Effects of Immobilization Stress on the Background Impulse Activity of Locus Coeruleus Neurons

We studied in rats changes in the impulse background activity (BA) of locus coeruleus (LC) neurons after short- and long-term immobilization stress; distributions of LC neurons by the level of regularity of their BA, dynamics of spike trains, and pattern of histograms of interspike intervals (ISI) were taken into account. We also calculated the means of the main BA statistical indices. Both short- and long-lasting immobilizations resulted in drops in the mean frequency of background discharges of LC neurons to about half of the initial value. Two-hour-long immobilization evoked statistically significant shifts in the distribution of LC neurons by the level of regularity of their BA, while after longer (15 h) immobilization this distribution nearly returned to the initial pattern. Short-lasting immobilization exerted no significant effect on the dynamic characteristics of BA; statistically significant changes in this respect developed only after longer stress. After 15-h-long immobilization, we also observed a noticeable increase in the number of neurons with polymodal ISI distributions. Therefore, stress results in significant modifications of the temporal parameters of the BA of LC neurons; characteristics of the BA of these neurons should be considered neuronal correlates of the stress state.     

Analysis of the Neuronal Background Impulse Activity in the Rat Locus Coeruleus

Using several techniques of statistical analysis, we studied in detail the extracellularly recorded background impulse activity (BA) of neuronal elements of the rat locus coeruleus (LC). Impulse trains generated by most LC neurons were stationary and demonstrated different levels of regularity; a nonstationary type of BA was observed in 17% of the neurons under study. Statistical parameters of the BA generated by LC neurons showed a wide variability. Distributions of the BA interspike intervals (ISI) of most LC neurons were characterized by more or less expressed bimodality or polymodality. Serial correlation analysis of the ISI durations both in stationary and nonstationary spike trains allowed us to differentiate five main types of the dynamics of ISI successions in the BA of LC neurons.     

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.     

Self-Initiated Motor Behavioral Act-Related Neuronal Activity in the Cat Locus Coeruleus

In experiments on awake cats, we recorded the activity of 61 putative noradrenergic neurons localized within the region of the locus coeruleus (LC) of the brainstem. The animals were trained to perform a self-initiated (voluntary) motor act aimed at obtaining a food reward by pushing a pedal by the forelimb. The intervals between pushings (stay of the limb on a platform before initiation of the movement) should not be shorter than 4 sec, and the duration of the movement itself should not exceed 1 sec. The following impulse reactions were most clearly manifested (i) related to the pre-starting events and performance of the voluntary movement, (ii) related to the presentations of the conditioning stimuli, which predicted giving out the food reward (a positive signal) or the absence of the latter (a negative signal) and (iii) related to the reward presentation. About 50% of the LC units under study had changed their activities before the movement was initiated. These reactions can be related to a cognitive component (determination of the movement initiation(, which is present in the experimental task. Most neurons responded by phasic activation to presentation of the conditioning signals, and this activation was more pronounced in the case of negative signals. Responses of the studied nerve cells are probably indicative of the involvement of the LC neuronal systems in the perception of the emotiogenic stimuli, as well as in the processes providing the maintenance of selective attention within different stages of targeted behavioral acts.     

Slow Oscillations of the Purkinje Cell Firing Rate Induced by Electrical Stimulation of the Locus Coeruleus in Rats

In anesthetized Wistar rats, we studied the effect of electrical stimulation of the locus coeruleus (LC) on the firing rates of Purkinje cells using spectral analysis. The frequency of extracellularly recorded activity of Purkinje cells was measured before and during the 1st, 5th, 6th, and 11th min after cessation of 10-sec-long LC stimulations. Spectral analysis of the Purkinje cell firing rates (imp./bin, the bin duration was 2^-8 sec) for 60- to 120-sec-long intervals was performed using fast Fourier transformation after digital conversion of unitary spikes. Mean power spectra of the Purkinje cell firing rates (derived from 8-sec-long consecutive epochs at a sampling rate of 256 sec^-1) showed an increase in the slow frequency range (0.1-1.0 Hz) after LC stimulation, particularly due to the slowest components (below 0.5 Hz). This effect lasted more than 1 min and usually less than 6 min after cessation of LC stimulation and could be interpreted as the development of slow oscillations in the Purkinje cell firing. Our results suggest that slow oscillations of the firing rate of cerebellar output neurons, induced by LC stimulation, reflect a specific coordination of the cerebellar neuronal activities (important for a central norepinephrine influence) in regulation of different pathological states.     

