Characterization of age-dependent decline in spontaneous running performance in the heart failure Tgαq*44 mice.

In this study we characterize the impact of aging on the spontaneous running performance of the Tgαq*44 mice (transgenic murine model of chronic heart failure) as compared to the wild-type FVB mice. In 166 mice we have recorded the following parameters of their physical activities in the running wheels: the total distance covered during the experiment (Dsum), the maximal distance covered in single-effort (Dmax), mean time spent on running per 24 h (Tmean), mean running speed (νmean), the maximum instantaneous speed of run (νmax) and the number of efforts (i.e. the number of running events undertaken by the mice) during 54 days, in four age groups ~4, ~10, ~12 and ≥12.5 months of age. The level of spontaneous running performance of the FVB mice remained essentially unchanged, but a strong impact of aging in the Tgαq*44 mice on their running performance was found. Namely, the Dsum, Dmax, Tmean and νmean in the Tgαq*44 mice at the age of ≥12.5 months decreased by ~50%, when compared to its level corresponding level at the age of ~4 months, with far lesser effect of aging on their Vmax. Surprisingly, the number of attempts to perform running by the Tgαq*44 mice at the age of 4 - 12 months remained essentially unchanged. This suggests that the exercise intolerance of the aging heart failure (HF) mice seems to be more dependent on deterioration of heart and muscles function linked to HF than on a possible ageing-related impairment of the 'willngness' to initiate running, generated by the central nervous system.

Toxic effects of acrylamide on survival, development and haemocytes of Musca domestica.

The influence of acrylamide, a potentially toxic substance present in some types of food, on survival, postembryonic development and haemocytes, insect's blood cells, of the housefly was examined. Larvae were reared on media contaminated with acrylamide at concentrations of 82 microg/g, 164 microg/g or 246 microg/g. The length of larval and pupal stages as well as the survival of larvae and pupae was examined. To study the effects of acrylamide on haemocytes, the analysis of their index and morphology was performed in the third instar larva. The obtained data showed that the survival of larvae exposed to 82 microg/g and 164 microg/g concentrations of acrylamide decreased by 50% and 85%, respectively, whereas 246 microg/g concentration was lethal. In both groups of flies, larval and pupal stages were significantly lengthened by about 1.5 day in comparison with control. Moreover, acrylamide increased the number of prohaemocytes and intermediate cells while the number of plasmatocytes and granulocytes decreased. The size of plasmatocytes decreased in acrylamide-treated larvae when compared with these cells of control flies. The reduced survival of animals is probably due to affecting haemocytes involved in immune responses in insects. Moreover, the housefly's blood cells showed to be sensitive to toxin, which suggests their usefulness to test toxicity of substances present in food products.

Structural daily rhythms in GFP-labelled neurons in the visual system of Drosophila melanogaster.

In the visual system of Drosophila melanogaster, two classes of interneurons in the first optic neuropil, or lamina, the monopolar cells L1 and L2, show rhythmic circadian changes in the shape and size of their axons. In the present study we have used the GAL4-UAS system to target the GFP expression to the L2 cells in D. melanogaster and to examine morphological changes in the cell body, nucleus, axon and dendritic spines. Our results showed that in addition to changes in the caliber of its axon, L2 also shows daily changes in the morphology of its dendritic spines, differences which are most pronounced at the beginning of the night. There are also changes in the sizes of the cells' nuclei in the lamina cortex, which are largest at the beginning and in the middle of day, in females and males, respectively. In contrast to the axon and dendrites, L2's soma does not change size significantly during the day or night. The observed changes clearly indicate the cyclical modulation of the structure of the L2 interneurons. These changes seem to be regulated by a circadian clock, which exhibits certain differences between the sexes.

Heavy metal accumulation, heat shock protein expression and cytogenetic changes in Tetrix tenuicornis (L.) (Tetrigidae, Orthoptera) from polluted areas.

