetic neuropathy and affect the brain, spinal cord and peripheral nerves. In diabetic animals, plasma and tissue catecholamine levels have been reported to be increased, decreased or unchanged, and these disparities have been explained by differences in the tissues selected, severity or duration of diabetes. Dopamine, norepinephrine and epinephrine from different tissues were extracted by absorption onto alumina, and measured by high performance liquid chromatography with electrochemical detection. We found that diabetes alters catecholaminergic systems in a highly specific manner. The dopamine content is reduced in the dopaminergic nigrostriatal system only. Norepinephrine is differently altered in several areas of the sympathetic nervous system. It is increased in cardiac ventricles, and decreased in stellate ganglia and the blood serum. However, it is not altered in the central nervous system. Finally, epinephrine is only altered in the adrenal gland where it is increased, and in the serum where it is reduced. Our results suggest that diabetes reduces the activity of the nigrostriatal dopaminergic system. Changes found at the sympathoadrenal level could be explained by reduced norepinephrine and epinephrine synthesis, with increased storage due to a reduced release from synaptic vesicles.     

Ultrastructural changes in the MP3 neuron of the mollusk Lymnaea stagnalis after cryopreservation of the isolated brain

Investigation of a possibility of long-term storage of frozen (-196 degrees C) viable neurons and nervous tissue is one of the central present day problems. In this study ultrastructural changes in neurons of frozen-thawed snail brain were examined as a function of time. We studied the influence of cryopreservation, cryoprotectant (Me2SO), cooling to 4-6 degrees C, and a prolonged incubation in physiological solution at 4-6 degrees C on dictyosomes of Golgi apparatus, endoplasmic reticulum (ER) cisternae and mitochondria. It has been found that responses of these intracellular structures of cryopreserved neurons to the above influences are similar: dissociation of Golgi dictyosomes, swelling of endoplasmic reticulum cisternae and mitochondrial cristae. Both freezing-thawing and cryoprotectant were seen to cause an increase in the number of lysosomes, liposomes, myelin-like structures, and to form large vacuoles. The structural changes in molluscan neurons caused by cryopreservation with Me2SO (2 M) were reversible.     

Distribution of 125I-labeled rat growth hormone in regional brain areas and peripheral tissue of the rat.

The uptake of intraperitoneally injected 125I-labeled rat growth hormone into brain and peripheral tissues was measured in normal and hypophysectomized adult rats. A significant level of radioactivity was observed in the seven brain regions examined -- the telencephalon, diencephalon, midbrain, pons-medulla, cerebellum, pineal and pituitary glands. The pineal and pituitary glands, which are outside the blood-brain barrier, contained three to four times the concentration of radioactivity of the other brain regions. Compared to brain, the level of radioactivity was much higher in peripheral tissues (the diaphragm, kidney, serum and liver). For example, the serum contained ten times the level of radioactivity of most brain regions. For a given tissue, however, the normal and hypophysectomized rats showed a comparable amount of 125I-growth hormone. Trichloroacetic acid precipitates from each tissue sample showed that peripheral tissues had a higher proportion of radioactivity (35-48% of total tissue radioactivity) than the brain samples (13-26%). The data support the view that growth hormone, or a metabolite can enter the central nervous system and may directly affect on-going metabolic processes.     

Characterization of a membrane protein from cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata

Rabbits were immunized with cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata. The resultant antiserum had one major antibody activity against an antigen called the Torpedo vesicle antigen. This antigen could not be demonstrated in muscle, liver or blood and is therefore, suggested to be nervous-tissue specific. The vesicle antigen was quantified in various parts of the nervous system and in subcellular fractions of the electric organ of Torpedo marmorata and was found to be highly enriched in synaptic vesicle membranes. The antigen bound to concanavalin A, thereby demonstrating the presence of a carbohydrate moiety. By means of charge-shift electrophoresis, amphiphilicity was demonstrated, indicating that the Torpedo vesicle antigen is an intrinsic membrane protein. The antigen was immunochemically unrelated to other brain specific proteins such as 14-3-2, S-100, the glial fibrillary acidic protein and synaptin. Furthermore, it was unrelated to two other membrane proteins, the nicotinic acetylcholine receptor and acetylcholinesterase, present in Torpedo electric organ. The antiserum against Torpedo synaptic vesicles did not react with preparations of rat brain synaptic vesicles or ox adrenal medullary chromaffin granules.     

The role of blood-brain barrier in the development of childhood febrile seizures and temporal lobe epilepsy

The blood-brain barrier (BBB) of the central nervous system (CNS) is a physiological barrier that makes it possible to control the exchange of ions, molecules and cells between blood and brain tissue and prevent their free inflow into the brain. BBB is crucial for maintenance of brain homeostasis. The BBB damage accompanies many degenerative, neurological and inflammatory (infectious or noninfectious) diseases and pathological states. Current review reports about the BBB role in the development of childhood febrile seizures and temporal lobe epilepsy.