Electrophysiological and Pharmacological Analysis of Neuronal Activity in the Midbrain Medial Raphe Nucleus after Acute Immobilization Stress

In albino rats anesthetized with urethane, most medial raphe neurons are characterized by a low rate of the discharges. Acute immobilization stress resulted in a significant enhancement in the number of regularly discharging neurons. After immobilization, the number of neurons with a low-rate impulsation decreased, while the number of neurons with intensive firing increased. Treatment with fluoxetine enhanced the number of regularly firing neurons more than three times, and a predominant part of the neurons demonstrated a high level of background activity. It should be supposed that a significant part of the neurons with high-rate discharges are serotonergic, because their number also increased after fluoxetine administration.     

Connections Between NO Synthase-Containing Neurons of the Pedunculopontine Tegmental Nucleus and the Substantia Nigra in Rats

Experiments on rats with detection of the NADP-d-activity in neurons of the pedunculopontine tegmental nucleus (PPTg) and their processes demonstrated that between the substantia nigra (SN) and PPTg there are connections established by the neurons containing nitric oxide synthase (NOS). Two types of connections were observed. Axon-like collaterals of the neuronal pathways sent by PPTg neurons toward the forelimb penetrate the SN from its dorsal side. In addition, dendrites of the NOS-containing neurons localized within the rostral PPTg part penetrate the caudoventral region of the reticular SN (SNr). It is supposed that NO produced by these processes within the SN can exert modulating influences on synaptic transmission in this structure; when produced in excessive amounts under some pathological conditions, NO can be a factor evoking neurodegeneration in the midbrain.     

Simultaneous Determination of 5-Hydroxytryptophan and 3,4-Dihydroxyphenylalanine in Rat Brain by HPLC with Electrochemical Detection Following Electrical Stimulation of the Dorsal Raphe Nucleus

HPLC coupled with electrochemical detection was used to make concurrent measurements of the rate of accumulation of 5-hydroxytryptophan and 3,4-dihydroxyphenylalanine in selected brain regions (striatum, nucleus accumbens, septum, medial periventricular hypothalamus) and thoracic spinal cords of rats treated with NSD 1015, an inhibitor of aromatic-L-amino-acid decarboxylase. 5-Hydroxytryptophan and 3,4-dihydroxyphenylalanine accumulated in all brain regions 30 min after the intravenous infusion of various doses of NSD 1015; there were no significant differences in the responses to 12.5, 25, 50, and 100 mg/kg. After the intravenous administration of 25 mg/kg NSD 1015 the concentrations of 5-hydroxytryptophan and 3,4-dihydroxyphenylalanine increased linearly with time in all brain regions for at least 30 min. Electrical stimulation of 5-hydroxytryptamine neurons in the dorsal raphe nucleus for 30 min at 5 or 10 Hz increased 5-hydroxytryptophan accumulation in all brain regions but not in the spinal cord. Unexpectedly, this stimulation also increased the accumulation of 3,4-dihydroxyphenylalanine in the hypothalamus and spinal cord. These results suggest that 5-hydroxytryptophan accumulation following the administration of NSD 1015 is a valid index of 5-hydroxytryptamine neuronal activity in the brain.     

Role of Serotonin2A and Serotonin2B/2C Receptor Subtypes in the Control of Accumbal and Striatal Dopamine Release Elicited In Vivo by Dorsal Raphe Nucleus Electrical Stimulation

This study investigates, using in vivo microdialysis, the role of serotonin2A (5-HT2A) and 5-HT(2B/2C) receptors in the effect of dorsal raphe nucleus (DRN) electrical stimulation on dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA) extracellular levels monitored in the nucleus accumbens (NAC) and the striatum of halothane-anesthetized rats. Following DRN stimulation (300 microA, 1 ms, 20 Hz, 15 min) DA release was enhanced in the NAC and reduced in the striatum. The 5-HT2A antagonist SR 46349B (0.5 mg/kg) and the mixed 5-HT(2A/2B/2C) antagonist ritanserin (0.63 mg/kg) significantly reduced the effect of DRN stimulation on DA release in the NAC but not in the striatum. DA responses to DRN stimulation were not affected by the 5-HT(2B/2C) antagonist SB 206553 (5 mg/kg) in either region. None of these compounds was able to modify the enhancement of DOPAC and 5-HIAA outflow induced by DRN stimulation in either the NAC or the striatum. Finally, in both brain regions basal DA release was significantly increased only by SB 206553. These results indicate that 5-HT2A but not 5-HT(2B/2C) receptors participate in the facilitatory control exerted by endogenous 5-HT on accumbal DA release. Conversely, 5-HT(2B/2C) receptors tonically inhibit basal DA release in both brain regions.     

