Noradrenaline Release from Cultured Mouse Postganglionic Sympathetic Neurons

The possible existence of alpha2-autoreceptors, P2-autoreceptors, and adenosine A1- or A2A-receptors was studied in cultured thoracolumbar postganglionic sympathetic neurons from mice. The cells were preincubated with [3H]noradrenaline and then superfused. The selective alpha2-adrenoceptor agonist UK 14,304 reduced the electrically evoked overflow of tritium. When the cultures were stimulated by trains of increasing pulse number, ranging from a single pulse to 72 pulses at 3 Hz, the concentration-inhibition curve of UK 14,304 was shifted progressively to the right and the maximal inhibition obtainable became progressively smaller. Six alpha-adrenoceptor antagonists shifted the concentration-inhibition curve of UK 14,304 in a parallel manner to the right. Neither ATP (3-300 microM), adenosine (0.01-100 microM), the selective A1-receptor agonist cyclopentyladenosine (1-1,000 nM), nor the selective A2A-receptor agonist CGS-21680 (1-10,000 nM) changed the basal or the electrically evoked overflow of tritium. It is concluded that the cultured neurons possess presynaptic, release-inhibiting alpha2-autoreceptors. As in intact tissues, the effectiveness of presynaptic alpha2-adrenergic inhibition depends on the "strength" of the releasing stimulus. The pK(D) values of the six antagonists against UK 14,304 indicate that the autoreceptors belong to the pharmacological alpha2D and hence the genetic alpha(2A/D) subtype of alpha2-adrenoceptor. Neither P2-autoreceptors nor receptors for adenosine, the degradation product of ATP, were detected.     

Noradrenaline Storing Vesicles in Sympathetic Neurons and Their Putative Role in Neurotransmitter Release: An Historical Overview of Controversial Issues

More than 25 years have passed since the original demonstration that proteins such as chromogranin A and dopamine--hydroxylase, which are co-stored together with noradrenaline in large dense cored vesicles in adrenergic nerves, are released by exocytosis. Despite much evidence in favour, it was for a long time thought that large dense cored vesicles were not eminently involved in the release of noradrenaline. The present review attempts to demonstrate, making use of evidence from different approaches, that the release of noradrenaline from sympathetic neurons occurs ultimately from large dense cored vesicles. A model of the secretory cycle is proposed.     

Cellular Localization of the Molecular Forms of Acetylcholinesterase in Primary Cultures of Rat Sympathetic Neurons and Analysis of the Secreted Enzyme

The secretion and cellular localization of the molecular forms of acetylcholinesterase (AChE) were studied in primary cultures of rat sympathetic neurons. When cultured under conditions favoring a noradrenergic phenotype, these neurons synthesized and secreted large quantities of the tetrameric G4, and the dodecameric A12 forms, and minor amounts of the G1 and G2 forms. When these neurons adopted the cholinergic phenotype, i.e., in the presence of muscle-conditioned medium, the development of the cellular A12 form was completely inhibited. These neurons secreted only globular, mainly G4, AChE. Both cellular and secreted A12 AChE in adrenergic cultures aggregated at an ionic strength similar to that of the culture medium, raising the hypothesis that this form was associated with a polyanionic component of basal lamina. In noradrenergic neurons, 60-80% of the catalytic sites were exposed at the cell surface. In particular, 80% of G4 form, but only 60% of the A12 form, was external, demonstrating for the A12 form a sizeable intracellular pool. The hydrophobic character of the molecular forms was studied in relation to their cellular localization. As in muscle cells, most of the G4 form was membrane-bound. Whereas 76% of the cell surface A12 form was solubilized in the aqueous phase by high salt concentrations, only 50% of the intracellular A12 form was solubilized under these conditions. The rest of intracellular A12 could be solubilized by detergents and was thus either membrane-bound or entrapped in vesicles originating from, e.g., the Golgi apparatus.     

