Neo-Timm and selenium stainable glial cells of the rat telencephalon

The Neo-Timm and selenium methods predominantly stain the neuropil of the rat brain and have been found to visualize zinc in synaptic vesicles. A fraction of glial cells and neuronal somata is also stained, especially when the Neo-Timm method is used. In the present study the localization and appearance of stained glial cells in the rat telencephalon are described using the two methods and the effect of metal chelating agents on the stained glial cells is examined. Neo-Timm stained glial cells were observed in both white and grey matter, with a preponderance in the major fiber tracts of the telencephalon, and were seen to contain rather large silver grains in their cytoplasm. Chelation with diethyldithiocarbamate (DEDTC) or dithizone prevented this staining. Brains from rats treated intravitally with selenium contained only occasionally stained glial cells. However, when present they showed the same characteristics as the Neo-Timm stained glial cells, including the reaction to chelation. Although both the Neo-Timm and selenium methods primarily visualize zinc in the neuropil of the rat brain, the possibility that copper could contribute to the glial cell staining cannot be ruled out. This possibility is further discussed.     

Quantification of vesicular zinc in the rat brain

By means of the Neo-Timm method it has recently been shown that zinc is present in a fraction of the round clear synaptic vesicles of certain boutons located primarily in telencephalic structures (Pérez-Clausell and Danscher 1985). It is believed that this zinc belongs to a fraction of the total brain zinc which is histochemically active (Frederickson and Danscher 1988) in that it can be visualized by means of e.g. the Neo-Timm and selenium methods (autometallography). The present study is based on the suggestion that the autometallographically developed zinc patterns represent a histochemical quantitative expression of this fraction of the total brain zinc. The different colours of the zinc pattern reflect local variations in the concentration of zinc containing vesicles. Large boutons with a high content of stained vesicles will show up darkly because of fusion of adjoining silver grains while smaller boutons with fewer zinc containing vesicles give rise to yellow staining of various shades. We have exploited this difference in staining of pattern by applying computerized optic densitometry to light microscopic sections treated according to the Neo-Timm and the selenium methods, respectively.     

Quantification of vesicular zinc in the rat brain

Summary By means of the Neo-Timm method it has recently been shown that zinc is present in a fraction of the round clear synaptic vesicles of certain boutons located primarily in telencephalic structures (Pérez-Clausell and Danscher 1985). It is believed that this zinc belongs to a fraction of the total brain zinc which is histochemically active (Frederickson and Danscher 1988) in that it can be visualized by means of e.g. the Neo-Timm and selenium methods (autometallography). The present study is based on the suggestion that the autometallographically developed zinc patterns represent a histochemical quantitative expression of this fraction of the total brain zinc. The different colours of the zinc pattern reflect local variations in the concentration of zinc containing vesicles. Large boutons with a high content of stained vesicles will show up darkly because of fusion of adjoining silver grains while smaller boutons with fewer zinc containing vesicles give rise to yellow staining of various shades. We have exploited this difference in staining pattern by applying computerized optic densitometry to light microscopic sections treated according to the Neo-Timm and the selenium methods, respectively.     

Clioquinol Inhibits Zinc-Triggered Caspase Activation in the Hippocampal CA1 Region of a Global Ischemic Gerbil Model

Background

Excessive release of chelatable zinc from excitatory synaptic vesicles is involved in the pathogenesis of selective neuronal cell death following transient forebrain ischemia. The present study was designed to examine the neuroprotective effect of a membrane-permeable zinc chelator, clioquinol (CQ), in the CA1 region of the gerbil hippocampus after transient global ischemia.

Methodology/Principal Findings

The common carotid arteries were occluded bilaterally, and CQ (10 mg/kg, i.p.) was injected into gerbils once a day. The zinc chelating effect of CQ was examined with TSQ fluorescence and autometallography. Neuronal death, the expression levels of caspases and apoptosis inducing factor (AIF) were evaluated using TUNEL, in situ hybridization and Western blotting, respectively. We were able to show for the first time that CQ treatment attenuates the ischemia-induced zinc accumulation in the CA1 pyramidal neurons, accompanied by less neuronal loss in the CA1 field of the hippocampus after ischemia. Furthermore, the expression levels of caspase-3, -9, and AIF were significantly decreased in the hippocampus of CQ-treated gerbils.

