Space Marker Research Articles

Entry of water, metabolic substrates and extracellular space markers into various structures of mouse brain in vivo.

Une méthode nouvelle est présentée pour étudier des corrélations qui existent entre le cerveau et le comportement des animaux.

Cell membrane resorption in the rat exocrine pancreas cell after in vivo stimulation of the secretion, as studied by in vitro incubation with extracellular space markers.

Pancreatic secretion in the rat was stimulated in vivo by pilocarpine injection causing 90% of the storage granules to be discharged within 2 h. Incubation in vitro with [(14)C]sorbitol indicated that maximal ingestion of this extracellular space marker occurred 3 h after secretogogue injection. Morphological cell membrane measurements on cells with stimulated secretion revealed a simultaneous decrease in amount of membrane bordering the microvilli at the cell apex, lamellar processes, and infoldings present at the latero-basal face of these cells. In 3-h stimulated cells, having the average zymogen granule content characteristic for that phase of secretion, ferritin treatment in vitro showed that the infoldings and related fragmentation vesicles had ingested ferritin and could consequently be considered as being transport vehicles for redundant cell membrane. During stimulated secretion numerous vesicles and vacuoles appeared in the apical cytoplasm. Part of these structures were postulated to be related to the Golgi complex and were discussed in relation to secretory protein transport. Another part of these structures was assumed to have an endocytotic nature, although they never contained ferritin.

Sodium uptake by frog skin and its modification by inhibitors of transepithelial sodium transport.

1. The suitability of inulin and mannitol as markers of the diffusional space that exists between the surface of frog skin and the outer barrier to sodium transport has been compared in experiments designed to measure the rapid uptake of sodium.2. Inulin and mannitol both equilibrate finally with the same space at the outer surface of the frog skin, but the rate at which this equilibration occurs is considerably faster for mannitol.3. The dependence of the rapid uptake of sodium on the concentration of sodium in the external medium, using mannitol to correct for extracellular sodium, can be described by simple saturation kinetics with an apparent K(m) for sodium entry of 24 mM and a maximal rate of uptake of 1.28 muequiv/cm(2). hr.4. The effect of amiloride is to inhibit sodium uptake, the degree of inhibition depending both on the concentration of sodium in the external medium and on the level of transepithelial transport normally maintained by the skin.5. Ouabain inhibits sodium uptake when the tissue has been pre-incubated in sodium containing medium. It has no effect on sodium uptake if the pre-incubation takes place in sodium free medium.6. A short-circuit current and potential difference can be elicited across frog skin in the presence of ouabain, by increasing the concentration of sodium bathing the outside surface. This potential and short-circuit current is abolished by the presence of amiloride.7. These results provide direct evidence that amiloride acts to stop entry of sodium into the transport compartment and suggest that the ouabain inhibition of sodium uptake is mediated primarily through an increase of epithelial sodium concentration.

Compartmental analysis of efflux of extracellular space markers in rabbit detrusor muscle.

Mit Hilfe des Computers wurde die Abgabe von [14C]-markiertem Mannit, Succrose und Inulin aus isoliertem Kaninchen-Detrusormuskel analysiert. Alle drei Verbindungen werden aus mindestens drei Abschnitten abgegeben und aus einer gebundenen Fraktion gelost. Die Grosse des am raschesten von Mannit und Succrose beanspruchten Abschnittes entsprich derjenigen des Inulins.

Distribution of intraventricular horseradish peroxidase in normal and hydrocephalic cat brains.