Effect of intracerebral implantation of the embryonicLocus Coeruleus on the conditioned reflex avoidance reaction in rats with experimental atrophy of the frontotemporal brain cortex

We studied the effect of intracerebral grafting of the embryoniclocus coeruleus from donor rats on the conditioned reflex emotional avoidance reaction (EAR) and contents of adrenaline and noradrenaline in the brain structures and body tissues of recipient rats. Group 1 of the animals consisted of intact rats, group 2 included rats with electrolytic impairment and subsequent atrophy of the frontotemporal brain cortex regions, while in group 3 the rats with impaired cortex were subjected to parallel transplantation of the embryoniclocus coeruleus into the injured region. Group-3 animals manifested a considerable level of recovery of the conditioned reflex behavioral stereotype disturbed after the cortical injury. Biochemical analysis showed that transplantation of the embryoniclocus coeruleus exerts a rehabilitative/regulating influence on the functioning of the catecholaminergic systems in the group-3 rats.     

Effects of acoustic and immobilization stress on background impulse activity of neurons of the central and lateral nuclei of the Amygdala

It was shown that acoustic and immobilization stresses (developed due to a 2.5-h-long session of intensive, 100 dB, acoustic influence and a 2-h-long session of soft fixation of the body and extremities, respectively) result in significant modifications of the characteristics of background impulse activity of neurons of the nuclei of the rat amygdalar complex. Modifications were greater in the lateral nucleus of the amygdala. Possible roles of some structures of the monoaminergic cerebral systems in acute stress-related transformations of the impulse activity generated by neurons of the amygdalar complex are discussed. Neirofiziologiya/Neurophysiology, Vol. 38, No. 2, pp. 131–139, March–April, 2006.     

Effects of Bemitil on the Activity of Noradrenergic and Serotonergic Brainstem Neurons and on EEG of Awake Cats

In experiments on five awake cats, we studied the effects of bemitil, a drug possessing psychostimulatory, antidepressive, and actoprotector properties (peroral introduction, 50 mg/kg), on the activity of neurons of the aminergic cerebral systems. Eleven noradrenergic (NA-ergic) neurons of the locus coeruleus (LC) and 11 serotonergic (ST-ergic) neurons of the nuclei raphe (NR) were examined. A control experimental series was carried out on 8 NA-ergic neurons of the LC and 8 ST-ergic neurons of the NR. Bemitil was found to exert opposite effects on the impulse activity of NA-ergic and ST-ergic brainstem neurons; it suppressed impulsation of LC neurons and increased the spiking frequency of NR neurons within certain time intervals after its introduction. Analysis of EEG showed that bemitil decreased the spectral power of the delta and theta activities, which was accompanied by behavioral relaxation. Neirofiziologiya/Neurophysiology, Vol. 37, No. 3, pp. 235–243, May–June, 2005.     

Genetic Control of Biogenic-Amine Systems in Drosophila Under Normal and Stress Conditions

The contents of octopamine and its precursors (tyrosine and tyramine) were studied in adults of two lines of Drosophila virilis with contrasting stress responses. It was demonstrated that in individuals responding to stress by a hormonal stress reaction (line 101), the contents of octopamine and tyrosine are lower than in nonresponding flies (line 147). It was found that there is no difference between the lines in the level of tyramine under normal conditions. The dopamine response to stressor was also studied. Genetic analysis of these differences revealed that they are controlled by a single gene and that the gene is not sex-linked. The gene controlling the response was found to be linked to chromosome 6 of D. virilis.     