Conclusions/Significance

The present study indicates that the neuroprotective effect of CQ is related to downregulation of zinc-triggered caspase activation in the hippocampal CA1 region of gerbils with global ischemia.     

Electron microscopic analysis of glial cells in the rat telencephalon stained with the Neo-Timm and selenium methods

Two histochemical methods for visualization of zinc in synaptic vesicles, the Neo-Timm and selenium methods, have been shown to additionally stain glial cells and neuronal somata. In a previous light microscopic study the majority of stained glial cells were seen in the major fiber tracts of the rat telencephalon. The aim of the present study was to further characterize the stained glial cells with respect to glial cell type and ultrastructural localization of the silver grains responsible for the staining. Electron microscopic analysis of brains treated according to either method revealed that the vast majority of stained glial cells belonged to the dark oligodendroglial cell type. However, a smaller number of stained astrocytes was also seen, especially in the grey matter. The silver grains responsible for the staining were located in electron-dense rounded cytoplasmic organelles, suggestive of lysosomes.     

Histochemical localization of zinc ions in the epididymis of the rat

Summary In the present study, the autometallograpic zinc sulphide technique, an improved version of the original Timm sulphide-silver method, was used. This technique reveals a particular pool of ionic zinc that is chelatable by diethyldithiocarbamate. At the light microscopical level, no reaction for zinc was found in tissues of young prepubertal rats. In adult mating and non-mating rats low zinc staining was found in the head and intermediate epididymis whereas the tail of the epididymis demonstrated high levels of zinc ions. Sections from the epididymal tail revealed a compartmentalization, based on pronounced differences in staining intensity along the epididymal ducts. At higher magnification zinc ions were found in the apical part of the principal cell and in the lumen. At the ultrastructural level autometallographic grains were located in vesicles and in lysosome-like structures of the apical parts of the principal cells. The luminal grains were found either associated with sperm cells, with the surface of the large microvilli (stereocilia), or free in the seminal fluid. The variation in content of zinc ions in the epididymal epithelium and lumen suggests that zinc ions are secreted into the lumen from the epididymal tail and may somehow be involved in maturation of the sperm cells.     

Evidence that the ZNT3 protein controls the total amount of elemental zinc in synaptic vesicles.

The ZNT3 protein decorates the presynaptic vesicles of central neurons harboring vesicular zinc, and deletion of this protein removes staining for zinc. However, it has been unclear whether only histochemically reactive zinc is lacking or if, indeed, total elemental zinc is missing from neurons lacking the Slc30a3 gene, which encodes the ZNT3 protein. The limitations of conventional histochemical procedures have contributed to this enigma. However, a novel technique, microprobe synchrotron X-ray fluorescence, reveals that the normal 2- to 3-fold elevation of zinc concentration normally present in the hippocampal mossy fibers is absent in Slc30a3 knockout (ZNT3) mice. Thus, the ZNT3 protein evidently controls not only the "stainability" but also the actual mass of zinc in mossy-fiber synaptic vesicles. This work thus confirms the metal-transporting role of the ZNT3 protein in the brain.     

Synapses in the cerebral ganglion of adult Ciona intestinalis

Summary Electron microscopy of the synaptic morphology of synapses in the cerebral ganglion of the adult ascidian (sea squirt) Ciona intestinalis reveals that the synapses are restricted to the central neuropil of the ganglion. Many of the synapses show a polarity of structure such that pre and post synaptic parts can be identified. The vesicles in the presynaptic bag are of two main diameters 80 and 30 nm respectively. The large vesicles have electron dense contents that vary both in their capacity and dimensions.The pre and postsynaptic membranes are more electron dense than the surrounding membranes, but they are only slightly thicker. Both the pre and post synaptic membranes have electron dense dots some 10 nm in diameter associated with their cytoplasmic surfaces. Sometimes the presynaptic membrane has larger peg-like projections between the vesicles. Associated with the post synaptic membrane are tubules some 10 nm in diameter. These tubules may be the dots cut obliquely.The synaptic cleft material is more electron dense than the surrounding intercellular material, and in it there is a dense line made up of granules about 3–5 nm in diameter. This dense line is usually mid way between the pre and post synaptic membranes, but may be nearer the postsynaptic membrane.No tight junctions between adjacent nerve process profiles have been observed.I wish to thank Professors J. Z. Young, F. R. S. and E. G. Gray for much advice and encouragement, also Dr. R. Bellairs for the use of electron microscope facilities and Mr. R. Moss and Mrs. J. Hamilton for skillful technical assistance.     