The orthopteran insect Tetrix tenuicornis, collected from polluted and unpolluted areas, was used to study heavy metal accumulation and its impact on stress protein levels and on changes in the number and morphology of chromosomes in mitotic and meiotic cells. During two consecutive years, insects were collected from polluted areas of zinc-lead mine spoils near Boleslaw (Poland) and from unpolluted areas near Busko and Staszów (Poland). T. tenuicornis from the polluted area showed 1.5, 4.03, 4.32 and 41.73 times higher concentrations of copper (Cu), zinc (Zn), lead (Pb) and cadmium (Cd), respectively, than insects of the same species collected from unpolluted areas. Insects exposed to heavy metals showed only small changes, and rather a decrease in the concentration of constitutive and inducible heat shock proteins Hsp70, the level of which increases under stress conditions. A cytogenetic study of T. tenuicornis revealed intra-population anomalies in chromosome number and morphology in mitotic and meiotic cells and the presence of an additional B chromosome in germinal cells. In 50% of females collected from polluted areas, mosaic oogonial mitotic chromosome sets and diploid, hypo- or hypertetraploid, tetraploid, and octoploid chromosome numbers were detected. In turn, 14.6% of males showed a heterozygous deficiency of chromatin in L2 and M3 bivalents in addition to the presence of B chromosomes.

Involvement of V-ATPase in the regulation of cell size in the fly's visual system.

In the fly's visual system, two classes of lamina interneuron, L1 and L2, cyclically change both their size and shape in a rhythm that is circadian. Several neurotransmitters and the lamina's glial cells are known to be involved in regulating these rhythms. Moreover, vacuolar-type H+-ATPase (V-ATPase) in the optic lobe is thought also to participate in such regulation. We have detected V-ATPase-like immunoreactivity in the heads of both Drosophilla melanogaster and Musca domestica using antibodies raised against either the B- or H-subunits of V-ATPase from D. melanogaster or against the B-subunit from two other insect species Culex quinquefasciatus and Manduca sexta. In the visual systems of both fly species V-ATPase was localized immunocytochemically to the compound eye photoreceptors. In D. melanogaster immunoreactivity oscillated during the day and night and under constant darkness the signal was stronger during the subjective night than the subjective day. In turn, blocking V-ATPase by injecting a V-ATPase blocker, bafilomycin, in M. domestica increased the axon sizes of L1 and L2, but only when bafilomycin was applied during the night. As a result bafilomycin abolished the day/night difference in axon size in L1 and L2, their sizes being similar during the day and night.

The regulation of circadian rhythms in the fly's visual system: involvement of FMRFamide-like neuropeptides and their relationship to pigment dispersing factor in Musca domestica and Drosophila melanogaster.

The cross-sectional area of axon profiles in two classes of interneuron, L1 and L2, in the fly's lamina, exhibits a circadian rhythm of swelling and shrinking; axon caliber also changes after microinjecting putative lamina neurotransmitters. Among these, the neuropeptide pigment-dispersing factor, PDF, is proposed to transmit circadian information from the housefly's (Musca domestica) clock to L1 and L2, increasing axon caliber during the day. Testing whether other neurotransmitters may modulate this effect we have: (1) examined optic lobe cell immunoreactivity to FMRFamide peptides and its co-immunolocalization to PDF in M. domestica and Drosophila melanogaster, and to the product of the circadian clock gene PER in D. melanogaster; and (2) made microinjections of FMRFamide and related neuropeptides into the second neuropil, or medulla. In M. domestica, nine groups of optic lobe cells, several cells in the lateral and dorsal protocerebrum, and in the subesophageal ganglion, together contribute dense FMRFamide immunoreactive arborizations in almost all central brain and optic lobe neuropils. In D. melanogaster a similar pattern of labeling arises from fewer cells. Daytime microinjections show that another neuropeptide, similar to molluscan FMRFamide, shrinks M. domestica's L1 and L2 axons, thus opposing the action of PDF. We discuss evidence for a medulla site of action for a released FMRFamide-like peptide, either from: MeRF2 cells, acting directly on L1 and L2's medulla terminals; or MeRF1 cells, acting indirectly via medulla centrifugal cells C2 and C3.