Release of the NILE and Other Glycoproteins from Cultured PC 12 Rat Pheochromocytoma Cells and Sympathetic Neurons

Studies were carried out on the glycoproteins (GPs) released by cultured rat sympathetic neurons and by cultured PC12 rat pheochromocytoma cells with and without nerve growth factor (NGF) treatment. Cultures were prelabeled with [3H]fucose and then incubated for 4-8 h in fresh unlabeled medium. The material released into the medium was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. The patterns of labeled material released by all three types of cultures were similar. One of the major components released was of apparent Mr less than or equal to 230,000. Another major component of apparent Mr = 55,000 as well as minor components of apparent Mr less than or equal to 180,000, 140,000, 118,000, and 105,000 were also detected. An additional peptide of apparent Mr less than or equal to 210,000 was released only by the sympathetic neurons. The soluble released Mr less than or equal to 230,000 component appeared to be derived from a previously characterized neuronal integral membrane GP referred to as the NILE (NGF-inducible large external) GP. Evidence for this included recognition of the released component by a monospecific antiserum prepared against membrane-derived NILE GP. At least several of the other released GPs appeared to be derived from membrane-bound components with which they share immuno-crossreactivity. Since the soluble NILE and other released GPs had somewhat faster mobilities on SDS-polyacrylamide gels than their apparent membrane-bound correspondents, release could either be due to, or accompanied by, minor changes in molecular structure.     

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive ^1-3, and influences the types and distribution of these inputs ^4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models ⁷, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders ^8-10 and neurodegenerative diseases ^11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture ^14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) ^16,17 or Matrigel ¹⁸ to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons ¹⁹ that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies ^17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth ^17,18.Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.     

α2-Adrenoceptor-Mediated Inhibition of Electrically Evoked [3H]Noradrenaline Release from Chick Sympathetic Neurons: Role of Cyclic AMP

Abstract: This study explores the role of cyclic AMP in electrically evoked [³H]noradrenaline release and in the α₂-adrenergic modulation of this release in chick sympathetic neurons. Along with an increase in stimulation-evoked tritium overflow, applications of forskolin enhanced the formation of intracellular cyclic AMP. Both effects of forskolin were potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. The forskolin-induced increase in overflow was abolished by the Rp-diastereomer of cyclic AMP-thioate, an antagonist at cyclic AMP-dependent protein kinases, and 1,9-dideoxy-forskolin, an inactive analogue at adenylyl cyclase, had no effect on the evoked overflow. A 24-h pretreatment with either cholera toxin or forskolin reduced the subsequent forskolin-induced accumulation of cyclic AMP and inhibited the stimulation-evoked release. Basal cyclic AMP production, however, remained unaltered after forskolin treatment and was enhanced after 24 h of cholera toxin exposure. The α₂-adrenergic agonist bromoxidine did not affect the formation of cyclic AMP stimulated by forskolin but reduced electrically evoked release. However, effects of bromoxidine on ³H overflow were attenuated by forskolin as well as by 8-bromo-cyclic AMP. Effects of bromoxidine on [³H]noradrenaline release were paralleled by an inhibition of voltage-activated Ca²⁺ currents, primarily through a delayed time course of current activation. This effect was abolished when either forskolin or 8-bromo-cyclic AMP was included in the pipette solution. Both substances, however, failed to affect Ca²⁺ currents in the absence of bromoxidine. These results suggest that the signaling cascade of the α₂-adrenergic inhibition of noradrenaline release involves voltage-activated Ca²⁺ channels but not cyclic AMP. Elevated levels of cyclic AMP, however, antagonize this α₂-adrenergic reduction, apparently through a disinhibition of Ca²⁺ channels.     

