Functional evaluation of nerve-skeletal muscle constructs engineered in vitro

Summary Previously, we have engineered three-dimensional (3-D) skeletal muscle constructs that generate force and display a myosin heavy-chain (MHC) composition of fetal muscle. The purpose of this study was to evaluate the functional characteristics of 3-D skeletal muscle constructs cocultured with fetal nerve explants. We hypothesized that coculture of muscle constructs with neural cells would produce constructs with increased force and adult MHC isoforms. Following introduction of embryonic spinal cord explants to a layer of confluent muscle cells, the neural tissue integrated with the cultured muscle cells to form 3-D muscle constructs with extensions. Immunohistochemical labeling indicated that the extensions were neural tissue and that the junctions between the nerve extensions and the muscle constructs contained clusters of acetylcholine receptors. Compared to muscles cultured without nerve explants, constructs formed from nerve-muscle coculture showed spontaneous contractions with an increase in frequency and force. Upon field stimulation, both twitch (2-fold) and tetanus (1.7-fold) were greater in the nerve-muscle coculture system. Contractions could be elicited by electrically stimulating the neural extensions, although smaller forces are produced than with field stimulation. Severing the extension eliminated the response to electrical stimulation, excluding field stimulation, as a contributing factor. Nervemuscle constructs showed a tendency to have higher contents of adult and lower contents of fetal MHC isoforms, but the differences were not significant. In conclusion, we have successfully engineered a 3-D nerve-muscle construct that displays functional neuromuscular junctions and can be electrically stimulated to contract via the neural extensions projecting from the construct.     

Free radical generation by skeletal muscle of adult and old mice: effect of contractile activity

Oxidative modification of cellular components may contribute to tissue dysfunction during aging. In skeletal muscle, contractile activity increases the generation of reactive oxygen and nitrogen species (ROS). The question of whether contraction-induced ROS generation is further increased in skeletal muscle of the elderly is important since this influences recommendations on their exercise participation. Three different approaches were used to examine whether aging influences contraction-induced ROS generation. Hind limb muscles of adult and old mice underwent a 15-min period of isometric contractions and we examined ROS generation by isolated skeletal muscle mitochondria, ROS release into the muscle extracellular fluid using microdialysis techniques, and the muscle glutathione and protein thiol contents. Resting skeletal muscle of old mice compared with adult mice showed increased ROS release from isolated mitochondria, but no changes in the extracellular levels of superoxide, nitric oxide, hydrogen peroxide, hydroxyl radical activity or muscle glutathione and protein thiol contents. Skeletal muscle mitochondria isolated from both adult and old mice after contractile activity showed significant increases in hydrogen peroxide release compared with pre-contraction values. Contractions increased extracellular hydroxyl radical activity in adult and old mice, but had no significant effect on extracellular hydrogen peroxide or nitric oxide in either group. In adult mice only, contractile activity increased the skeletal muscle release of superoxide. A similar decrease in muscle glutathione and protein thiol contents was seen in adult and old mice following contractions. Thus, contractile activity increased skeletal muscle ROS generation in both adult and old mice with no evidence for an age-related exacerbation of ROS generation.     

Consequences of metapopulation collapse: comparison of genetic attributes between two Allegheny woodrat metapopulations

Disruptions in metapopulation connectivity due to demographic pressure can leave genetically isolated subpopulations susceptible to genetic drift, accumulation of deleterious alleles, and inbreeding depression. Such a scenario may be playing out within Allegheny woodrat (Neotoma magister) metapopulations as a series of synergistic extrinsic pressures have contributed to the rangewide decline of the species over the last 40 years. Our goal was to elucidate the effects of demographic collapse on metapopulation function by using 11 microsatellites markers to quantify differences in patterns of connectivity and genetic diversity between a demographically stable metapopulation and one in severe demographic decline. The demographically diminished metapopulation had lower levels of genetic diversity than the stable metapopulation at all levels evaluated (metapopulation-, subpopulation-, and individual-scales). In contrast to patterns of connectivity observed within the stable metapopulation, peripheral subpopulations in the diminished metapopulation had become completely isolated and were drifting toward genetic fixation, likely as a result of the extirpation of stepping-stone subpopulations. The declining genetic parameters observed within these isolated peripheral subpopulations suggest that inbreeding depression may be contributing significantly to their demographic decline. Allegheny woodrats readily express the genetic consequences of metapopulation decline due to the low effective population sizes of subpopulations and the species’ limited dispersal capacity. Differences in genetic parameters observed between demographically stable and diminished Allegheny woodrat metapopulations emphasize the risks posed to metapopulation function and associated genetic processes introduced with demographic decline.     

A giant rhinocerotoid (Mammalia, Perissodactyla) from the Late Oligocene of north-central Anatolia (Turkey)

A giant rhinocerotoid is described for the first time south of the Black Sea, in Turkey. The single specimen, a fragmentary radius referred to Paraceratherium sp., originates from conglomerates nearby at Gözükizilli, in the Çankiri–Çorum Tertiary basin. These layers correspond to the Lower member of the Kizilirmak Formation. The same locality (Gözükizilli-2) yields also the small rhinocerotid Protaceratherium sp., cf. P. albigense (Roman, 1912). Three other mammal localities (Gözükizilli-1, in the Lower Member of the formation, with several rodent species; Tepe 641 and Kizilirmak, in the Upper Member, with a diversified micro- and macro-mammal fauna) allow us to refer the Kizilirmak Formation as a whole to the Late Oligocene. All the observed taxa have strong Asian and/or European affinities, which precludes any geographical insulation for this part of Anatolia during the Late Oligocene.  © 2008 The Linnean Society of London, Zoological Journal of the Linnean Society, 2008, 152 , 581–592.     