Stimulation of somatic embryo formation by mutagens and darkness in culture of immature zygotic embryos of Arabidopsis thaliana (L.) Heynh.

The aim of the study was to evaluate the influence of light conditions,physical state of the induction medium and the mutagenic treatment on theembryogenic ability of Arabidopsis thaliana (L.) immaturezygotic embryos differing in developmental stage. The efficiency of directsomatic embryogenesis (DSE) was analysed in a culture of immature zygoticembryos at an early (ES) and a late (LS) developmental stage. The efficiency ofDSE was scored as a percentage of the explants producing somatic embryos. Theexperiments indicated that the physical state of the induction medium (solid orliquid) did not influence the embryogenic ability of the cultured explants. Inthe cultures on both solid and liquid induction medium, the ES explantsproducedsomatic embryos with a frequency of 25.8–37.3% i.e. 2.5–3-timeslower than LS explants. However, an increase in the embryogenic ability of ESexplants (up to 69.8%) was observed when DSE was induced in darkness. Moreover,the stimulation of DSE efficiency in culture of ES explants was also observedafter mutagenic treatment. The chemical mutagens, MNH and EMS, applied forexplant treatment, both stimulated efficiency of somatic embryo formation inculture of ES explants. The most effective DSE induction was observed when MNHand EMS were applied in doses of 0.125–1.0 mM × 3h and0.05–0.2% × 18h, respectively. In these treatment combinations thefrequency of ES explants forming somatic embryos was found to be about 2 timeshigher than in the control culture.     

Isolation and characterization of the two structural genes coding for phosphofructokinase in yeast

Summary Yeast phosphofructokinase is an octamer composed of two different kinds of subunit. The genes coding for these subunits have been isolated by means of functional complementation in a pfk1 pfk2 double mutant. As a source of DNA the genomic library of Nasmyth and Tatchell (1980) constructed in the yeast multicopy vector YEp13 was used. Plasmids containing the information of one or the other gene were identified by back-transformation into pfk single mutants (pfk1 PFK2, PFK1 pfk2). Restriction maps of the respective insertions are provided. The genomic organization was confirmed by Southern analysis. Northern analysis showed hybridization to mRNAs of about 3.6 kb for both genes, corresponding to the molecular weight of the protein subunits. Transformation with one of the plasmids did not lead to an increase in phosphofructokinase activity. Subcloning of both genes in one multicopy vector (YEp13) and reintroduction into the yeast cell resulted in a 3.5-fold higher specific activity compared to the wild type. Overproduction of the protein subunits in this transformant was confirmed by SDS-polyacrylamide electrophoresis of crude extracts stained with Coomassie-blue. This was not accompanied by an increased ethanol production. The sequences encoding the two subunits were shown to share homology.     

The influences of ecto- and endoderm in determining the axial polarity ofHydra attenuata Pall. (Cnidaria,Hydrozoa)

Summary Ectoderm and endoderm of the gastric column ofHydra attenuata Pall. were separated from each other and reassembled with either the same (controls) or opposite polar orientation. The controls always regenerated a head and basal disc according to the original polarity. In those specimens in which the polarity of ecta- and endoderm was opposite 33 specimens out of 41 reconstituted a single polyp whose body axis was clearly identifiable. Of these cases 8 followed the polarity of the ectoderm, 3 obeyed that of the endoderm, and 22 formed axes perpendicular to the original longitudinal axis. In 5 cases 2 specimens regenerated from the reassembled specimens. It is thus demonstrated that axial polarity ofHydra is determined by both the ecto- and the endoderm.This work was carried out with the support of the Swiss National Science Foundation (Grant Nr. 3.317-0.78)     

Carbon and Sulfur Metabolism in Representatives of Two Clusters of Bacteria of the Genus Leucothrix: A Comparative Study

Major pathways of carbon and sulfur metabolisms were studied in representatives of two clusters of bacteria: Leucothrix thiophila (cluster I, strains 2WS, 4WS, and 6WS) and Leucothrix sp. (cluster II, strains 1WS, 3WS, and 5WS). All strains were capable of chemoorganoheterotrophic growth, as well as of chemolithoheterotrophic growth in the presence of reduced sulfur compounds. The bacteria were found to possess a complete set of the enzymes of the tricarboxylic acid cycle and glyoxylate cycle. The dehydrogenase activity in cells of cluster I strains was an order of magnitude lower than in cluster II strains and in other known heterotrophic bacteria. Cells of bacteria of both clusters exhibited high activity levels of enzymes involved in the energy metabolism of sulfur. The oxidation of sulfur compounds and the operation of the electron-transport chain were shown to be related. Cluster II bacteria more efficiently use organic compounds in their energy metabolism, whereas cluster I bacteria are characterized by more efficient utilization of reduced sulfur compounds. During sulfite oxidation, cluster I bacteria can synthesize ATP both via substrate-level phosphorylation and oxidative phosphorylation, whereas cluster II bacteria synthesize ATP only via the latter process.     