Modulatory Inputs on Sympathetic Neurons in the Rostral Ventrolateral Medulla in the Rat

1. The first part of this study looks at spontaneously active neurons located in the rostral ventrolateral medulla (RVLM) with projections to the thoracic spinal cord. Sixteen neurons were intracellularly recorded in vivo. Four out of 16 neurons were antidromically activated from the thoracic spinal cord (axonal conduction velocities varied from 1.8 m/s to 9.5 m/s).2. The simultaneous averages of the neuronal membrane potential and arterial blood pressure triggered by the pulsatile arterial wave or the EKG-R wave demonstrated changes in membrane potential (hyperpolarization or depolarization) locked to the cardiac cycle in four neurons in this group. These neurons (three of them bulbospinal) were further tested for barosensitivity by characterizing the responses to electrical stimulation of the aortic depressor nerve. Four neurons responded with inhibitory hyperpolarizing responses characterized as inhibitory postsynaptic potentials (IPSP) to aortic nerve stimulation (onset latency: 32.3 ± 5.0 ms; mean ± SEM).3. In two neurons in the RVLM, one of them characterized as barosensitive, electrical stimulation of the opposite RVLM (0.5 Hz, 1.0 ms pulse duration, 25–100 A) elicited excitatory postsynaptic potentials (EPSPs) with latencies of 9.07 and 10.5 ms. At resting membrane potential, the onset latency of the evoked EPSPs did not change with increasing stimulus intensities. Some of the recorded neurons were intracellularly labelled with biocytin for visualization. They were found in the RVLM.4. These experiments in vivo would support the idea of a functional commissural pathway between the RVLM of both sides.5. Anatomical data have shown that some of those commissural bundle fibers originate in the C1 adrenergic neuronal group in the RVLM. In the second part of this study, we used an intracellular recording technique in vitro to investigate the effects of the indirect adrenergic agonist tyramine on neurons in the RVLM with electrophysiological properties similar to premotor sympathetic neurons in vivo.6. Tyramine (0.5–1 mM) produced a pronounced inhibitory effect with hyperpolarization and increase in membrane input resistance on two neurons characterized as regularly firing (R), and on one neuron characterized as irregularly firing (I). This effect was preceded by a transient depolarization with increases in firing rate.7. These results would indicate that neurons in the RVLM recorded in vitro and with properties similar to premotor sympathetic neurons can be modulated by catecholamines released from terminals probably making synaptic contacts.     

Translocation and Activation of Protein Kinase C in Striatal Neurons in Primary Culture: Relationship to Phorbol Dibutyrate Actions on the Inositol Phosphate Generating System and Neurotransmitter Release

The actions of the tumor-promoting phorbol ester phorbol dibutyrate were examined, under identical physiological conditions, on three distinct cellular processes in striatal neurons: the distribution of protein kinase C, the carbachol-stimulated generation of [3H]inositol monophosphate, and the KCl-evoked release of gamma-[3H]aminobutyric acid ([3H]GABA). Phorbol dibutyrate induced a rapid (complete in 5 min), dose-dependent, entirely reversible (t0.5 = 15 min) translocation of protein kinase C from cytosol to membrane. On longer exposure to phorbol dibutyrate, membrane-associated protein kinase C returned toward the control level, and total cellular enzyme activity declined markedly. Phorbol dibutyrate also induced the dose-dependent attenuation of carbachol-stimulated [3H]inositol monophosphate production and potentiation of KCl-evoked release of [3H]GABA. The translocation of protein kinase C and the potentiation of KCl-evoked [3H]GABA release were both rapidly reversed following washout of phorbol dibutyrate. In addition, for both processes, the effect of a 1-h exposure to phorbol dibutyrate was markedly less than that observed following a 5-min exposure to the agent. In direct contrast, inhibition of carbachol-stimulated [3H]inositol monophosphate production was not rapidly reversed following washout of phorbol dibutyrate and was actually more pronounced following a 1-h exposure, compared with a 5-min exposure. These findings indicate that some, but not all, of the actions of phorbol dibutyrate are closely associated with the translocation of protein kinase C in striatal neurons in primary culture.     