The distribution of intraventricularly injected horseradish peroxidase (HRP) was examined in the ependymal and subependymal tissue of both normal and experimental hydrocephalic cats by light and electron microscopy. At different time intervals after the intracisternal injection of kaolin, the hydrocephalic ventricles were perfused at various intraventricular pressures with mock cerebrospinal fluid (CSF) containing HRP. The uptake and localization of HRP by periventricular brain tissue was studied with respect to transventricular absorption of cerebrospinal fluid. The results of both light and electron microscopic studies have shown that in normal, acute or chronic hydrocephalic cats perfused at either high or low pressure the distribution of HRP is similar. The enzyme was localized mainly to the intercellular spaces, between ependymal cells, glial cells, and in perivascular spaces (in and across the basement membrane of subependymal blood vessels). HRP did not penetrate endothelial tight junctions even in chronic hydrocephalic cats undergoing significant rates of transventricular absorption of CSF. It is concluded that since the distribution of HRP in brain is determined by diffusion, its use as a tracer for the bulk movement of CSF through nervous tissue is limited. It is, however, an excellent extracellular space marker in electron microscopy.

Uptake of nicotine and extracellular space markers by isolated rat ganglia in relation to receptor activation.

1. Uptake of (3)H-nicotine by isolated rat superior cervical sympathetic (SCG) and nodose (NG) ganglia was measured in vitro. Depolarization of the ganglia by nicotine was measured electrically.2. Nicotine depolarized the SCG but not the NG. The mean ED50 for depolarization was 5.3 x 10(-6)M.3. Both ganglia accumulated nicotine when incubated in 3.1 x 10(-5)M(3)H-nicotine: after 30 min incubation the ratios of tissue to medium concentrations were (mean +/- S.E. of mean): SCG, 3.49 +/- 0.13; NG, 2.50 +/- 0.09.4. Total water contents, estimated by drying to constant weight, were: SCG, 83.8 +/- 0.12%; NG, 80.1 +/- 0.21%. Extracellular spaces, measured as (3)H-mannitol space, were: SCG, 38.8 +/- 1.3; NG, 40.3 +/- 0.8% wet weight. These values were not significantly altered by nicotine.5. Correction for tissue fluid spaces indicated that the ratio of the mean intracellular fluid concentration to the extracellular fluid concentration for (3)H-nicotine at 3.1 x 10(-5)M were: SCG, 7.4; NG, 5.6. The ratios were not altered in any consistent manner on varying the nicotine concentration between 3.1 x 10(-7) and 1.6 x 10(-4)M.6. When the nicotine concentration was sufficiently great (6.2 x 10(-6)M or more) to evoke large SCG depolarizations, hexamethonium (2.5 x 10(-3)M) reduced (3)H-nicotine uptake by the SCG by up to 19% without affecting uptake by the NG, and thereby reduced the uptake difference between the two ganglia. With nicotine concentrations <6.2 x 10(-6)M, hexamethonium did not modify uptake by either ganglion.7. It was concluded that nicotine may be concentrated within neurones, and that such intracellular accumulation may be augmented during depolarization induced by nicotine.

Intracellular accumulation of free fatty acids in isolated white adipose cells

A simple, rapid, and accurate method was developed for measuring intracellular FFA levels in isolated white adipose cells using sucrose-(14)C or inulin carboxyl-(14)C as nontransportable, nonutilizable markers of the extracellular space. Following incubation, medium and cells were separated by centrifugation and the infranatant medium was removed by aspiration. The volume of medium trapped between cells was determined by measuring the amount of sucrose-(14)C or inulin carboxyl-(14)C retained in the floating packed adipose cells. In this way the FFA content of the adipose cells could be corrected for contamination by FFA bound to extracellular albumin. With this technique the initial events in hormone-activated lipolysis were studied under conditions of maximal and constant rates of triglyceride hydrolysis. The FFA content of isolated adipocytes of fed rats was 0.5 micro mole/g cell lipid. On addition of norepinephrine in the presence of medium albumin, the concentration of intracellular FFA rapidly increased and reached a plateau at a concentration of 2-2.5 micro moles/g cell lipid. In the presence of medium albumin an initial lag in glycerol release occurred and this was attributed to partial hydrolysis of triglyceride with retention of lower glycerides. After 5 min of incubation FFA and glycerol output was constant. In the absence of medium albumin norepinephrine-stimulated lipolysis was reduced more than 90% and extracellular FFA release was not detected. Nevertheless, intracellular FFA accumulation was identical to that seen in the presence of albumin. The data suggest that most of this intracellular pool of FFA is bound to cytoplasmic constituents.