Chelation of hippocampal zinc enhances long‐term potentiation and synaptic tagging/capture in CA1 pyramidal neurons of aged rats: implications to aging and memory

Aging is associated with decline in cognitive functions, prominently in the memory consolidation and association capabilities. Hippocampus plays a crucial role in the formation and maintenance of long‐term associative memories, and a significant body of evidence shows that impairments in hippocampal function correlate with aging‐related memory loss. A number of studies have implicated alterations in hippocampal synaptic plasticity, such as long‐term potentiation (LTP), in age‐related cognitive decline although exact mechanisms underlying are not completely clear. Zinc deficiency and the resultant adverse effects on cognition have been well studied. However, the role of excess of zinc in synaptic plasticity, especially in aging, is not addressed well. Here, we have investigated the hippocampal zinc levels and the impairments in synaptic plasticity, such as LTP and synaptic tagging and capture (STC), in the CA1 region of acute hippocampal slices from 82‐ to 84‐week‐old male Wistar rats. We report increased zinc levels in the hippocampus of aged rats and also deficits in the tetani‐induced and dopaminergic agonist‐induced late‐LTP and STC. The observed deficits in synaptic plasticity were restored upon chelation of zinc using a cell‐permeable chelator. These data suggest that functional plasticity and associativity can be successfully established in aged neural networks by chelating zinc with cell‐permeable chelating agents.     

Osmium tetroxide-zinc iodide reactive sites in the photoreceptor cells of the retina of the rat

Summary The osmium tetroxide-zinc iodide fixative of Champy-Maillet has been used to study the rat's retina at the electron microscope level. Electron opaque deposits were observed all along the photoreceptor cells and concentrated in the outer segments of rods and cones and in the nerve endings. In the outer segments that deposits are located in the inter and intra disk spaces as well as between the disk and outer membranes. In the outer plexiform layer reactive sites include synaptic vesicles and mitochondria; other minor reactive sites are described in the inner segment and inner plexiform layer.Electron opaque deposits were not seen if potassium iodide substitutes zinc iodide in the fixative. However, if osmium tetroxide-potassium iodide fixed retinae are immersed in osmium tetroxide-zinc iodide the characteristic electron-dense material is evidenced at those same sites. The effect of other several fixatives were studied with a similar double fixation procedure. Our finding points to the histochemical demonstration of an unidentified component (s) of the retina which shows a striking specificity of localization and which is made evident when zinc iodide is used in the Champy-Maillet mixture.This work has been supported by grants of the Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina and U.S. Air Force AF-AFOSR 67-0963 A.We are greatly indebted to Miss Haydée Agoff and to Mr. Alberto Saenz for their skillful technical assistance.     

The fine structure of cephalopod blood vessels III. Vessel innervation

Summary The cephalic aorta of Octopus vulgaris has a fairly complete endothelium lining the lumen, a thick complete basement membrane, a layer of circularly orientated and a layer of longitudinally orientated muscle fibres. Presumptive synaptic endings are of two types. In the circular muscle, axons containing vesicles, contact club-shaped projections of the muscle. The gap between the pre- and postsynaptic membranes is less than 100 Å and in some places apparently forms a tight junction. The second type of ending has been found in the longitudinal muscle; here axons full of vesicles end on the muscle. The ending is enclosed by a mesaxon of muscle and the synaptic gap is approximately 100 Å. In the smaller blood vessels, axons end on myofilament-containing pericytes of blood vessels (equivalent to small arterioles). The endings contain vesicles and have a synaptic gap of 100 Å. Only some of the pericytes seem to be innervated and transmission between one pericyte to another may be mediated by specialized junctions between the cells. The smaller non-myofilament containing vessels (equivalent to capillaries) are not thought to be innervated.We would like to thank Professor J. Z. Young and Dr. E. G. Gray for advice and encouragement, Mrs. Jane Astafiev for drawing Figs. 1 and 12, Mr. S. Waterman for photography, and Miss Cheryl Martin for secretarial assistance.     