Localization of the clock controlling circadian rhythms in the first neuropile of the optic lobe in the housefly.

The visual system of a fly expresses several circadian rhythms that have been detected in the photoreceptors of the compound eye and in the first neuropile, the lamina, of the underlying optic lobe. In the lamina, axons of two classes of interneuron, L1 and L2, exhibit cyclical size changes, swelling by day and shrinking by night. These rhythmic size changes may be generated by circadian oscillators located inside and/or outside the optic lobe. To localize such oscillators, we have examined changes in the axonal cross-sectional areas of L1 and L2 within the lamina of the housefly (Musca domestica) under conditions of 12 h of light and 12 h of darkness (LD12:12), constant darkness (DD) or continuous light (LL) 24 h after the medulla was severed from the rest of the brain. After the lesion, the axon size changes of L1 and L2 were maintained only in LD conditions, but were weaker than in control flies. In DD and LL conditions, they were eliminated. This indicates that circadian rhythms in the lamina of a fly are generated central to the lamina and medulla neuropiles of the optic lobe. Cyclical changes of light and darkness in LD conditions are still able, however, to induce a weak daily rhythm in the axon sizes of L1 and L2.

Daily rhythmic changes of cell size and shape in the first optic neuropil in Drosophila melanogaster.

Daily rhythms of changes in axon size and shape are seen in two types of monopolar cell-L1 and L2-that are unique cells within each of the modules or cartridges of the first optic neuropil or lamina in the fly's optic lobe. In the fruit fly Drosophila, L1 and L2's axons swell at the beginning of both day and night, with larger size increases occurring at the beginning of night. Later, they shrink during the day and night, respectively. Simultaneously, they change shape from an inverted conical form during the day to a cylindrical one at night. This is because the axonal cross section of L1 increases during the night, especially at proximal depths of the lamina, closest to the brain, whereas the axon of L2 increases in size at distal lamina depths. The cross-sectional areas of the L1 cell and of an individual cartridge both change under constant darkness (DD), indicating the circadian origin of changes observed under day/night (LD) conditions. We sought to see whether such changes impart a net change to the entire lamina's volume or shape that is visible by light microscopy, but oscillations in the volume or the curvature of the whole lamina neuropil are found neither in LD nor in DD. These size changes are discussed in relation to previous findings in the housefly Musca, with respect to differences in L1 and L2 between the two species, and to differences in the time course of their circadian changes.

[Molecular mechanisms of the biological clock]. / Molekularne mechanizmy zegara biologicznego.

Considerable progress has been made in elucidating the mechanisms underlying the generation of circadian rhythmicity. This review describes recent observations and hypothesis on the nature of circadian biological clock in different organisms, such as cyanobacteria (Synechococcus; in which the first noneukaryotic clock was described), Neurospora and Drosophila (representing respectively a fungal and insect system of crucial value for the molecular dissection of circadian timing systems), as well as mammals. Being specific for particular group of organisms, a common model of the circadian biological clock can be depicted which involves a negative transcription/translation-based feedback loop, containing positive and negative elements, and clock genes, as well as clock controlled genes that are directly related to rhythmic metabolism and behavior.

Neurotransmitter regulation of circadian structural changes in the fly's visual system.