The effect of loading and extraction temperatures, composition of medium, and molecular size on retention of extracellular space marker by mouse cerebrum slices.

Mouse cerebrum slices were incubated for 1 hour at either 37° or 0° in media containing the ‘extracellular space markers’, inulin, sucrose, D-mannitol, D-sorbitol, sulfate, or thiocyanate. They were then transferred to marker-free medium, and the marker was extracted for 1 hour. With both loading temperatures, some marker was retained at 37°, and an equal or greater amount was retained at 0°. The greater the molecular weight of the carbohydrate marker, the higher the NaCl concentration needed for greater retention upon extraction at 0° than at 37°. Slices loaded at 0° required a higher salt concentration than those loaded at 37° for greater retention to occur upon extraction at 0°. Experiments in media made hypertonic with D-mannitol, D-sorbitol, or choline chloride instead of additional NaCl show that the effect of additional salt is not simply due to increased tonicity. Unlike the other markers, thiocyanate did not show a retention difference. Although the results correlate poorly with ‘second marker sp...

The effect of tonicity, d-mannitol, d-sorbitol, and choline chloride on the water content of mouse brain slices, and on the compartmentation of the space indicated by ‘extracellular space markers’

(1) Mouse cerebrum slices were incubated with ‘extracellular space markers’ in either HEPES-( N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) buffered media with various concentrations of NaCl, or standard HEPES-buffered medium with 400 mM choline chloride, 800 mM d-mannitol, or 800 mM d-sorbitol added. Nominal tonicities of the media ranged from 170 to 1100 mosM. (2) Tissue swells extensively in media ranging from hypotonic to hypertonic containing various salt concentrations. Swelling is less in the choline chloride medium, and is slight in the d-mannitol or d-sorbitol medium. (3) In media with various salt concentrations, the penetration of inulin, sucrose, d-mannitol, d-sorbitol, and sulfate into tissue is greater at 37°C than at 0°C, showing the presence of a second marker (inulin) space for these substances; i.e., a tissue compartment which is accessible to the marker substance at 37°C but not at 0dgC (Cohen et al. 6). At both temperatures, the smaller the marker molecule, the larger the indicated ‘extracellular space’; and the greater the NaCl concentration, the greater the marker penetration. The size of the second space is only slightly dependent on the marker, or on the salt concentration. For some markers, the second space is reduced or absent in the sugar alcohol or choline chloride medium. (4) Several ‘extracellular space markers’ may enter some cellular compartment. The 800 mM sugar alcohols increase the permeability of cells to markers. Choline chloride, d-mannitol, and d-sorbitol, in high concentrations, enter most tissue water but are not actively concentrated. (5) The entry of thiocyanate is anomalous, being consistently greater at 0°C than 37°C. (6) In high NaCl medium, d-glutamate at 0°C penetrates tissue to the same extent as inulin at 37°C thus indicating the continuing existence at 0°C of a tissue compartment corresponding to the 37°C inulin space. Several other amino acids give larger tissue spaces at 0°C, with concentrative uptake of l-histidine and l-leucine occurring by 20 min incubation, and glycine by 60 min incubation. (7) It has been suggested that the second marker space may consist of a ramifying system of microtubules extending throughout a cell body and communicating with the extracellular space through a micropore. The effects of the incubation medium on the second space and on the total tissue water may be due in part to changes in the size of these tubules.

The temperature-dependent compartmentation of the ‘extracellular space’ in mouse brain slices as revealed by the markers: Inulin, sucrose, d-mannitol, d-sorbitol and sulfate

Summary A new HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid)-buffered medium for incubating brain slices is described. The penetration of the ‘extracellular space markers’, inulin, sucrose, d -mannitol, d -sorbitol and sulfate into mouse cerebrum slices is greater at 37°C than at 0°C, indicating the existence of asecond marker (inulin) space for these substances. This second space is a tissue compartment which is accessible to the marker substance at 37°C but not at 0°C. At both temperatures, the smaller the marker molecule the larger the indicated extracellular space. A second inulin space also exists in cat and guinea pig brain slices. The penetration of d -glutamate into mouse cerebrum slices at 0°C is equal to the penetration of inulin at 37°C. This shows the continuing existence at 0°C of a tissue compartment corresponding to the 37°C inulin space. Other amino acids studied at 0°C penetrate more than d -glutamate. l -Arginine and l -histidine show concentrative uptake after 80 min incubation. The second marker space may consist of a ramifying system of tubules extending throughout a cell body and connecting to the extracellular space by a micropore.