On the distribution of axonal terminals containing spheroidal and flattened synaptic vesicles in the hippocampus and dentate gyrus of the rat and cat

Summary A quantitative analysis has been made of the distribution of presynaptic profiles containing round (or spheroidal) and flattened (or ellipsoidal) synaptic vesicles in the apical and basal dendritic zones and in the layer of pyramidal cell somata of fields CA₁ and CA₃ of the hippocampus, and in the molecular and granular layers of the dentate gyrus of the rat and cat.In the apical and basal dendritic zones of fields CA₁ and CA₃ the overwhelming majority of the synapses are of the asymmetrical variety, the axon terminals ending principally upon dendritic spines, and to a lesser extent upon the shafts and secondary or tertiary branches of the dendrites. Between 1 and 8% of the axon terminals in these zones contained flattened vesicles: all of these formed symmetrical contacts upon medium-sized or large dendritic shafts. In the molecular layer of the dentate gyrus a slightly higher percentage of flattened vesicle containing profiles was observed (10%); again these formed symmetrical contacts upon dendritic shafts. In the stratum pyramidale of the hippocampal fields and the stratum granulosum of the dentate gyrus of the rat, flattened vesicle containing synapses are two or three times more numerous than those with spheroidal vesicles. In the cat hippocampus the axosomatic synapses are about equally distributed between those containing round, and those with flattened vesicles.The finding that at the focus of post-synaptic inhibition, at the level of the pyramidal cell somata, the majority of the axon terminals contains flattened synaptic vesicles, whereas in the region of termination of the extrinsic, commissural and long association pathways (all of which are excitatory) virtually all the synapses contain round vesicles, strongly supports the view that endings containing flattened vesicles mediate post-synaptic inhibition in the hippocampal formation.Supported in part by Grant EY-00599 from the National Eye Institute.We should like to thank Mr. Paul Myers and Mr. Milburn W. Rhoades for their technical assistance, and Mrs. Doris Stevenson for secretarial help.     

Synaptic organization in the lateral geniculate nucleus of the monkey

Summary The synaptic organization in the lateral geniculate nucleus of the monkey has been studied by electron microscopy.The axon terminals in the lateral geniculate nucleus can be identified by the synaptic vesicles that they contain and by the specialized contacts that they make with adjacent neural processes. Two types of axon terminal have been recognized. The first type is relatively large (from 3–20 ) and contains relatively pale mitochondria, a great many vesicles and, in normal material, a small bundle of neurofilaments. These terminals have been called LP terminals. The second type is smaller (1–3 ), contains darker mitochondria, synaptic vesicles, and no neurofilaments. These have been called SD terminals.Both types of terminal make specialized axo-somatic and axo-dendritic synaptic contacts, but the axo-somatic contacts are relatively rare. In addition the LP terminals frequently make specialized contacts with the SD terminals, that is, axo-axonal contacts, and at these contacts the asymmetry of the membranes is such that the LP terminal must be regarded as pre-synaptic to the SD terminal.The majority of the synaptic contacts are identical to those that have been described previously (Gray, 1959 and 1963a) but, in addition, a new type of contact has been found. This is characterized by neurofilaments that lie close to the post-synaptic membrane, and by an irregular post-synaptic thickening. Such filamentous contacts have been found only where an LP terminal contacts a dendrite or a soma.The degeneration that follows removal of one eye demonstrates that the LP terminals are terminals of optic nerve fibres. The origin of the SD terminals is not known.The glial cells often form thin lamellae around the neural processes and tend to isolate synaptic complexes. These lamellae occasionally show a complex concentric organization similar to that of myelin.It is a pleasure to thank Prof. J. Z. Young for advice and encouragement and Dr. E. G. Gray for the considerable help he has given us. Dr. J. L. de C. Downer gave us much help with the care of the animals and with the operations. We also wish to thank Mr. K. Watkins for technical assistance and Mr. S. Waterman for the photography.     