The visual system of the fly's compound eye undergoes a number of cyclical day/night changes that have a circadian basis. Such responses are seen in the synaptic terminals of the photoreceptors and in their large monopolar-cell interneurons in the first optic neuropile, or lamina. These changes include, in the photoreceptor terminals, rhythms in the numbers of synapses and the vertical migration of screening pigment; and, in the monopolar cells L1 and L2, a rhythm in the transients of the electroretinogram and in the cyclical swelling of L1 and L2 lamina axons, as well as of the epithelial glia that surround these. Some of these changes are seen in both the housefly and the fruit fly, but the time-course of such changes differs between the two species. Many of the changes are influenced by the injection of various transmitter candidates, in a direction that can be reconciled with the possibility of normal endogenous release of two substances, 5HT from the neurites of 5HT-immunoreactive neurons, and pigment dispersing factor peptide from the neurites of PDH cells. Consistent with this interpretation, the immunoreactive varicosities of PDH cells exhibit size changes attributable to their cyclical release of peptide, or to its cyclical synthesis and/or transport from the PDH cell somata. Thus, neurotransmitter substances not only have rapid electrophysiological actions in the optic lobe, but also longer-lasting, presumably indirect, neuromodulatory actions, which are manifest as structural changes among the lamina's neurons and synapses. These actions involve an interplay between aminergic and peptidergic systems, but the exact role and especially the site of action of each has still to be elucidated.

Neurotransmitters alter the numbers of synapses and organelles in photoreceptor terminals in the lamina of the housefly, Musca domestica.

Various organelles in the lamina terminals of housefly photoreceptors exhibit daily rhythms having a circadian basis. These include changes in the numbers of photoreceptor tetrad and L2 feedback synapses, and longitudinal movements of screening pigment. Circadian information has previously been suggested to spread from the clock to the lamina via widefield cells expressing either 5-hydroxytryptamine or pigment-dispersing hormone-like immunoreactivity. We examined the action of these neuromodulators, and other candidate neurotransmitters, 4 h after injecting either the transmitter or a control into the medulla. We counted electron microscope profiles of organelles that normally exhibit circadian changes, and two types of invagination into photoreceptor terminals, capitate projections and inter-receptor invaginations. No single substance mediated the changes observed. Injected pigment-dispersing hormone peptide decreased the number of pigment granules, implicating this peptide in screening pigment migration, but produced no changes in synapse-related organelles. alpha-Aminobutyric acid exclusively decreased the number of L2 feedback synapses. Responses to other transmitters were specific, and often large, but generally not statistically significant. Histamine, for example, may decrease the number of tetrads, possibly by direct autoregulation. The results suggest that there is likely to be more than one effector in the circadian pathways to the lamina.

Neurites of period-expressing PDH cells in the fly's optic lobe exhibit circadian oscillations in morphology.

Circadian rhythms have been shown both in the expression of the period (per) gene in 'lateral neurons' and in cells of the outermost neuropil, or lamina, of the fly's optic lobe. Some lateral neurons also exhibit PDH peptide-like immunoreactivity, arborizing widely throughout the optic lobe. Using confocal microscopy in the housefly, we analysed the size and spacing of PDH neurite varicosities, sites of possible peptide release exhibiting circadian rhythmicity. During the subjective day in constant darkness, there were fewer, larger varicosities than during subjective night. The endogenous rhythm was masked by the light exposure that occurred under a day-night cycle and continuous light conditions. Our findings indicate that PDH neurites convey circadian information out from the pacemaker, where they could regulate the circadian rhythms that have been described in the lamina, possibly via cyclical release of their peptide.

Circadian rhythms in screening pigment and invaginating organelles in photoreceptor terminals of the housefly's first optic neuropile.

Screening pigment granules occur in the synaptic terminals of photoreceptors in the fly's (Musca domestica, L.) compound eye. The granules resemble ommochrome granules in the overlying photoreceptor cell body. There are also two types of invagination into receptor terminals: capitate projections (from glial cells) and invaginations from neighboring receptor terminals. The number of profiles of these organelles in the first optic neuropile, the lamina, have been counted using single-section quantitative electron microscopic methods. Pigment granules are concentrated proximally in the terminal, toward the brain. The numbers change, increasing during the night (1 h after lights off) up to values more than twice the number 1 h after lights on, apparently by longitudinal migration of granules from the cell body into the terminal. Files entrained to day/night conditions and then held under constant darkness continue to exhibit changes in the numbers of profiles. Even though overall there were 80-90% fewer granule profiles than under day/night conditions, the numbers attained a peak many times higher at the end of the subjective day. Thus, the changes are endogenous, showing circadian rhythmicity. Although their significance is unknown, these changes parallel previously described circadian rhythms in the receptor terminals and their lamina monopolar-cell targets. The invaginations from receptor terminals were more numerous under day/night conditions than under constant darkness, and cycled in constant darkness, peaking at the end of subjective night. Capitate projections, by contrast, failed to change significantly under the experimental conditions analyzed, a lack of responsiveness they share with photoreceptor tetrad synapses.

