Keeping track of the literature isn’t easy, so Outside JEB is a monthly feature that reports the most exciting developments in experimental biology. Short articles that have been selected and written by a team of active research scientists highlight the papers that JEB readers can’t afford to miss.
POSTURE CONTROL
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Outside JEB
TO STAND OR SLOUCH? Muscle movements in animals are highly complex. Controlled by an impressive array of neural networks, they co-ordinate movements and keep us trundling along. But many of these movements are only possible because animals are capable of maintaining an erect posture, preventing themselves from collapsing in a heap on the floor. Morgane Le Bon-Jego, working with colleagues in Bordeaux and Marseille, France, is interested in a reflex mechanism called the resistance reflex, which helps to maintain a creature’s posture. Knowing that the hormone serotonin can modify posture, they decided to find out exactly how it affects the resistance reflex. To do this, they carried out a series of experiments on the crayfish Procambarus clarkii to see how this reflex is affected by serotonin. Le Bon-Jego already knew that the resistance reflex relies on the chordotonal organs in the legs. Arthropods’ chorodontal organs are located in the limb joints and code the joints’ movement and position, depending on how stretched or relaxed they are. When a leg is moved, the chordotonal organ activates the motor neurons to the opposing muscle in the limb, which contracts and maintains the limb’s status quo, keeping the creature upright. Curious to know how serotonin affects the resistance reflex, the team made recordings from the crayfish’s leg motor neurons whilst they stretched and relaxed the chordotonal organ to simulate leg movements. They compared neural activity in the motor neurons with the opposing muscle before and after they applied serotonin to the crustacean’s nervous system.
firing in the motor neurons to the opposing muscle increased. This was caused by serotonin activating pathways with many connections onto the motor neurons. Serotonin appears to boost the signal in the motor neurons, enhancing the resistance reflex. Secondly, the team looked at the motor neurons’ properties and found that the addition of serotonin makes the neurons more likely to fire, which means they respond more readily to inputs from the pathways activated by serotonin in the first experiment. The team have produced an elegant mathematical model to show how the motor neurons combine and compute all the small effects of serotonin on the nervous system. The overall effect of adding serotonin is an increase in motor neuron activity, which therefore enhances the resistance reflex. There is, however, a final twist to the tale. There was quite a bit of variation in the response of the crayfishes’ resistance reflex to serotonin, which the team speculate could be explained by the social status of the animals. Dominant animals reign supreme, holding themselves in a tall upright posture to emphasise their status. To maintain this posture, it could be that their reflexes are highly sensitive to higher levels of serotonin, whilst subordinate animals are less sensitive and have a tendency to slouch. Although the current experiments cannot prove this hypothesis, they do show that standing still can be as finely controlled as highly co-ordinated movements of many pairs of legs. 10.1242/jeb.00984
Le Bon-Jego, M., Cattaert, D. and Pearlstein, E. (2004). Serotonin enhances the resistance reflex of the locomotor network of the crayfish through multiple modulatory effects that act cooperatively. J. Neurosci. 24, 398-411.
When the team simulated an upward movement of the leg by stretching the chordotonal organ in the presence of serotonin, the size and frequency of spikes THE JOURNAL OF EXPERIMENTAL BIOLOGY 207 (11)
Laura Blackburn University of Cambridge, UK
[email protected]
Outside JEB
THE COST OF COLOR When it comes to genomes, most mammals don’t stint on their sense of smell. A substantial proportion of the mammalian genome is devoted to olfaction, and conservative estimates indicate that odorant receptor genes number in the hundreds. However, the significance of the size of an animal’s odorant receptor genome is not clear because the receptors are not very narrowly tuned, and one receptor can be stimulated by several different odorants. Nevertheless, in humans, many of the odorant receptor genes have become pseudogenes and are nonfunctional, which fits nicely with our impression that we do not rely as heavily on our sense of smell as some other mammals do. A new study by Yoav Gilad and colleagues examines the occurrence of odorant receptor pseudogenes across primates and demonstrates an interesting correlation between odorant receptor pseudogenes and color vision. First, Gilad and his coworkers demonstrated that the proportion of odorant receptor genes that are pseudogenes varies considerably across primates. The authors examined the sequences of 100 odorant receptor genes from 19 primate species and found that about 20% of the odorant receptor genes are pseudogenes in six species of New World monkeys and in lemurs. This proportion jumps to around 30% in the New World black howler monkeys, in six species of Old World monkeys and in four species of non-human apes that were examined. In humans, more than 50% of the odorant receptor genes examined were pseudogenes. Curiously, the species in which many of the odorant receptor genes are pseudogenes also have enhanced color vision.