Analysis of Adenosine Immunoreactivity, Uptake, and Release in Purified Cultures of Developing Chick Embryo Retinal Neurons and Photoreceptors

We have investigated the presence of endogenous adenosine and of mechanisms for adenosine uptake and release in chick embryo retinal neurons and photoreceptors grown in purified cultures in the absence of glial cells. Simultaneous autoradiographic and immunocytochemical analysis showed that endogenous adenosine and the uptake mechanism for this nucleoside colocalize in practically all the photoreceptors, but only in approximately 20% of the neurons. Approximately 25% of the neurons showed either immunocytochemical labeling or autoradiographic labeling, while greater than 50% of the neurons were unlabeled with both techniques. [3H]Adenosine uptake was saturable and could be inhibited by nitrobenzylthioinosine and dipyridamole and by pretreatment of the [3H]adenosine with adenosine deaminase. Although these observations indicate that the uptake is specific for adenosine, only 35% of accumulated radioactivity was associated with adenosine, with the remaining 65% representing inosine, hypoxanthine, and nucleotides plus uric acid. Adenosine as well as several of its metabolites were released by the cells under basal as well as K(+)-stimulated conditions. Potassium-enhanced release was blocked by 10 mM CoCl2 or in Ca2(+)-free, Mg2(+)-rich solutions. The results indicate that retinal cells that synthesize, store, and release adenosine differentiate early during embryogenesis and are therefore consistent with a hypothetical role for adenosine in retinal development.     

Alanine Uptake and Release by Sympathetic Ganglia of Chicken Embryos

Uptake and release of alanine were measured in lumbar sympathetic chains excised from embryos of white leghorn chickens, 14-15 days old, and incubated in a modified Eagle's minimum essential medium. In the presence of [U-14C]glucose, glucose carbon accumulated in alanine in the medium at a rate that increased when unlabeled alanine was added and sometimes exceeded the rate of appearance in lactate. When combined with uptake data, the increase in appearance of labeled alanine in the medium could be accounted for quantitatively by interference with its reuptake, without assuming a change in the unidirectional output of labeled alanine, provided allowance was made for the measured properties of exchange between the extracellular space and the surrounding medium. According to this model, the constant unidirectional outflux of labeled alanine was about 50 mumol/g dry weight/h. When [U-14C]alanine was added to medium containing unlabeled glucose, the alanine was consumed at a rate that increased as the concentration of alanine in the medium was elevated. The uptake rate was found to fit a modified Michaelis-Menten equation with a Umax of about 120 mumol/g dry weight/h, a Km of 0.5-1.0 mM, and a Kd of 0.75 ml/g dry weight/h. By chemical measurement of changes in alanine concentration in the medium during incubation, the uptake rate was shown to equal the output rate when about 0.2 mM alanine was present. Much of the alanine consumed in the presence of glucose was metabolized to CO2, raising the total CO2 output above the rate obtained with glucose alone. When alanine was present at a concentration of 10-20 mM, it contributed almost as much carbon to CO2 as did the glucose. A higher percentage of the carbon from alanine was incorporated into tissue constituents than was carbon from either glucose or lactate. It is concluded that alanine can be significant both as a product and as a substrate, but that its role as substrate would not be great at typical concentrations of alanine in blood.     

Integrons: Past,Present, and Future

SUMMARY

Integrons are versatile gene acquisition systems commonly found in bacterial genomes. They are ancient elements that are a hot spot for genomic complexity, generating phenotypic diversity and shaping adaptive responses. In recent times, they have had a major role in the acquisition, expression, and dissemination of antibiotic resistance genes. Assessing the ongoing threats posed by integrons requires an understanding of their origins and evolutionary history. This review examines the functions and activities of integrons before the antibiotic era. It shows how antibiotic use selected particular integrons from among the environmental pool of these elements, such that integrons carrying resistance genes are now present in the majority of Gram-negative pathogens. Finally, it examines the potential consequences of widespread pollution with the novel integrons that have been assembled via the agency of human antibiotic use and speculates on the potential uses of integrons as platforms for biotechnology.     