Heat shock proteins (HSP70) as biomarkers in ecotoxicological studies.

Hsp70, so-called stress proteins, were studied in the centipede Lithobius mutabilis when exposed in laboratory tests to different concentrations of the insecticide dimethoate (DMT; 0, 0.012, 0.111 mg kg-1 dwt), the detergent linear alkilobenzene sulfonate (LAS; 0, 16, 80, 400, 2000, 10,000 mg kg-1 dwt), and copper (Cu; 0, 56, 167, 500, 1000, 1500 mg kg-1 dwt) and in the field in captured animals from polluted (2 and 4 km from a zinc-and-lead smelter) and unpolluted (35 and 40 km from the smelter) areas. Hsp70 in centipedes were also tested for seasonal differences (March and September) in field-captured animals and for a temperature effect under laboratory conditions (5, 15, and 25 degrees C). Moreover, hsp70 were examined in housefly (Musca domestica) pupae after rearing larvae on food medium contaminated with DMT or LAS. Hsp70 were found in all animals tested, including controls, and their levels were not clearly related to the laboratory treatment with chemicals or temperature or to the degree of contamination in polluted areas. In centipedes from unpolluted areas, a significant seasonal difference in hsp70 content was found. The problems with using hsp70 as a universal biomarker in ecotoxicological studies are discussed.

Daily rhythms in cells of the fly's optic lobe: taking time out from the circadian clock.

Considerable progress has recently been reported in locating the cellular basis and molecular mechanisms of the circadian clock in the fruitfly, Drosophila melanogaster. To advance beyond the clock, towards the outputs that lie between the clock itself and the circadian rhythms in behaviour that it regulates, will present new challenges. This is because most behaviours are generated by complex neuronal circuits, which are themselves difficult to unravel. Recently described anatomical changes in the optic lobe of the related housefly, Musca domestica, exhibit a circadian rhythm that is, by contrast, relatively easy to assay. This rhythm is apparently controlled by at least two sets of diffuse modulatory neurones. One of these, immunoreactive to the peptide pigment-dispersing hormone, also expresses in Drosophila the product of the period (per) gene, the most widely studied of the so-called clock genes that are essential for the correct expression of circadian rhythmicity. The second, called LBO5HT, is immunoreactive to 5-HT, a widely invoked transmitter system in insect circadian rhythms. The identification of these elements, and a widening cascade of events which their actions apparently trigger, opens up new opportunities to examine old problems in the regulation of circadian rhythms in the nervous system.

Neurotransmitters regulate rhythmic size changes amongst cells in the fly's optic lobe.

Axon calibre in monopolar cells L1 and L2 of the fly's lamina can change dynamically. Swelling by day, L2 exhibits a daily rhythm of changing size apparently mediated by wide-field LBO5HT and PDH cells. L1/L2 axon profiles were measured planimetrically in the housefly, Musca domestica, from 1 microns cross sections. Four hours after injecting 80-100 nl of 1.25 x 10(-4) M 5-HT into the optic lobe, L1's axon swelled but L2's did not, whereas 2.2 x 10(-5) M of PDH enlarged both axons. Similar to 5-HT, 1.63 x 10(-4) M histamine (the photoreceptor transmitter) enlarged L1 but not L2, mimicking light exposure, while 1.7 x 10(-4) M glutamate and 1.94 x 10(-4) M GABA both decreased L1 and L2. 2.5 x 10(-4) M of 5,7-dihydroxytryptamine decreased L2 and, somewhat, L1, an effect attributable to the loss of LBO5HT neurites. Twenty four hours after cutting LBO5HT and PDH commissural pathways, L1 and L2 both shrank. Apparently, L2's size depends on either LBO5HT or sufficient 5-HT, and L1 and L2 have different response ranges to 5-HT. Responses to PDH imply that daytime PDH release drives a circadian rhythm, enlarging L1 and L2.