Biochemical and ultrastructural properties of a mitochondrial inner membrane fraction deficient in outer membrane and matrix activities.

Treatment of the inner membrane matrix fraction of rat liver mitochondria with the nonionic detergent Lubrol WX solubilized about 70% of the total protein and 90% or more of the following matrix activities: malate dehydrogenase, glutamate dehydrogenase, and isocitrate dehydrogenase (NADP). The Lubrol-insoluble fraction was enriched in cytochromes, phospholipids, and a Mg(++)-stimulated ATPase activity. Less than 2% of the total mitochondrial activity of monoamine oxidase, an outer membrane marker, or adenylate kinase, an intracristal space marker could be detected in this inner membrane fraction. Electron micrographs of negatively stained preparations showed vesicles (</=0.4 micro diameter) literally saturated on the periphery with the 90 A ATPase particles. These inner membrane vesicles, which appeared for the most part to be inverted with respect to the normal inner membrane configuration in intact mitochondria, retained the succinicoxidase portion of the electron-transport chain, an intact phosphorylation site II with a high affinity for ADP, and the capacity to accumulate Ca(++). A number of biochemical properties characteristic of intact mitochondria and the inner membrane matrix fraction, however, were either absent or markedly deficient in the inner membrane vesicles. These included stimulation of respiration by either ADP or 2,4-dinitrophenol, oligomycin-sensitive ADP-ATP exchange activity, atractyloside sensitivity of adenine nucleotide requiring reactions, and a stimulation of the Mg(++)-ATPase by 2,4-dinitrophenol.

Selective disruption of the sarcotubular system in frog sartorius muscle. A quantitative study with exogenous peroxidase as a marker.

Skeletal muscles which have been soaked for 1 hr in a glycerol-Ringer solution and then returned to normal Ringer solution have a disrupted sarcotubular system. The effect is associated with the return to Ringer's since muscles have normal fine structure while still in glycerol-Ringer's. Karnovsky's peroxidase method was found to be a very reliable marker of extracellular space, filling 98.5% of the tubules in normal muscle. It was interesting to note that only 84.1% of the sarcomeres in normal muscle have transverse tubules. The sarcotubular system was essentially absent from glycerol-treated muscle fibers, only 2 % of the tubular system remaining connected to the extracellular space; the intact remnants were stumps extending only a few micra into the fiber. Thus, glycerol-treated muscle fibers provide a preparation of skeletal muscle with little sarcotubular system. Since the sarcoplasmic reticulum is not destroyed and the sarcolemma and myofilaments are intact in this preparation, of the properties of the sarcolemma may thus be separated from those of the tubular system.

Extracellular fluid, ionic distribution and exchange in isolated frog brain.

