Pharmacological analysis of ionotropic glutamate receptor function in neuronal circuits of the zebrafish olfactory bulb

Although synaptic functions of ionotropic glutamate receptors in the olfactory bulb have been studied in vitro, their roles in pattern processing in the intact system remain controversial. We therefore examined the functions of ionotropic glutamate receptors during odor processing in the intact olfactory bulb of zebrafish using pharmacological manipulations. Odor responses of mitral cells and interneurons were recorded by electrophysiology and 2-photon Ca(2+) imaging. The combined blockade of AMPA/kainate and NMDA receptors abolished odor-evoked excitation of mitral cells. The blockade of AMPA/kainate receptors alone, in contrast, increased the mean response of mitral cells and decreased the mean response of interneurons. The blockade of NMDA receptors caused little or no change in the mean responses of mitral cells and interneurons. However, antagonists of both receptor types had diverse effects on the magnitude and time course of individual mitral cell and interneuron responses and, thus, changed spatio-temporal activity patterns across neuronal populations. Oscillatory synchronization was abolished or reduced by AMPA/kainate and NMDA receptor antagonists, respectively. These results indicate that (1) interneuron responses depend mainly on AMPA/kainate receptor input during an odor response, (2) interactions among mitral cells and interneurons regulate the total olfactory bulb output activity, (3) AMPA/kainate receptors participate in the synchronization of odor-dependent neuronal ensembles, and (4) ionotropic glutamate receptor-containing synaptic circuits shape odor-specific patterns of olfactory bulb output activity. These mechanisms are likely to be important for the processing of odor-encoding activity patterns in the olfactory bulb.     

Developmental changes of nerve growth factor and its mRNA in the rat hippocampus: Comparison with choline acetyltransferase

Previous experiments have demonstrated that in the septo-hippocampal system choline acetyltransferase (ChAT) is induced by nerve growth factor (NGF) (Gnahn et al. (1983) Dev. Brain Res. 9, 45-52) and that hippocampal NGF and mRNANGF levels are correlated with the density of cholinergic innervation (Korsching et al. (1985) EMBO J. 4, 1389-1393). In the present investigation we have compared the developmental changes of ChAT, NGF, and mRNANGF levels in this system. During the postnatal development of the hippocampus the time courses of NGF and ChAT were well correlated including the most rapid increase between P12 and P14. This increase in hippocampal NGF was preceded by a corresponding increase in mRNANGF. The developmental changes in hippocampal NGF levels were also closely reflected by corresponding changes in the septum. This, together with previous observations (Korsching et al., 1985) that the adult septum, in spite of relatively high NGF levels, does not contain measurable quantities of mRNANGF, suggests that the NGF levels in the septum are determined by the quantity of NGF transported retrogradely from the field of innervation rather than by local synthesis. During the prenatal period hippocampal NGF levels were relatively high, whereas the mRNANGF was below the level of detection. Since the ingrowth of septal fibers, and with that also the removal of NGF by retrograde transport, begins around birth, the relatively high prenatal NGF levels probably result from an accumulation produced by a small copy number of mRNANGF prior to the removal of NGF by retrograde axonal transport. It is concluded that the correlation of the developmental changes in NGF and mRNANGF with the ChAT activity in the hippocampus further supports the concept of a physiological role of NGF in the central nervous system.     

Nitrous oxide and methane fluxes of a pristine slope mire in the German National Park Harz Mountains

Pristine peatlands covered by Histosols (bogs and fens) with high water table and a restricted oxygen (O₂) availability are known to have low emissions of nitrous oxide (N₂O) but may be a significant source for atmospheric methane (CH₄) which are both important greenhouse gases. For the first time N₂O and CH₄ fluxes of a pristine slope mire in the German Harz Mountains have been monitored. Previously reported peatlands are characterised by anaerobic conditions due to high water table levels. Slope mires monitored here receive O₂ through slope water inflow. Gas fluxes have been monitored deploying closed chamber method on a central non-forested area and a forested area at the periphery of the slope mire. By means of groundwater piezometers water table levels, ammonium and nitrate contents as well as hydro-chemical variables like oxygen content and redox potential of the mire pore water have been concurrently measured with trace gas fluxes at both monitoring sites of the slope mire. The slope mire took up small amounts of atmospheric methane at a rate of −0.02 ± 0.01 kg C ha⁻¹ year⁻¹ revealing no significant difference between the forested and non-forested site. Higher uptake rates were observed during low water table level. In contrast to pristine peatlands influx of oxygen containing pore water into slope mire does limit reduction processes and resultant CH₄ emission. N₂O fluxes of the forested and non-forested sites of the slope mire did not differ and amounted to 0.25 ± 0.44 kg N ha⁻¹ year⁻¹. Higher emissions were observed at low water table levels and during thawing periods. In spite of favourable conditions N₂O fluxes of the slope mire have been comparable to those of pristine peatlands.     