Endocytosis: Past,Present, and Future

Endocytosis may have been a driving force behind the evolution of eukaryotic cells. It plays critical roles in cell biology (e.g., signal transduction) and in organismal physiology (e.g., tissue morphogenesis).Endocytosis, the process of cellular ingestion, may have been the driving force behind evolution of the eucaryotic cell (de Duve 2007). Acquiring the ability to internalize macromolecules and digest them intracellularly would have allowed primordial cells to move out from their food sources and pursue a predatory existence; one that might have led to the development of endosymbiotic relationships with mitochondria and plastids. Thus, it is fitting that endocytosis was first discovered and named as the processes of cell “eating” and “drinking.” In 1883, the developmental biologist Ilya Metchnikoff coined the term phagocytosis, from the Greek “phagos” (to eat) and “cyte” (cell), after observing motile cells in transparent starfish larva surround and engulf small splinters that he had inserted (Tauber 2003). Decades later, in 1931, Warren H. Lewis, one of the earliest cell “cinematographers” coined the term pinocytosis, from the Greek “pinean” (to drink), after observing the uptake of surrounding media into large vesicles in cultured macrophages, sarcoma cells, and fibroblasts by time-lapse imaging (Lewis 1931; Corner 1967).Importantly, these pioneering studies also revealed that the function of endocytosis goes well beyond eating and drinking. Indeed, Metchnikoff, considered one of the founders of modern immunology, realized that the phagocytic behavior of the mesodermal amoeboid cells he had observed under the microscope could serve as a general defense system against invasive parasites, in the larva as in man. This revolutionary concept, termed the phagocytic theory, earned Metchnikoff the 1908 Nobel Prize in Physiology or Medicine for his work on phagocytic immunity, which he shared with Paul Ehrlich who discovered the complementary mechanisms of humoral immunity that led to the identification of antibodies (Vaughan 1965; Tauber 2003; Schmalstieg and Goldman 2008). The phagocytic theory was a milestone in immunology and cell biology, and formally gave birth to the field of endocytosis.Key discoveries over the intervening years, aided in large part by the advent of electron microscopy, revealed multiple pathways for endocytosis in mammalian cells that fulfill multiple critical cellular functions (Fig. 1). These mechanistically and morphologically distinct pathways, and their discoverers, include clathrin-mediated endocytosis (Roth and Porter 1964), caveolae uptake (Palade 1953; Yamada 1955), cholesterol-sensitive clathrin- and caveolae-independent pathways (Moya et al. 1985; Hansen et al. 1991; Lamaze et al. 2001), and, more recently, the large capacity CLIC/GEEC pathway (Kirkham et al. 2005). In place of Metchnikoff’s splinters, many of these discoveries resulted from the detection and tracking of internalized HRP-, ferritin-, or gold-conjugated ligands by electron microscopy. These electron-dense tracers allowed researchers to identify cellular structures associated with the uptake and intracellular sorting of receptor-bound ligands. A particularly striking example is the pioneering work of Roth and Porter, who in 1964 observed the uptake of yolk proteins into mosquito oocytes. To synchronize uptake, they killed female mosquitos at timed intervals after a blood feed and observed the sequential appearance of electron-dense yolk granules in coated pits, coated and uncoated vesicles, and progressively larger vesicles. Their remarkable observations accurately described coated vesicle budding, uncoating, homo- and heterotypic fusion events, as well as the emergence of tubules from early endosomes (Fig. 2), all of which are now known hallmarks of the early endocytic trafficking events.Open in a separate windowFigure 1.Time line for discoveries of endocytic pathways and their discoverers. Boxes are color-coded by