How has enhanced color vision evolved in primates? In most mammals, the retina contains rod cells, which are extremely sensitive and allow for vision at low light levels, and cone cells, which are less sensitive and allow for daytime vision. Humans have three classes of cone cells containing slightly different opsin molecules that are maximally sensitive to light at short, medium or long wavelengths, creating the perception of blue, green and red light. When stimulated in different proportions, these opsins give us the ability to perceive fine gradations in color across the spectrum of visible light. By contrast, most mammals have dichromatic vision: their cone cells have only two opsin genes, one that makes an opsin sensitive to short (blue) wavelengths and another that makes one sensitive to longer (red or green) wavelengths. Many New World monkeys have two different alleles at this second locus, one of which makes an opsin that is most sensitive at green wavelengths, and the other at red wavelengths. These two alleles are believed to have evolved into separate loci two times, once in the New World howler monkeys and once again in Old World monkeys and apes, giving us trichromatic vision. Gilad and his co-workers suggest that this improved color vision led to decreased reliance on olfaction, which in turn may have led to relaxed selection pressure to maintain functional odorant receptor genes. If so, their work provides a mechanistic explanation for the long-held belief in neurobiology that an ecological change that increases use of one sensory system tends to lead to decreased function in other senses.
LOCOMOTOR ENERGETICS
SENSORY CHANGES
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SWINGING IS MORE COSTLY THAN WE THOUGHT If you concentrate on what your legs are actually doing during a stride, there are really only two fundamental actions: (1) limbs must exert force against the ground to support and propel the body and (2) limbs must swing through the air to be repositioned for the next support/ propulsion phase. These two actions occur in discrete phases of a stride cycle called stance and swing, respectively. Although it is clear that the metabolic cost of legged locomotion derives mostly from the recruitment and actions of limb muscles during the stride, what has remained unclear is the relative cost of stance- versus swing-related muscle activity. Does the cost of swinging one’s limbs contribute significantly to the overall cost of locomotion?
10.1242/jeb.00985
Gilad, Y., Wiebe, V., Przeworski, M., Lancet, D. and Pääbo, S. (2004). Loss of olfactory receptor genes coincides with the acquisition of full trichromatic vision in primates. PLoS Biol. 2, 120-125.
Heather L. Eisthen Michigan State University
[email protected]
One school of thought posits that the energetic cost of swinging the limbs is minimal. Early evidence for this idea came from load-carrying experiments by Dick Taylor and colleagues. Their results suggested that forces exerted by the limb against the ground during stance largely determined the energetic cost of locomotion, rendering swing-related muscle actions energetically irrelevant. However, recent work by Rich Marsh and colleagues at Northeastern University suggests that we should not yet relegate the swing phase to obscurity in the context of locomotor energetics. So how does one go about trying to partition the costs of stance versus swing during a stride? The Northeastern group reasoned that, during aerobic exercise, measuring blood flow to active muscles
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Outside JEB might be the best means of estimating their energy use. To measure muscle blood flow, colored, microscopic (15 µm) polystyrene spheres were injected into the left ventricle of champion runners: guinea fowl. The animals were then exercised on a treadmill over a 5-fold range of speeds. During these locomotor trials, spheres travelled through the bird’s circulatory system until they eventually became lodged in capillary beds due to their size. Assuming the spheres were well mixed within the blood, the number found lodged in a muscle’s capillary beds should be proportional to the blood flow to that muscle and, presumably, the amount of energy used by that muscle while the bird was running. After the exercise trials, the bird’s limb muscles were dissected out and digested to get at the trapped spheres. Taking advantage of the dye carried within the microspheres, Marsh’s group used spectrophotometry to assay how many microspheres had lodged in each limb muscle. Looking at muscles that were electrically active during the stride, the team found three-quarters of the spheres lodged in muscles active mainly during stance. Thus, a full quarter of the blood flowing into the limb musculature during running goes to muscles that are active during the swing, suggesting that the swing phase accounts for approximately 25% of the energy used in a stride. Their experiments also showed that this fraction, which is higher than many of us might have guessed a priori, remains constant regardless of speed. As those of us interested in locomotor mechanics and energetics know, it is often the seemingly simple questions that are the hardest to answer. Fortunately, innovative approaches such as those used by Marsh and colleagues are giving us new insights into the locomotor energetics of an individual stride and, importantly, have shown that swinging the limbs is indeed more costly than we thought. 10.1242/jeb.00987
Marsh, R. L., Ellerby, D. J., Carr, J. A., Henry, H. T. and Buchanan, C. I. (2004). Partitioning the energetics of walking and running: swinging the limbs is expensive. Science 303, 80-83.