Anaerobic Cometabolic Conversion of Benzothiophene by a Sulfate-Reducing Enrichment Culture and in a Tar-Oil-Contaminated Aquifer

Anaerobic cometabolic conversion of benzothiophene was studied with a sulfate-reducing enrichment culture growing with naphthalene as the sole source of carbon and energy. The sulfate-reducing bacteria were not able to grow with benzothiophene as the primary substrate. Metabolite analysis was performed with culture supernatants obtained by cometabolization experiments and revealed the formation of three isomeric carboxybenzothiophenes. Two isomers were identified as 2-carboxybenzothiophene and 5-carboxybenzothiophene. In some experiments, further reduced dihydrocarboxybenzothiophene was identified. No other products of benzothiophene degradation could be determined. In isotope-labeling experiments with a [¹³C]bicarbonate-buffered culture medium, carboxybenzothiophenes which were significantly enriched in the ¹³C content of the carboxyl group were formed, indicating the addition of a C₁ unit from bicarbonate to benzothiophene as the initial activation reaction. This finding was consistent with the results of earlier studies on anaerobic naphthalene degradation with the same culture, and we therefore propose that benzothiophene was cometabolically converted by the same enzyme system. Groundwater analyses of the tar-oil-contaminated aquifer from which the naphthalene-degrading enrichment culture was isolated exhibited the same carboxybenzothiophene isomers as the culture supernatants. In addition, the benzothiophene degradation products, in particular, dihydrocarboxybenzothiophene, were significantly enriched in the contaminated groundwater to concentrations almost the same as those of the parent compound, benzothiophene. The identification of identical metabolites of benzothiophene conversion in the sulfate-reducing enrichment culture and in the contaminated aquifer indicated that the same enzymatic reactions were responsible for the conversion of benzothiophene in situ.     

Population dynamics of an introduced bacterium degrading chlorinated benzenes in a soil column and in sewage sludge

The capacity of the -Proteobacterium Pseudomonas sp. strain P51, which degrades chlorinated benzenes, to metabolize 1,2,4-trichlorobenzene (TCB) under environmental conditions was tested by its release into two experimental systems. The first system consisted of laboratory scale microcosms which were operated with and without the addition of TCB and which were inoculated with sludge from a wastewater treatment plant. The second system consisted of a non sterile, water saturated soil column. We determined survival of strain P51 after its introduction and its ability to degrade TCB. The population dynamics was followed by selective plating and applying the polymerase chain reaction (PCR) to detect strain P51 and the chlorobenzene ( tcb) genes on catabolic plasmid pP51. The results showed a completely different behaviour of strain P51 in the two habitats under the applied conditions. In the soil column the P51 bacteria inoculated the entire area and their population reached 2 × 10⁶ cells/g soil. The population remained active since TCB was degraded to concentrations below the detection limit of 30 g/l. In the sludge microcosms, the number of strain P51 cells immediately decreased from 4 × 10⁷ cells/ml to 10⁵ cells/ml over a period of 2 days after inoculation, and then the strain disappeared to levels below our detection limit (10³–10⁴ cells/ml). In the reactor without TCB the population of P51 maintained a stable value of 10⁵ cells/ml during 8 days but then also decreased to levels below the detection limit. In addition, no significant TCB degradation was found in the sludge reactors. The influence of presence of TCB on maintenance of strain P51 in the two habitats is discussed. This work demonstrates the possibility to successfully apply preselected strains to degrade otherwise poorly degradable substances in complex mixed microbial communities. However, survival and activity may depend strongly on the type of system into which the strain is introduced.     