Proteasomal Activity in Synaptosomes Obtained from the Cerebral Structures of Rats Subjected to Long-Lasting Immobilization Stress

We studied the proteasomal activity in synaptosomes obtained from tissues of the cerebral cortex, cerebellum, and hippocampus, as well as in the cytoplasm of cells of these brain structures, of rats subjected to long-lasting immobilization stress. It was demonstrated that the chymotrypsin-like activity of proteasomes in synaptosomes of the cerebral cortex and hippocampus of stressed animals was significantly higher (380 and 560%, respectively) as compared with that observed in control rats. The chymotrypsin-like and peptidylglutamyl peptide hydrolase activities of proteasomes in the cytoplasm of cortical cells under stress conditions also increased (210 and 180%, respectively). These data show that the activity of a multicatalytic proteolytic complex is sharply increased in synaptic terminals of cells of the cerebral cortex and hippocampus of stressed animals. The above complex plays a crucial role in the utilization of short-lived proteins whose molecules form receptors and ion channels; the amount of such proteins is especially great in synaptic terminals.     

Optimum Assay Conditions of the Activity of Phytochelatin Synthase from Tobacco Cells

We determined the characteristics of phytochelatin synthase from tobacco (Nicotiana tabacum cv. Bright Yellow-2) cells, especially the conditions for the enzyme stability. From the results, we proposed the optimum assay conditions of the enzyme activity.     

Cold Stress Stimulates Intracellular Calcification by the Coccolithophore, Emiliania huxleyi (Haptophyceae) Under Phosphate-Deficient Conditions

Intracellular calcification by the coccolith-producing haptophyte Emiliania huxleyi (NIES 837) is regulated by various environmental factors. This study focused on the relationship between cold and phosphate-deficient stresses to elucidate how those factors control coccolith production. (45)Ca incorporation into coccoliths was more than 97% of the total (45)Ca incorporation by whole cells. In a batch culture, orthophosphate in the medium (final concentration, 28.7 muM) was rapidly depleted within 3 days, and then extracellular alkaline phosphatase (AP) activity, an indicator of phosphate deprivation, increased during the stationary growth phase. The increase in AP activity was slightly higher at 20 degrees C than at 12 degrees C. The calcification started to increase earlier than AP activity, and the increase was much higher at 12 degrees C than at 20 degrees C. Such enhancement of calcification was suppressed by the addition of phosphate, while AP activity was also suppressed after a transient increase. These results suggest that phosphate deprivation is a trigger for calcification and that a rather long induction period is needed for calcification compared to the increase in AP activity. While calcification was greatly stimulated by cold stress, other cellular activities such as growth, phosphate utilization, and the induction of AP activity were suppressed. The stimulation of coccolith production by cold stress was minimal under phosphate-sufficient conditions. The high calcification activity estimated by (45)Ca incorporation was confirmed by morphological observations of coccoliths on the cell surface under bright-field and polarization microscopy. These results indicate that phosphate deprivation is the primary factor for stimulating coccolith production, and cold stress is a secondary acceleration factor that stimulates calcification under conditions of phosphate deprivation.     

Stress response to cold in Trichinella species

Trichinella-type larvae (L(1)) are found in the muscles of infected wild animals and domestic pigs and are the cause of trichinosis in man and other animals throughout the world. These parasites are exposed to low temperatures during their life cycle. On this premise, three Trichinella species of different types of habitat (the arctic T. nativa, the cosmopolitan T. spiralis, and the tropical T. nelsoni) were selected to examine the effect of a shift in temperature, from 37 to 4 degrees C, on long-term survival. Evaluation was then made of whether these effects were related to differential protein synthesis and/or heat shock protein (Hsp) expression. Test samples at 0, 2, 4, or 8 h and 1, 5, or 9 days after the temperature shift were obtained and subjected to Hsp determination by Western blotting. Total protein changes were explored by SDS-PAGE followed by densitometric analysis of the gels. During the "acclimatization phase" (at 2, 4, and 8 h), a different total protein and a depressed Hsp expression pattern were shown in each Trichinella species. Following acclimatization, Hsp70, but not Hsp60 or Hsp90, markedly increased above control levels in the three species, indicating a role for this Hsp as a classic stress protein. The synthesis of a 50-kDa Hsp was significantly induced in T. spiralis larvae, suggesting its potential function as a cold shock protein in this species.     