Patterns and Pathways in the Post-Establishment Spread of Non-Indigenous Aquatic Species: The Slowing Invasion of North American Inland Lakes by the Zebra Mussel

The zebra mussel, Dreissena polymorpha, has spread through eastern North American aquatic ecosystems during the past 15 years. Whereas spread among navigable waterways was rapid, the invasion of isolated watersheds has progressed more slowly and less predictably. We examined the patterns of overland spread over multiple spatial and temporal extents including individual lake districts, states, and multi-state regions in the USA and found that only a small proportion (<8%) of suitable inland lakes have been invaded, with the rate of invasion appearing to be slowing. Of the 293 lakes known to be invaded, 97% are located in states adjacent to the Laurentian Great Lakes with over half located in Michigan. Only six states have more than 10 invaded lakes and only in Michigan and Indiana have more than 10% of suitable lakes become invaded. At smaller spatial extents, invaded lakes are often clustered within a lake-rich region across southern Michigan and northern Indiana. This clustering appears primarily due to multiple overland invasions originating from the Great Lakes followed to a lesser extent by subsequent secondary overland and downstream dispersal. Downstream spread appears responsible for only one third of the inland invasions. Temporally, invasions peaked in the late 1990s, with only 13 new invasions (0.4% of suitable lakes) reported in 2003 in the four-state region surrounding Lake Michigan. Peak rates of invasion occurred 4–6 years earlier in Michigan relative to Indiana and Wisconsin, but this time lag is likely due to differences in the establishment of Great Lake source populations rather than ‘stepping stone’ dispersal across the landscape.     

Uptake of serotonin, 5-hydroxytryptophan and tryptophan by giant serotonin-containing neurones and other neurones in the central nervous system of the snail (Helix pomatia)

Summary Following exposure to tritiated 5-HTP, silver grains were observed over the perikarya of the GSCs (Giant serotonin cells) of Helix pomatia and other known serotonin-containing neurones in light and electron microscope autoradiograms. There was no indication that the 5-HTP was taken up by nerve endings or by non-nervous structures. The distribution of radioactivity was completely different in autoradiograms of tissue exposed to tritiated serotonin. Silver grains, often in very high concentrations, were observed only over certain fine axon branches and processes thought to be nerve endings. Electron microscope autoradiography showed that these processes contained small dense-cored vesicles, morphologically identical to those thought to sequester serotonin in the perikarya of the GSCs. The accumulation of tritiated tryptophan was less specific; all the neurone perikarya showed an accumulation of radioactivity after exposure to this substance.We are grateful to Professor J. F. Lamb for the use of the Scintillation Spectrometer.     

Region- and cell type-selective expression of the evolutionarily conserved Nolz-1/zfp503 gene in the developing mouse hindbrain

Nolz-1/Zfp503, a zinc finger-containing gene, is a mammalian member of the SP1-related nocA/elb/tlp-1 gene family. Previous studies have shown that Nolz-1 homologs are important for patterning the rhombomeres in zebrafish hindbrain. We therefore studied the expression pattern of Nolz-1 in the developing mouse hindbrain. Nolz-1 mRNA expression was detected in the prospective rhombomere 3, 5 and caudal regions as early as E8.75. After E11.5, Nolz-1-positive cells were organized as distinct cell clusters, and they were largely non-overlapped with either Pax2-positive or Phox2b-positive domains. Most interestingly, we found that Nolz-1 was specifically expressed by Phox2b-negative/Isl1/2-positive somatic motor neurons, but not by Phox2b-positive/Isl1/2-positive branchial and visceral motor neurons, suggesting that Nolz-1 may regulate development of somatic motor neurons in the hindbrain. In addition to be expressed in differentiating post-mitotic neurons, Nolz-1 was also expressed by progenitor cells in the ventricular zone located in the dorsal part of aqueduct and the alar plates of hindbrain, which suggests a regulatory role of Nolz-1 in the germinal zone. Taken together, based on its domain- and cell type-selective pattern, Nolz-1 may involve in regulation of various developmental processes, including regional patterning and cell-type specification and differentiation in the developing mouse hindbrain.     