pathway. *, Nobel laureate. HRP, horseradish peroxidase; CCVs, clathrin-coated vesicles; CCPs, clathrin-coated pits; EGFR, epidermal growth factor receptor; PM, plasma membrane; ER, endoplasmic reticulum; CLIC/GEEC, clathrin-independent carriers/GPI-enriched endocytic compartments.Open in a separate windowFigure 2.Fiftieth anniversary of the discovery of clathrin-mediated endocytosis by Roth and Porter (1964). The image is the hand-drawn summary of observations made by electron microscopic examination of the trafficking of yolk proteins in a mosquito oocyte. Note the many details, later confirmed and mechanistically studied over the intervening 50 years. These include the growth, invagination, and pinching off of coated pits (1,2), which concentrate cargo taken up by coated vesicles (3), the rapid uncoating of nascent-coated vesicles (4), homotypic fusion of nascent endocytic vesicles in the cell periphery (5), the formation of tubules from early endosomes (7), and changes in the intraluminal properties of larger endosomes (6). Finally, yolk proteins are stored in granules as crystalline bodies (8). (From Roth and Porter 1964; reprinted, with express permission, from Rockefeller University Press © 1964, The Journal of Cell Biology 20: 313–332, doi: 10.1083/jcb.20.2.313.)Another milestone in the field of endocytosis was the discovery of the lysosome by Christian de Duve (Appelmans et al. 1955). Whereas the finding of phagocytosis and other endocytic pathways was possible through microscopy, the discovery of lysosomes originated from a biochemical approach (Courtoy 2007), which benefited from the invention of the ultracentrifuge. de Duve and coworkers observed that preparations of acid phosphatase obtained by subcellular fractionation had an unusual behavior: contrary to most enzymatic activities, the activity of acid phosphatase increased rather than decreased with time, freezing–thawing of the fractions and the presence of detergents. Interestingly, the same was true for other hydrolases, which depended on acidic pH for their optimal activity. This led him to postulate that the acid hydrolases were contained in acidified membrane-bound vesicles. In collaboration with Alex Novikoff, he visualized these vesicles, the lysosomes, by electron microscopy (Beaufay et al. 1956) and later showed their content of acid phosphatase (Farquhar et al. 1972). In 1974, de Duve was awarded the Nobel Prize for Physiology or Medicine for his seminal finding of the lysosomes and peroxisomes. He shared it with Albert Claude and George E. Palade “for their discoveries concerning the structural and functional organization of the cell.” The importance of this work lies also in the significant therapeutic applications that followed. The discovery by Elizabeth Neufeld and collaborators of uptake of lysosomal enzymes by cells provided the foundation for enzyme replacement therapy for lysosomal storage disorders (Neufeld 2011).In the 1970s, research in endocytosis entered the molecular era. Using de Duve and Albert Claude-like methods of subcellular fractionation, Barbara M. Pearse purified clathrin-coated vesicles from pig brain (Pearse 1975). A year later, she isolated a major protein species of 180 kDa, which she named clathrin “to indicate the lattice-like structures which it forms” (Pearse 1976). It was a breakthrough that inaugurated the molecular dissection of clathrin-mediated endocytosis.Over the intervening years, the continued application of microscopy (which now spans from electron cryotomography to live cell, high-resolution fluorescence microscopy), genetics (in particular, in yeast, Caenorhabditis elegans and Drosophila melanogaster), biochemistry (including cell-free reconstitution of endocytic membrane trafficking events), as well as molecular and structural biology have revealed a great deal about the cellular machineries and mechanisms that govern trafficking along the endocytic pathway. A partial, and because of space limitations, necessarily incomplete list of milestones (