Intracellular and extracellular labeling of neurons in fresh slice preparations combined with multiple tract tracing

We describe methods for studying axo-dendritic projections, one of the forms of neural connection involved in the complex circuits of the central nervous system, including brainstem auditory pathways. This form of neural connection is often difficult to visualize by conventional tract tracing techniques. Retrogradely identified cells were filled intracellularly with a mixture of fluorescent Lucifer yellow and nonfluorescent HRP in live slice preparations to reveal the detailed morphological features of these cells with special attention to the distal dendrite that may receive projections from suspected or known input axons. Extracellular or intracellular labeling of cells with axons that project to the distal dendrite of the identified cells was accomplished in the same live slice preparation. Using a live slice rather than a fixed slice allows accurate, visually controlled placement of anterograde tracer, which requires living axons for transport, into the source of input to the identified cells within the slice. Live slices also permit one to characterize the identified cells electrophysiologically. Intracellular labeling of cells in a potential source of local input to the identified cells also provides conclusive information concerning with connections of the cells involved.     

Elevationally biased avian predation as a contributor to the spatial distribution of geometrid moth outbreaks in sub‐arctic mountain birch forest

1. Population dynamics and interactions that vary over a species' range are of particular importance in the context of latitudinal clines in biological diversity. Winter moth (Operophtera brumata) and autumnal moth (Epirrita autumnata) are two species of eruptive geometrids that vary widely in outbreak tendency over their range, which generally increases from south to north and with elevation. 2. The predation pressure on geometrid larvae and pupae over an elevational gradient was tested. The effects of background larval density and bird occupancy of monitoring nest boxes on predation rates were also tested. Predation on larvae was tested through exclusion treatments at 20 replicate stations over four elevations at one site, while pupae were set out to measure predation at two elevations at three sites. 3. Larval densities were reduced by bird predation at three lower elevations, but not at the highest elevation, and predation rates were 1.9 times higher at the lowest elevation than at the highest elevation. The rate of predation on larvae was not related to background larval density or nest box occupancy, although there were more eggs and chicks at the lowest elevation. There were no consistent differences in predation on pupae by elevation. 4. These results suggest that elevational variation in avian predation pressure on larvae may help to drive elevational differences in outbreak tendency, and that birds may play a more important role in geometrid population dynamics than the focus on invertebrate and soil predators of previous work would suggest.     

A possible role of erythrocytes in storing and distributing arachidonic acid in rat

Young rats weighing 120g and having a packed red cell volume of 37.4% were maintained on a fat free diet. In eight weeks their body weights and packed red cell volume increased to 425g and 45.4%, respectively. However, the proportion of arachidonic acid (20:4) in the total fatty acids was unaltered in erythrocytes but was lowered in other tissues. In adipose tissue, which contained only trace levels of 20:4, about one third of the fatty acid was linoleic acid (18:2). Feeding fat free diet caused a depletion of most of 18:2 in the adipose tissue. Thus, during growth, when 18:2 is excluded from the diet, erythropoiesis is not inhibited. Furthermore, 20:4 produced from stored 18:2 may be used for the production of erythrocytes which retain the tetraenoic acid effectively.     

A Critical Review of the “Undoing Hypothesis”: Do Positive Emotions Undo the Effects of Stress?

Research in affective science has shown that trait positive affect reduces the risk of developing disease and is protective against the progression of certain diseases. Research also suggests that trait positive affect predisposes individuals to more frequent experiences of positive emotional states. The undoing effect has been proposed as a potential mechanism explaining how positive emotions influence health outcomes. According to this hypothesis, the experience of state positive emotions or positive affect contributes to faster recovery from the body’s physiological response to stress. This paper critically reviews literature concerning the undoing hypothesis. Several common methodological weaknesses were identified, including conceptual and design issues. Based on the reviewed studies two separate conclusions were drawn. First, there was insufficient evidence to conclude that the occurrence of positive emotional states speeds physiological recovery. Second, there was insufficient evidence to evaluate the undoing hypothesis as a potential mechanism through which state positive emotions affect health. Future research should focus on identifying the key parameters responsible for demonstrating the undoing effect when it is observed in order to understand how the experience of positive emotions may exert positive effects on health outcomes.     

Comparison of point counts and territory mapping for detecting effects of forest management on songbirds

Point counts are commonly used to assess changes in bird abundance, including analytical approaches such as distance sampling that estimate density. Point‐count methods have come under increasing scrutiny because effects of detection probability and field error are difficult to quantify. For seven forest songbirds, we compared fixed‐radii counts (50 m and 100 m) and density estimates obtained from distance sampling to known numbers of birds determined by territory mapping. We applied point‐count analytic approaches to a typical forest management question and compared results to those obtained by territory mapping. We used a before–after control impact (BACI) analysis with a data set collected across seven study areas in the central Appalachians from 2006 to 2010. Using a 50‐m fixed radius, variance in error was at least 1.5 times that of the other methods, whereas a 100‐m fixed radius underestimated actual density by >3 territories per 10 ha for the most abundant species. Distance sampling improved accuracy and precision compared to fixed‐radius counts, although estimates were affected by birds counted outside 10‐ha units. In the BACI analysis, territory mapping detected an overall treatment effect for five of the seven species, and effects were generally consistent each year. In contrast, all point‐count methods failed to detect two treatment effects due to variance and error in annual estimates. Overall, our results highlight the need for adequate sample sizes to reduce variance, and skilled observers to reduce the level of error in point‐count data. Ultimately, the advantages and disadvantages of different survey methods should be considered in the context of overall study design and objectives, allowing for trade‐offs among effort, accuracy, and power to detect treatment effects.