Immobilization of the epoxide hydrolase from Aspergillus niger

Three methods for the immobilization of the epoxide hydrolase from the fungus Aspergillus niger were tested. The highest immobilization yield (90%) and retention of activity (65%) were obtained by adsorption onto DEAE-cellulose compared to adsorption onto hydrophobic porous polypropylene and covalent linkage using Eupergit resin. The enzymatic properties of the immobilized enzyme were similar to those of the free enzyme with respect to the effect of temperature and pH on both activity and stability as well as the effect of solvent (DMF) on activity. The kinetic parameters were affected leading to lower K _M(app) and higher Vm _(app).     

Immobilization of Zymomonas mobilis 2716, for the protection of cellular activity

Alginate-immobilized Zymomonas mobilis cells produced 17.8% (v/v) ethanol in less than 24 h, with an ethanol yield of 97%, compared with 88% for free cells, using a fed-batch cultivation technique. The substrate, glucose, was added intermittently in powder form to foster nucleation of the CO₂ formed. Repeated-batch cultivation led to complete utilization of approximately 200 g glucose/l in 7.5 h with a 98% conversion efficiency to ethanol. Free cells used the glucose less efficiently (conversion efficiency of 78%), and even after 100 h the glucose was not fully consumed. Freeze-substitution electron microscopy studies showed that immobilized cells generally displayed lesser blebbing and membrane disruption than free cells. These studies further suggest that membrane blebbing may be due to an effect of high initial glucose levels, and not due to the accumulation of end-products ethanol and CO₂.L.A. Kirk, H.W. Doelle and R.I. Webb are with the Department of Microbiology, University of Queensland, Brisbane, QId 4072, Australia. R.I. Webb is also with the Microscopy and Microanalysis Centre, University of Queensland, Brisbane, QId 4072, Australia;     

Thermotolerance of Photosystem 2 of Three Alpine Plant Species Under Field Conditions

The species specific response of photosystem 2 (PS2) efficiency and its thermotolerance to diurnal and seasonal alterations in leaf temperature, irradiance, and water relations were investigated under alpine field conditions (1 950 m) and in response to an in situ long-term heat treatment (+3 K). Three plant species were compared using the naturally occurring microstratification of alpine environments, i.e. under contrasting leaf temperatures but under similar macroclimatic conditions. Thermotolerance of PS2 showed a high variability in all three species of up to 9.6 K. Diumal changes (increases or even decreases) in PS2 thermotolerance occurred frequently with a maximum increase of +4.8 K in Loiseleuria procumbens. Increasing leaf temperatures and photosynthetic photon flux density influenced thermotolerance adjustments. Under long-term heating (+3 K) of L. procumbens canopies with infra-red lamps, the maxima of the critical (T_c) and the lethal (T_p) temperature of PS2 increased by at least 1 K. Thermotolerance of the leaf tissue (LT₅₀) increased significantly by +0.6 K. The effects of slight water stress on thermotolerance of PS2 were species specific. High temperature thresholds for photoinhibition were significantly different between species and increased by 9 K from the species in the coldest microhabitat to the species in the warmest. Experimental heating of L. procumbens canopies by +3 K caused a significant (p>0.01) upward shift of the high temperature threshold for photoinhibition by +3 K. Each species appeared to be very well adapted to the thermal conditions of its microhabitat as under the most frequently experienced daytime leaf temperatures no photoinhibition occurred. The observed fine scale thermal adjustment of PS2 in response to increased leaf temperatures shows the potential to optimise photosynthesis under varying environmental conditions as long as the upper thermal limits are not exceeded.     