Cholinergische Synapsen im Oberschlundganglion der Waldameise (Formica lugubris Zett.)

Summary The corpora pedunculata of the wood ant (Formica lugubris Zett.) consist of two sharply defined layers: The perikaryon layer and the subjacent neuropil. Synaptic endings are found exclusively in the neuropil. The synapses consist of a central, presynaptic end knob of 1.2–2.5 diameter and a relatively large number of surrounding postsynaptic processes of 0.3–1.1 diameter. These junctions are analogous to axodendritic synapses of the vertebrates. The presynaptic process contains mitochondria and a multitude of light vesicles (300–600 Å diameter). Larger vesicles 700–1000 Å with a dark center are seen more rarely. The synaptic cleft has a diameter of approximately 130 Å and varies somewhat with different fixation methods. With glutaraldehyde-osmium fixation, this relatively wide gap is maintained only in circumscribed areas of the junction, while in adjacent areas it tends to contract and an external compound membrane is formed. The postsynaptic region is characterized by the presence of a subsynaptic network which is revealed only by suitable fixation methods. This and the persistent synaptic cleft are the main structural differentiations found in junctional areas thus far.Cholinesterase is located with the aid of thiolacetic acid (Barnett) and Eserin control studies. The enzyme is found in the cytoplasm immediately adjacent to the pre- and postsynaptic membranes. In two thirds of our observations the reaction is far more concentrated postsynaptically than presynaptically. In one third, the distribution is reversed. Only an insignificant amount of cholinesterase is present within the synaptic cleft. There is no evidence that cholinesterase is evenly distributed along the entire junctional region. In contrast, only small circumscribed areas show a positive reaction and these coincide with the extent of the synaptic cleft and the subsynaptic network. Such areas seem to correspond to the active junctional areas.

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Unterstützt durch einen Kredit (Nr.2575) des Schweizer Nationalfonds für Wissenschaftliche Forschung.

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Herrn Prof. Dr. W. Bargmann zum 60. Geburtstag gewidmet.     

Photoperiodic and osmotic influences on the ultrastructure of the hypothalamic neurosecretory system of the White-crowned sparrow,Zonotrichia leucophrys gambelii

Summary An attempt was made to correlate functional changes in the neurohypophysis of the White-crowned Sparrow, Zonotrichia leucophrys gambelii, with morphologic features on the light- and electron-microscope levels. The aldehyde-fuchsin-staining anterior median eminence possesses essentially the same ultrastructural features as the non-staining posterior median eminence. The axon terminals are characterized by the presence of a large number of small vesicles (approximately 400 Å in diameter) and occasional electron-dense granules. The more-or-less depleted anterior median eminence occasionally evident in the photosensitive bird showing testicular development is indistinguishable ultrastructurally from the more intensely staining median eminence generally characteristic of the photorefractory bird. In the median eminence, stainability and functional state do not seem to be correlated with changes in the type, size or number of vesicles. A slight increase in the number of granules was noted in the photorefractory bird but this was considered insufficient basis to account for differences in stainability.The pars nervosa, on the other hand, responded to osmotic stimuli (saline drinking water) by loss of stainability and decrease in numbers of elementary neurosecretory granules. Small vesicles are also present in the pars nervosa axon terminals, but are intermingled with neurosecretory granules in normal birds. Acute-osmotic birds, however, had axon terminals almost entirely occupied by small vesicles.It is to be emphasized that the pars nervosa and the median eminence are two structurally very different regions of the neurohypophysis. The basis for aldehyde-fuchsin staining in the median eminence appears to differ from that in the pars nervosa. The implications of these findings are considered in regard to hypothalamic control over gonadotropic activity in the White-crowned Sparrow.Dedicated to Professor Dr. W. Bargmann in honor of his 60th birthday.This investigation was supported by grant GB-2484 from the National Science Foundation to Professor Bern, grant GB-2819 from the National Science Foundation to Professor Mewaldt, and grant NB-01353 from the National Institutes of Health to Professor Farner. The authors wish to express their appreciation of the technical assistance of Mrs. Irene Brown, Mr. John Butchart, Sally S. Kibby, Mrs. Carol Nicoll, and Mr. John Striffler. Mrs. Emily Reid kindly prepared the histograms.     