Isoproterenol inhibition of horse serum cholinesterase is connected with subunit dissociation

Tetrameric cholinesterase from horse serum undergoes concentration-dependent dissociation. The dimer is highly stable so that even on SDS polyacrylamide gels subunit dissociation to the 80-kDa polypeptide chains is incomplete. Glutaraldehyde cross-linking confirms this finding, giving rise to a tetramer: dimer ratio of approximately 1:1. The beta-adrenergic agent isoproterenol acts as an inhibitor of the enzyme with respect to butyrylthiocholine hydrolysis; inhibition kinetics point to a dissociative effect of the ligand as the underlying mechanism (S?ylemez, Z. & Ozer, I. (1985) Comp. Biochem. Physiol. 81c, 433-437). Evidence from sedimentation analysis confirms this hypothetical mechanism: the sedimentation coefficient in the presence of saturating concentrations of both the substrate butyrylthiocholine and the inhibitor isoproterenol shows a 35 +/- 5% decrease; in high speed sedimentation equilibria the weight average molecular mass is shifted from the tetramer (Mr = 312 +/- 12 kDa) to the dimer (Mr = 160 +/- 10 kDa). The transition is complete at isoproterenol concentrations below saturation. Applying glutaraldehyde cross-linking to monitor the particle distribution at varying isoproterenol concentrations confirms the change in quaternary structure in a qualitative way. Enzyme concentrations applied in the present experiments are in the range of the concentration of cholinesterase in horse serum. Therefore the dissociative mechanism of isoproterenol on the enzyme may be of biological significance.     

Dolichyl phosphate-dependent glycosyltransferases utilize truncated cofactors.

Synthetic truncated dolichyl phosphates of chain lengths from four to thirteen isoprene units (Jaenicke L. and Siegmund H.-U., Chem. Phys. Lipids 51 (1989) 159-170) were assayed for their cofactor activity in the enzymatic transfer of hexoses and hexosamines. The enzymes were microsomal preparations from the green alga Volvox carteri, baker's yeast, and mammalian liver cells. Under saturating conditions, the acceptor activities of the truncated dolichyl phosphates increased from zero to full strength as compared to the mixture of long-chain dolichyl phosphates from natural sources with growing chain length from five to nine isoprene units. Km determinations confirmed the results. Of the geometric isomers of dolichyl 7-phosphate (35 carbon atoms), the 14-trans compound has unchanged acceptor activity; all-trans dolichyl 7-phosphate, however, was almost inactive. The data suggest that hydrophobicity may be an important, but not the only criterion for the binding of the isoprene moiety to the active sites of the transferase enzyme(s) and that the geometry of more than only one double bond in the dolichols is recognized.     

Plasma normetanephrine: A biochemical marker of sympathetic nerve function in man

The concentration of the catecholamines in plasma are regarded by many investigators as biochemical markers of sympathoadrenal activity in man. This study was designed to: 1) assess the relationship of normetanephrine (NMN), the metabolite of norepinephrine (NE) most likely to represent degration of newly synthesized and released “active” norepinephrine, to that of norepinephrine in plasma of normal volunteers and 2) to determine regional variations in plasma normetanephrine concentrations at venous and arterial sites of patients undergoing cardiac catherization. The findings suggest that 1) plasma normetanephrine reflects sympathoadrenal activity and neurotransmitter production at both peripheral neuronal and adrenal medullary sites and 2) there is net removal of normetanephrine by liver and kidney. Plasma normetanephrine should provide assistance in the biochemical assessment of sympathoadrenal tone in man and in patients with various autonomic disorders.     

Effects of superoxide radicals on transport (Na+K) adenosine triphosphatase and protection by superoxide dismutase

Membrane (Na+K)ATPase isolated from rat brain was preincubated in a medium in which superoxide radicals were generated enzymatically. Exposure to superoxide radicals caused an irreversible inactivation, which could be prevented by further addition of superoxide dismutase. (Na+K)ATPase was also protected by addition of allopurinol, a xanthine oxidase inhibitor, during preincubation. The K-activated nitrophenylphosphatase associated with (Na+K)ATPase was also found to be inactivated by preincubation with superoxide radicals, which could be prevented by superoxide dismutase.