Rhizosphere Properties of Poaceae Genotypes Under P-limiting Conditions

Plant genotypes differ in P efficiency, i.e. their capacity to grow in soil with low P availability. Plant properties such as root and root hair length, release of P mineralising and mobilising compounds by the roots and P requirement for optimal growth are known to influence P efficiency. In order to improve the understanding of the role of rhizosphere properties in plant P uptake, we grew three Poaceae genotypes [two wheat (Triticum aestivum L.) genotypes (the P-efficient Goldmark and the P-inefficient Janz), and the Australian native grass Austrostipa densiflora L.] to maturity in an acidic loamy sand with low P availability. Addition of 120 mg P as FePO₄ kg⁻¹ (P120) improved the growth of all three genotypes. In both P0 and P120, growth and P uptake were smaller in Janz than in Goldmark. During the vegetative phase, growth and P uptake of Austrostipa were smaller than in Goldmark in P0 but greater in P120. These differences can be explained by plant properties such as root growth, specific P uptake, mobilisation of inorganic and organic P by root exudates and P utilisation efficiency. In P120, P availability in the rhizosphere was least in Janz and greatest in Austrostipa. Microbial biomass P in the rhizosphere was least in Janz. Acid phosphatase activity was greatest in the rhizosphere of Austrostipa and least in Janz. Plant growth and P uptake were positively correlated with microbial P, acid phosphatase activity and resin P in the rhizosphere, suggesting that microorganisms contribute to uptake of P by plants in this soil. Microbial community composition in the rhizosphere [analysed by fatty acid methylester (FAME) analysis and denaturing gradient gel electrophoresis (DGGE)] differed among genotypes, changed during plant development and was affected by P addition to the soil. Genotype-specific microbial community composition in the rhizosphere may have contributed to the observed differential capacity of plants to grow at low P availability.     

Selection of Competitive Strains of Rhizobium Nodulating Phaseolus Vulgaris and Adapted to Environmental Conditions in Egypt,Using the Gus-Reporter Gene Technique

Two Rhizobium etli strains, EBRI 2 and EBRI 26, isolated from Egypt were tested for nodulation competitiveness on beans using Rhizobium tropici CIAT 899G as the competing strain. The insertion of the gus-reporter transposon mTn5ssgusA30 did not alter the nodulation or nitrogen fixation capacity of mutant strain CIAT 899G compared to the wild type. At neutral pH, R. etli strains EBRI 2 and EBRI 26 were more competitive than CIAT 899G with the bean cultivar Saxa. These two strains gave nodule occupancies of 52.1 and 61.1% competing with equal cell numbers of CIAT 899G. Nodule occupancies from these two native strains increased with the bean cultivar Giza 6 from Egypt to 66 and 67.5%. Based on these results, cultivar Giza 6 was used to select the most competitive strains under stress of salinity or alkalinity as a major problem for a large part of Egyptian soils. Under stress of salinity (0.2% NaCl or 34.2 mM NaCl), the salt-sensitive strain EBRI 2 was more competitive than the salt-resistant strain EBRI 26. Strain EBRI 2 gave 87.4% but strain EBRI 26 gave 63.7% nodule occupancy against CIAT 899G. The same trend of results was observed under stress of alkalinity (pH 8). Strain EBRI 2 occupied 83% while Strain EBRI 26 occupied 53.2%.     

冷胁迫条件下拟南芥Rubisco相互作用蛋白质的比较分析

1,5-二磷酸核酮糖羧化酶/加氧酶(Rubisco,EC 4.1.1.39)在生物适应环境变化的过程中起到重要的作用.位于叶绿体中与冷胁迫密切相关的非常重要的复合酶——Rubisco,其相互作用的蛋白质至今没有系统的研究.对拟南芥进行4种处理:a.持续在20℃生长(对照);b.4℃4 h冷胁迫;c.4℃24 h冷胁迫;d.4℃24 h冷胁迫后放入20℃恢复24 h.然后利用免疫共沉淀、十二烷基硫酸钠聚丙烯酰胺凝胶电泳及基质辅助激光解析电离飞行时间质谱技术,在冷胁迫条件下研究了拟南芥光合抑制与Rubisco相互作用蛋白质解聚之间的关系.在鉴定出的5个与冷胁迫相关的Rubisco相互作用蛋白质中,AAA-型ATP酶家族蛋白和糖基转移酶对Rubisco活性及植物适应冷胁迫起着重要的作用.研究结果表明,Rubisco复合酶体系的解聚可能是低温胁迫下拟南芥光合速率降低的主要原因.