Gary B. Gillis Mount Holyoke College
[email protected]
CHIMERA CONSTRUCTION
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A NEW SPIN ON CHICKEN Are you tired of the same old chicken dish day in and day out? Well, new chicken chimeras may one day get you over your chicken blues. A chimera is either an organism, organ or organ part that consists of two or more tissues with different genetic compositions. It can be produced either by organ transplantation, grafting or, in the case of the present study, genetic engineering. But while mutagenesis and transgenic techniques have become routine in animals such as mice, zebrafish and cattle, this process is technically challenging in avian species, despite the many potential advantages in using avian embryos to study development and physiological systems. In the present study published by Yongmei Xi, Masa-Aki Hattori and colleagues, they used a novel approach to create an avian chimera: the peafowl–chicken.
observing the tissue under a fluorescent microscope, they discovered that they had successfully produced a chimeric embryo. They found glowing green patches of peafowl cells in several different embryonic locations, including the head, gonad, heart and ectoderm. And Hattori’s team also characterised the embryo samples with PCR, using primers for specific genetic markers and the GFP gene, confirming the presence of peafowl cells in the developing embryo. This analysis revealed that the peafowl cells were indeed present throughout the chicken’s embryonic tissues. The team had successfully produced a peafowl–chicken chimeric embryo. While there is potential for the incorporated peafowl cells to participate in the development of the chicken embryo, future studies are necessary to determine the long-term survival of these embryos and the functionality of the grafts. However, this study does describe a novel approach for chimera construction and tracking embryonic development. The development of unique chimeras could potentially lead to interesting new model systems for the study of disease and many physiological processes. And as for our diet: peafowl–chicken Kiev, anyone? 10.1242/jeb.00988
Xi, Y., Nada, Y., Soh, T., Fujihara, N., Hattori, M. (2004). Green fluorescent protein gene-transfected peafowl somatic cells participate in the development of chicken embryos. J. Exp. Zool. 301A, 139-149.
Hattori and colleagues incorporated a green fluorescent protein (GFP) into peafowl cells so that they glowed green and could be easily traced during migration through chicken tissues. The GFP-expressing peafowl cells were then mixed and fused by electroporation to isolated white leghorn chicken blastodermal cells, which are tiny reproductive cells from which the chicken embryo develops. The now fused peafowl–chicken cells were subsequently injected into freshly laid, fertile white leghorn chicken eggs, and the manipulated chicken eggs sealed with parafilm and allowed to develop at 37.6°C. The team analyzed the contribution of the peafowl cells to the embryonic development of the chicken by tracing the migration of cells carrying the marker GFP genes in the developing chimeric embryo. By sampling the injected embryos at different stages of development and
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M. Danielle McDonald RSMAS, University of Miami
[email protected]
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MICROGRAVITY
atrophy suspends rats by the tail to prevent the animals from bearing weight on their hindlimbs. Denervation of muscles is another method for causing muscle atrophy. In order to determine if these two protocols are good surrogates for space travel and to characterize the effects of microgravity at the molecular level, Takeshi Nikawa and collaborators used a DNA microarray to examine the expression of 26 000 genes in the muscle of 8-day-old rats exposed to tail suspension, denervation and space travel for 16 days in the space shuttle Columbia.
SPACED OUT MITOCHONDRIA Studies of physiological systems under extreme conditions teach us important lessons regarding the basics of life. One fascinating extreme condition is space travel. Space travel affects physiological functions in many ways, and one of its key signatures is muscle atrophy. Muscle atrophy occurs when there is an imbalance in the rate of protein synthesis and degradation. However, the molecular mechanisms that lead to muscle wasting under microgravity remain a mystery. Given the difficulty of carrying out experiments in space, experimental protocols have been developed to study the effects of weightlessness on the ground. One common protocol to induce muscle
All three treatments led to significant muscle atrophy, with tail suspension causing the most dramatic loss in muscle mass, namely an 86% reduction compared with controls. For the three protocols, muscle atrophy was accompanied by profound changes in cellular gene expression patterns. Indeed, the expression of genes involved in cellular functions ranging from metabolism to cell structure was affected. However, the cellular gene expression pattern of the muscle from space-flown rats was unique. Space travel resulted in a distinct change in the expression of mitochondrial and cytoskeletal genes as well as in the expression of genes related to protein degradation. Therefore, the two protocols used on the ground to mimic microgravity hint at the existence of different signalling pathways leading to muscle atrophy and, also, they do not reproduce the molecular
changes observed in space. The authors observed that the unique decrease in the expression of cytoskeletal genes in the muscle of space-flown rats was accompanied by an aberrant distribution of mitochondria. Furthermore, space travel resulted in the upregulation of oxidative stress genes. The authors propose a new model to explain muscle atrophy during space travel, where mitochondria function as gravity sensors. Microgravity would decrease the expression of cytoskeletal proteins resulting in disturbances in the distribution of mitochondria within the cells. Finding themselves in unexpected territories, the mitochondria would spill reactive oxygen species, which cause oxidative stress, and produce inappropriate amounts of ATP resulting in muscle atrophy. Overall, this paper lays the foundation to test the mitochondrial theory of muscle atrophy. 10.1242/jeb.00986
Nikawa, T., Ishidoh, K., Hirasaka, K., Ishihara, I., Ikemoto, M., Kano, M., Kominami, E., Nonaka, I., Ogawa, T., Adams, G. R. et al. (2004). Skeletal muscle gene expression in space-flown rats. FASEB J. 18, 522-524.
© 2004 The Company of Biologists Limited
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Julie St-Pierre Dana-Farber Cancer Institute and Harvard Medical School
[email protected]