Zinc chelation reduces traumatic brain injury-induced neurogenesis in the subgranular zone of the hippocampal dentate gyrus

Numerous studies have demonstrated that traumatic brain injury (TBI) increases hippocampal neurogenesis in the rodent brain. However, the mechanisms underlying increased neurogenesis after TBI remain unknown. Continuous neurogenesis occurs in the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) in the adult brain. The mechanism that maintains active neurogenesis in the hippocampal area is not known. A high level of vesicular zinc is localized in the presynaptic terminals of the SGZ (mossy fiber). The mossy fiber of dentate granular cells contains high levels of chelatable zinc in their terminal vesicles, which can be released into the extracellular space during neuronal activity. Previously, our lab presented findings indicating that a possible correlation may exist between synaptic zinc localization and high rates of neurogenesis in this area after hypoglycemia or epilepsy. Using a weight drop animal model to mimic human TBI, we tested our hypothesis that zinc plays a key role in modulating hippocampal neurogenesis after TBI. Thus, we injected a zinc chelator, clioquinol (CQ, 30 mg/kg), into the intraperitoneal space to reduce brain zinc availability twice per day for 1 week. Neuronal death was evaluated with Fluoro Jade-B and NeuN staining to determine whether CQ has neuroprotective effects after TBI. The number of degenerating neurons (FJB (+)) and live neurons (NeuN (+)) was similar in vehicle and in CQ-treated rats at 1 week after TBI. Neurogenesis was evaluated using BrdU, Ki67 and doublecortin (DCX) immunostaining 1 week after TBI. The number of BrdU, Ki67 and DCX positive cell was increased after TBI. However, the number of BrdU, Ki67 and DCX positive cells was significantly decreased by CQ treatment. The present study shows that zinc chelation did not prevent neurodegeneration but did reduce TBI-induced progenitor cell proliferation and neurogenesis. Therefore, this study suggests that zinc has an essential role for modulating hippocampal neurogenesis after TBI.     

Glucose uptake into plasma membrane vesicles from the maternal surface of human placenta

Summary Glucose uptake into plasma membrane vesicles from the maternal surface of the human placenta was measured with the Millipore filtration technique. Uptake ofd-glucose was dependent on the osmolarity of the incubation medium surrounding the vesicles. Uptake ofd-glucose exceeded that ofl-glucose. The uptake ofd-glucose was not enhanced by placing 100mm NaCl or NaSCN in the medium outside the vesicles (none inside) at the onset of uptake determinations.d-glucose transport was inhibited by cytochalasin B; phloretin, phlorizin, and 1-fluoro-2,4-dinitrobenzene.d-glucose uptake was inhibited by 2-deoxy-d-glucose, 3-O-methyl-d-glucose and to a lesser extent byd-galactose. It was not inhibited by -methyl-d-glucoside. Cytochalasin B binding to the vesicles was 30% inhibited in the presence of 80mm d-glucose. The results indicate that the system for facilitated transport ofd-glucose at the maternal face of the placenta is distinctly different from that on the brush-border membrane of intestine or renal tubule and more closely resembles that of human erythrocyte.     