On the Feedback Between Theory and Experiment in Elucidating the Molecular Mechanisms Underlying Neurotransmitter Release

This review describes the development of the molecular level Ca²⁺-voltage hypothesis. Theoretical considerations and feedback between theory and experiments played a key role in its development. The theory, backed by experiments, states that at fast synapses, membrane potential by means of presynaptic inhibitory autoreceptors controls initiation and termination of neurotransmitter release. A molecular kinetic scheme which depicts initiation and termination of evoked release is discussed. This scheme is able to account for both spontaneous release and evoked release. The physiological implications of this scheme are enumerated.     

Neurotransmitter Metabolism in Rat Brain Synaptosomes: Effect of Anoxia and pH

Synaptosomes isolated from the rat cerebral cortex by means of a discontinuous Ficoll gradient carry out net, sodium-dependent, veratridine-sensitive accumulation of gamma-aminobutyric acid (GABA), serotonin, norepinephrine, and dopamine. The intrasynaptosomal contents of the four neurotransmitters are: 30.4 nmol/mg protein, 17.4 pmol/mg protein, 13.5 pmol/mg protein, and 21.2 pmol/mg protein, respectively. Anaerobic preincubation of synaptosomes causes an irreversible decrease in the rates of neurotransmitter accumulation but does not affect the rates of their release. The inhibitory effect of anaerobiosis is enhanced by increased concentration of [H+] (decreased pH) in the medium. The most sensitive is the uptake of dopamine, the least that of serotonin. The rates of neurotransmitter efflux are unaffected by anaerobiosis. Synaptosomes leak catecholamines, GABA, and serotonin into the medium when subjected to anaerobiosis, and reintroduction of oxygen is accompanied by a rapid reaccumulation of all four neurotransmitters. It is concluded that: (1) Responses of synaptosomes to anaerobiosis are remarkably similar to the behavior of intact brain in hypoxia and ischemia. (2) Neurotransmitter uptake systems are more sensitive to short periods of anaerobiosis than either the energy metabolism or ion transport. (3) Some neurotransmitter uptake systems are more easily damaged by anaerobiosis than others.     

Bone Allografts: Past, Present and Future

Bone allograft transplantation has been performed in humans for more than one hundred and twenty years. During the first one hundred years (1880–1980), the major problem in bone allograft transplantation was availability. Most of the bone grafts used during this time were autografts. Allografts were not available due to a lack of legislation protecting procurers and processers. In addition, surgical procedures requiring allografts were not being performed. During the next twenty years (1980–2000), as allografis began to be used, the major issue was safety. Diseases transmitted during this period included AIDS and hepatitis. Avoidance of disease transmission became paramount. Sensitive blood tests and extensive efforts by bone banks to develop ways to clean. bone and clear it of infectious agents helped provide safe transplants. With concerns of availability and safety receding, the major issue in the future (2000–? ) will be the efficacy of the transplant. How allograft bone remodels in the host, how it incorporates and heals to host bone and how it integrates with the host skeleton will be the most important concerns of bone bankers and tissue transplant surgeons. Future research efforts will be applied to bone allograft transplantation to ensure that bone transplants heal quickly and sufficiently to be able to function as part of the weight-bearing skeletal system.     

Rice Research： Past, Present and Future

Rice （Oryza sativa L.） is a major crop in the world and provides the staple food for over half of the world＇s population. From thousands of years of cultivation and breeding to recent genomics, rice has been the focus of agriculture and plant research.     

Nitrogen Cycles: Past, Present, and Future

This paper contrasts the natural and anthropogenic controls on the conversion of unreactive N₂ to more reactive forms of nitrogen (Nr). A variety of data sets are used to construct global N budgets for 1860 and the early 1990s and to make projections for the global N budget in 2050. Regional N budgets for Asia, North America, and other major regions for the early 1990s, as well as the marine N budget, are presented to Highlight the dominant fluxes of nitrogen in each region. Important findings are that human activities increasingly dominate the N budget at the global and at most regional scales, the terrestrial and open ocean N budgets are essentially disconnected, and the fixed forms of N are accumulating in most environmental reservoirs. The largest uncertainties in our understanding of the N budget at most scales are the rates of natural biological nitrogen fixation, the amount of Nr storage in most environmental reservoirs, and the production rates of N₂ by denitrification.