Effects of chromium supplementation on food energy utilization and the trace-element composition in the liver and heart of glucose-exposed young mice

Essentiality of selenium (Se) for Japanese quail,Coturnix coturnix japonica, was confirmed using a formulated semipurified low-Se diet (basal) (0.05 ppm). Selenium-deficiency symptoms appeared in quails on this diet within 15 d, which corresponded to low levels of hemolysate glutathione peroxidase (GSH-Px) activity. Selenium administration at 0.05 and 2.0 ppm levels resulted in an increase of hemolysate GSH-Px activity by 64 and 116%, respectively, in both short- and long-term experiments. Growth over a 2-mo period increased the hemolysate GSH-Px activity by 120% at each level of dietary Se. A differential response was exhibited by hepatic mitochondrial and soluble GSH-Px activity to Se supplementation, the former increasing progressively with increments of Se at 0.05, 2.0, and 4.0 ppm by 45, 70 and 150%, respectively. The soluble GSH-Px activities of tissues, such as liver, kidney, and testis, and RBC membrane-bound activity remained unchanged in long-term studies at different levels of Se. Replenishment of Se to quails maintained on low-Se diets reflected no change in RBC membrane-bound and liver-soluble GSH-Px activities, although the activity in hemolysate increased consistently with Se. The GSH-Px activity in hemolysate was restored to the levels comparable to those of long-term studies only at Se administration at the 2.0-ppm level. The differential response of mitochondrial and soluble GSH-Px activities to Se and other related observations on mitochondrial functions suggest an additional role for Se in mitochondrial membrane processes and glutathione-related metabolic regulations.     

Case studies on the use of biotechnologies and on biosafety provisions in four African countries

This review is based on a study commissioned by the European Commission on the evaluation of scientific, technical and institutional challenges, priorities and bottlenecks for biotechnologies and regional harmonisation of biosafety in Africa. Biotechnology was considered within four domains: agricultural biotechnologies (‘Green’), industrial biotechnologies and biotechnologies for environmental remediation (‘White’), biotechnologies in aquaculture (‘Blue’) and biotechnologies for healthcare (‘Red’). An important consideration was the decline in partnerships between the EU and developing countries because of the original public antipathy to some green biotechnologies, particularly genetically modified organisms (GMOs) and food from GM crops in Europe. The study focus reported here was West Africa (Ghana, Senegal, Mali and Burkina Faso).The overall conclusion was that whereas high-quality research was proceeding in the countries visited, funding is not sustained and there is little evidence of practical application of biotechnology and benefit to farmers and the wider community. Research and development that was being carried out on genetically modified crop varieties was concentrating on improving food security and therefore unlikely to have significant impact on EU markets and consumers. However, there is much non-controversial green biotechnology such as molecular diagnostics for plant and animal disease and marker-assisted selection for breeding that has great potential application. Regarding white biotechnology, it is currently occupying only a very small industrial niche in West Africa, basically in the sole sector of the production of liquid biofuels (i.e., bio-ethanol) from indigenous and locally planted biomass (very often non-food crops). The presence of diffused small-scale fish production is the basis to develop and apply new (Blue) aquaculture technologies and, where the research conditions and the production sector can permit, to increase this type of production and the economy of this depressed areas. However, the problems bound to environmental protection must not be forgotten; priority should be given to monitor the risks of introduction of foreign species. Red biotechnologies potentially bring a vast domain of powerful tools and processes to achieve better human health, most notably improved diagnostics by molecular techniques, better targeting of pathogens and a better knowledge of their sensitivities to drugs to permit better treatment.Biosafety regulatory frameworks had been initiated in several countries, starting with primary biosafety law. However, disparate attitudes to the purpose of biosafety regulation (e.g., fostering informed decision-making versus ‘giving the green-light for a flood of GMOs’) currently prevent a needed consensus for sub-regional harmonisation. To date, most R&D funding has come from North America with some commercial interests from Asia, but African biotechnology workers expressed strong desire for (re-)engagement with interested parties from the European Union. Although in some of the visited countries there are very well qualified personnel in molecular biology and biosafety/regulation, the main message received is that human resources and capacity building in-house are still needed. This could be achieved through home-based courses and capacity-building including funds for post-degree research to motivate and retain trained staff.