An ultrastructural study of the carotid body of horse and dog

Summary Carotid body tissue from horse and dog has been investigated ultrastructurally. Several cell types are recognized: glomus cells which are regarded as chemoreceptors, sustentacular cells which enclose the glomus cells, and nerve fibers.The glomus cells contain electron dense granules which are interpreted as packages of biogenic monoamines. There are both dark and light glomus cells, the former containing more granules and ribosomes. Invaginations of the plasma membranes as well as free coated vesicles are often seen in the cytoplasm of glomus cells. Nerves within the glomus lobules are generally wrapped by sustentacular cells, but nerve endings are also seen in close contact with the glomus cells. Some endings contain synaptic vesicles as well as a great concentration of mitochondria. The corresponding fibers are thought to be efferent. Another type of contact is interpreted as en passant synapses of afferent fibers.The author wishes to express his gratitude to Professor L. Nicander who initiated this project and took most of the micrographs and to Professor Nils Obel and associate Professor Gustav Björk at the Royal Veterinary College for their valuable help with the surgical procedure and to Dr. Martin Ritzén of the Royal Medical College for making the tests for biogenic monoamines.     

Diltiazem at high concentration increases the ionic permeability of biological membranes

Summary The effects of diltiazem, a drug which inhibits the calcium channels in cardiac muscle as well as the light-sensitive channels in photoreceptor cells, were studied on ionic fluxes in both membrane and intact cell preparations. Diltiazem nonselectively increased the ionic permeability to both anions and cations in photoreceptor rod outer segment and synaptic membrane vesicles as well as in intact erythrocytes. Under our conditions, the estimated threshold for the diltiazem effect varied between 12.5 and 200 m. In each case the concentration dependence exhibited the sigmoidal shape characteristic of positive cooperativity. The effect of diltiazem on ionic fluxes from phospholipid vesicles were strongly influenced by phospholipid composition and membrane charge. By contrast, diltiazem inhibited the efflux of⁸⁶Rb from photoreceptor cells of intact aspartate-isolated retina, an effect opposite to that of diltiazem on ionic permeabilities in photoreceptor membrane vesicle preparations.These data raise serious doubts on the specificity of diltiazem as a calcium channel blocker or as a cGMP channel blocker when used at concentrations higher than 10 m.     

Is the Mg2+-ATP-dependent proton pumping activity of the synaptic vesicles a factor involved in the cerebral hypoxia?

The changes in the Mg²⁺-dependent V-type ATPase activity and the Mg²⁺-ATP-dependent H⁺ pumping activity of the synaptic vesicles from the cerebral cortex of rats submitted to intermittent chronic (4 weeks) mild or severe hypoxia were evaluated. The adaptation to the chronic severe hypoxia increases both the ATPase and the H⁺ pumping activities which are inhibited by NEM with an exponential relationship between the IC₅₀ values and the in vivo O₂ concentration. The Mg²⁺-dependent increase in H⁺ pumping activity of synaptic vesicles from the rats subjected to in vivo chronic hypoxia may be antagonized by nigericin (dissipating ΔpH) and by FCCP (dissipating ΔpH and Δ_ΨSV). In contrast, valinomycin (dissipating the Δ_ΨSV and facilitating an enhancement in ΔpH) increases in vitro the H⁺ pumping activity that is inhibited by the addition of high concentration of K gluconate (reducing the rate of K⁺ efflux). The preincubation of vesicles from hypoxic rats with FCCP, but not with nigericin, inhibits the valinomycin-increased H⁺ pumping activity.l-glutamate increases the H⁺ pumping activity in synaptic vesicles from the cerebral cortex of chronic hypoxic rats, whereas other amino acids (i.e.,l-aspartate andl-homocysteate) and glutamate analogs (i.e., quisqualate and ibotenate) are ineffective. The adaptation to both chronic intermittent severe hypoxia and in vivo treatment with posatireline causes a decrease in the Mg²⁺-ATPase activity consistent with the decrease in the H⁺ pumping one of the synaptic vesicles. The addition of nigericin into incubation medium magnifies the decrease in the H⁺ pumping activity, while the addition of FCCP is ineffective, suggesting that the treatment with posatireline interferes with the Δ_ΨSV component in the of the synaptic vesicles from rats submitted to chronic hypoxia. The results of the in vivo and in vitro experiments suggest that in the synaptic vesicles from hypoxic rats the Δ_ΨSV component in may be most effective in increasing the Mg²⁺-ATP-dependent H⁺ pumping activity.