ARTICLESExtracellular fluid, ionic distribution and exchange in isolated frog brainMW Bradbury, M Villamil, and CR KleemanMW Bradbury, M Villamil, and CR KleemanPublished Online:01 Mar 1968https://doi.org/10.1152/ajplegacy.1968.214.3.643MoreSectionsPDF (2 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByIs isosmotic MgSO4 isotonic with Condylactis gigantea tentacles? Comparison of their ionosmotic response to H2O–diluted seawater before and after exposure to isosmotic MgSO4–sodium seawater mixturesJournal of Experimental Marine Biology and Ecology, Vol. 297, No. 1History and Basic ConceptsIntracellular pH in the Toad Bufo Marinus Following Hypercapnia1 November 1991 | Journal of Experimental Biology, Vol. 161, No. 1History of the Blood-Brain Barrier ConceptCerebrovascular Permeability Coefficients to Sodium, Potassium, and Chloride5 October 2006 | Journal of Neurochemistry, Vol. 46, No. 6Uptake of 36 Cl and 22 Na by the Brain-Cerebrospinal Fluid System: Comparison of the Permeability of the Blood-Brain and Blood-Cerebrospinal Fluid BarriersJournal of Neurochemistry, Vol. 37, No. 1Interregional differences in brain intracellular pH and water compartmentation during acute normoxic and hypoxic hypocapnia in the anesthetized dogBrain Research, Vol. 214, No. 2Dynamics of formation and resolution of vasogenic brain OedemaActa Neurochirurgica, Vol. 57, No. 1-2Morphogenetic Role of Glycosaminoglycans (Acid Mucopolysaccharides) in Brain and Other TissuesViability of some metabolic processes in the isolated toad brain adapted to two osmotic environmentsJournal of Neurobiology, Vol. 6, No. 2Electrolyte- and fluid-spaces of rat brainin situ after infusion with dinitrophenolJournal of Neurobiology, Vol. 6, No. 1Ion and Energy Metabolism of the Brain at the Cellular LevelConditions affecting the extracellular space in the frog's forebrainThe Anatomical Record, Vol. 178, No. 3Water and electrolyte composition of the medulla oblongata of normal and hypertensive ratsLife Sciences, Vol. 12, No. 1The Estimation of Extracellular Space of Brain Tissue in VitroThe Extracellular Space in the Vertebrate Central Nervous SystemBrain SlicesThe effect of loading and extraction temperatures, composition of medium, and molecular size on retention of extracellular space marker by mouse cerebrum slices7 July 2009 | International Journal of Neuroscience, Vol. 1, No. 4The effect of tonicity, d-mannitol, d-sorbitol, and choline chloride on the water content of mouse brain slices, and on the compartmentation of the space indicated by ‘extracellular space markers’Brain Research, Vol. 23, No. 1The temperature-dependent compartmentation of the ‘extracellular space’ in mouse brain slices as revealed by the markers: Inulin, sucrose,d-mannitol,d-sorbitol and sulfateBrain Research, Vol. 21, No. 3The thiosulphate (35S) space in the CNS of rats after ventriculo-cisternal perfusionPfl�gers Archiv European Journal of Physiology, Vol. 316, No. 1Échanges Cationiques Au Niveau des Neurones Et des Cellules Gliales du Cerveau - Part 227 September 2008 | Archives Internationales de Physiologie et de Biochimie, Vol. 78, No. 4 More from this issue > Volume 214Issue 3March 1968Pages 643-651 Copyright & PermissionsCopyright © 1968 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1968.214.3.643PubMed5638994History Published online 1 March 1968 Published in print 1 March 1968 Metrics

Changes in cortical extracellular space during spreading depression investigated with the electron microscope.

Changes in cortical extracellular space during spreading depression investigated with the electron microscope.A Van Harreveld, and F I KhattabA Van Harreveld, and F I KhattabPublished Online:01 Jul 1967https://doi.org/10.1152/jn.1967.30.4.911MoreSectionsPDF (4 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Download PDF FiguresReferencesRelatedInformation Cited BySpreading depolarization is not an epiphenomenon but the principal mechanism of the cytotoxic edema in various gray matter structures of the brain during strokeNeuropharmacology, Vol. 134Simulation of spreading depolarization trajectories in cerebral cortex: Correlation of velocity and susceptibility in patients with aneurysmal subarachnoid hemorrhageNeuroImage: Clinical, Vol. 16Spreading Depression, Spreading Depolarizations, and the Cerebral VasculatureCenk Ayata and Martin Lauritzen1 July 2015 | Physiological Reviews, Vol. 95, No. 3Activity-Dependent Layer-Specific Changes in the Extracellular Chloride Concentration and Chloride Driving Force in the Rat HippocampusD. 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