Monopolar cell axons in the first optic neuropil of the housefly, Musca domestica L., undergo daily fluctuations in diameter that have a circadian basis.

Two types of monopolar cell interneurons, each with a single representative in every unit cartridge of the first optic neuropil, or lamina, of the housefly's optic lobe, have axons that undergo cyclical changes in diameter. The axons are largest during the beginning of day in a normal LD light cycle and smallest during the middle of the night, changes that were however significant only for one of the cells (L2). The axon cross-sectional area and its cyclical change for both L1 and L2 were both larger in the proximal lamina. The changes are not a simple consequence of relative osmotic change. Dehydration paradoxically increases axon size, and also fails to alter the day/night rhythm of axon size changes. Under conditions of constant darkness, both axons decrease in size, and one of the cells (L2) retains its cyclical size changes, being larger in the subjective day than in the subjective night. Under conditions of constant light, both axons increase in size, and L2 again shows a cyclical size change, just as under conditions of constant darkness. These changes seen under constant conditions are, by definition, circadian in origin. The effects of exposure to light or darkness can partially reset these circadian changes. One extra hour of light during the day increases the size of L1 and L2, whereas 1 hr of extra dark during the night does not decrease their size. It takes 13 hr of light to reverse the rhythm in size. The mechanism for all these changes is unclear but may involve ionic fluxes, possibly that are secondary to osmotic shifts and probably that involve at least two independent processes.

Daily and circadian rhythms of synaptic frequency in the first visual neuropile of the housefly's (Musca domestica L.) optic lobe.

Photoreceptors of the fly's compound eye generally show no very obvious daily or circadian rhythms, a lack which prompted us to examine whether their function might be regulated not in the retina, but at the site of transmission in the first visual neuropile, or lamina. Here, photoreceptor terminals (R1-R6) are reciprocally interconnected with one class of lamina monopolar cell, L2: L2 receives input from R1-R6 at so-called tetrad synapses, and in turn is presynaptic to R1-R6 at feedback synapses. We have calculated the mean frequencies of these synaptic profiles in electron micrographs of single lamina sections. L2 feedback synapses were more numerous at night than during the day, whereas the number of tetrads showed only small modulations between day and night. These changes persisted amongst feedback synapses in flies held in constant darkness, and are thus circadian. In contrast to the slow modulations during a 24 h cycle, the number of L2 feedback synapses after 1 h light pulse in flies held in constant darkness showed no clear change, whereas it increased the number of tetrad profiles. These findings support the occurrence of cyclical daily and circadian changes amongst the two lamina synaptic populations, with tetrads showing rather weak modulations in frequency, but more pronounced responses to the light pulse than feedback synapses.

Serotonin, dopamine, noradrenaline and their metabolites: levels in the brain of the house cricket (Acheta domesticus L.) during a 24-hour period and after administration of quipazine--a 5-HT2 receptor agonist.

1. The levels of 5-HT, DA, NA and DA metabolites (NADA, DOPAC) measured by HPLC (with electrochemical detection) in the brain of the house cricket did not change over a 24-hr period. The level of 5-HIAA, a 5-HT metabolite, was below the limit of detection. 2. The 5-HT and DOPAC levels decreased and NADA increased after quipazine injection but DA and NA levels did not change after it. 3. [3H]Ketanserin was used to identify serotonin receptors bound to sites in the house cricket brain with a KD of 5 nM and a concentration of Bmax 180 fmol/mg protein.