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.
DESMIN FUNCTION
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DESMIN LOSS MAKES MICE LAZY Surely we have all experienced muscle soreness following exercise. This is mainly caused by membrane damage of the muscle fibres. Skeletal muscle fibres are organised in myofibrils, which in turn are composed of repeating structural units arranged in series, the sarcomeres. Desmin is an intermediate filament protein that is located on the periphery of the sarcomeres, linking individual myofibrils laterally. Its strategic location suggests that it plays a major protective role in muscle structural integrity, as well as force transmission. It is known that when desmin is not expressed, the myofibrils have greater mobility and are more prone to misalignment. Nevertheless, the protective role of desmin during exercise-induced mechanical stress has not yet been ascertained. In a paper recently published in J. Appl. Physiol., Kurt Haubold and collaborators examined the athletic skills of mice lacking desmin, in order to investigate the role of this protein in maintenance of muscle fibre integrity. The authors of this study placed normal mice, and animals that lacked desmin, in cages with access to running wheels attached to bicycle computers. After recording the times and distances that the desmin-null animals ran over a three-week period, the team compared their performances with their wild-type cousins. Mice lacking desmin were significantly more indolent than wild-type mice, since they spent less time running and ran shorter distances. Moreover, the average speed of the lazy mice decreased 20% throughout the experiment, as they were unable to adapt to voluntary running. The animals also lacked endurance and performed less well under stress during treadmill tests at increased speeds. For example, the mice
that did not express desmin only maintained a speed of 20 m min–1 for less than 12 min, while most of their wild-type counterparts sustained that speed for at least 25 min. In addition, they could not run as fast as the wild-type mice in the stress test: less than half of desmin-null mice managed to run at 25 m min–1, whilst scampering along at high speed was no sweat for the wild-type mice. The relatively poor exercising ability of mice lacking desmin shows that this protein is essential for force generation and transmission through muscle fibres. Knowing that exercise-induced muscle membrane damage can result in increased levels of serum creatine kinase activity, the team tested desmin’s protective properties by looking for evidence of damage-induced creatine kinase efflux while the animals exercised. However, the mice didn’t show elevated levels of serum creatine kinase following exercise on the wheel. So it seems that impaired voluntary exercise performance in desmin-null mice is not caused by muscle membrane damage. The team also investigated the protective role of desmin against muscle damage by measuring creatine kinase levels following downhill running on a treadmill at a 16° incline. Downhill running induces eccentric muscle contractions and is a potential cause of muscle membrane damage. Surprisingly, mice lacking desmin enjoyed downhill running as much as the wild-type ones and had significantly lower serum creatine kinase activity. Again, it appears that the loss of desmin does not make muscles more susceptible to damage. The authors hypothesise that desmin loss impairs mitochondrial function and negatively influences aerobic exercise performance. These results indicate that desmin’s most important function associated with exercise is not the protection of muscle fibres from mechanical insults, but lateral force transmission and maintenance of myofibrillar organisation. And there is always the possibility that mutations in the desmin-encoding gene may have a similar effect in humans and adversely affect our exercise performance (well, that’s my excuse, anyway)! 10.1242/jeb.00750
Haubold, K., Allen, D., Capetanaki, Y. and Leinwand, L. (2003). Loss of desmin leads to impaired voluntary wheel running and treadmill exercise performance. J. Appl. Physiol. 95, 1617-1622.
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Jorge M. O. Fernandes University of St Andrews
[email protected]
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ENERGETICS
feeding hummingbirds changed dramatically with major changes in nectar temperature. Birds sipping nectar chilled to 4°C burnt up to 27% more energy than those feeding on 39°C nectar. And the costs rocketed when birds supped on lowsugar nectar – individuals fed on dilute 4°C nectar increased their metabolic rates to levels equal to those generated by a 15°C drop in body temperature. Most surprisingly, the costs of food warming were similar whether birds perched or hovered to feed.
HUMMINGBIRDS FORK OUT FOR A WARM BEVERAGE Mention food warming, and most of us conjure up images of slow Sunday roasts or convenient microwave dinners. For Chris Lotz and his colleagues at the University of Wyoming, however, this phrase holds another meaning entirely. Expert in the field of animal energetics, Lotz’s most recent study reveals the high price that rufous hummingbirds pay for a warm drink. All warm-blooded animals need to heat the food they have eaten to maintain their body temperature. But when pickings are low in energy, vast amounts of food must be consumed, at a hefty energetic cost. Nectar-feeding birds are a case in point – consuming up to five times their body mass each day when feeding on low-calorie liquids. And to make matters worse, mountain-dwelling rufous hummingbirds dine on chilly nectar that is often 10°C or less. Determined to shed light on the cost of food warming, Lotz and his team created a mathematical model to investigate the energetic costs of nectar heating in the rufous hummingbirds of the Rocky Mountains. The scientists worked out that the amount of energy required to heat nectar depends upon three things: how much nectar is consumed, the specific heat of the sugar solution and the difference between body temperature and nectar temperature. Armed with theoretical answers to questions of food warming costs, the researchers then tested the model’s predictions by measuring changes in the metabolic rates of captive birds drinking nectar at different temperatures. As forecast by their model, Lotz’s team discovered that the metabolic rates of
By quantifying the influence of nectar temperature and sugar content on food warming costs, Lotz has found that these costs are an important part of hummingbird energy budgets. From this springboard, future research is likely to reveal that the cost of warming a meal is an important component in the energy budgets of other animals that feed on cold, poor-quality food. Perhaps we should all spare a thought for the humble hummingbird next time we pop a pizza in the oven. 10.1242/jeb.00753
Lotz, C. N., Martínez del Rio, C. and Nicolson, S. W. (2003). Hummingbirds pay a high cost for a warm drink. J. Comp. Physiol. B 173, 455-462.
Fiona Gowland University of Aberdeen
[email protected]
MEMORY
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TRY NOT TO BREATHE Learning about how the nervous system works isn’t easy, and learning about learning and memory formation is particularly tricky. Fortunately, studies on molluscs and other invertebrates have proved particularly useful in dissecting the neural and molecular machinery for learning and memory formation. But once formed, memories are not necessarily permanent and might need consolidating over time. A recently recalled memory may be particularly fragile and may need reconsolidation. In a recent paper published in J. Neurosci., Susan Sangha, Ken Lukowiak and colleagues investigated the mechanisms underlying this reconsolidation. Turning to their favourite experimental animal, the pond snail Lymnaea stagnalis, they asked if memories are so fragile after being recalled that, without reconsolidation, they will be erased. Lymnaea can breathe through their pseudosome (or respiratory orifice) as well as through their skin. Sangha and colleagues put this talent to use in their experiments. The snails were placed in beakers of water through which N2 was bubbled to make it hypoxic. The snails climb to the surface to breathe through their pseudosome when they run out of oxygen. By gently prodding the pseudosome they can be taught to breathe through their skins and can even remember this lesson for days or weeks. This memory is stored in the same network of neurones that produces the breathing. But what happens when encountering the same set of circumstances jogs the snail’s memory? When exposed to the same hypoxic conditions, the snails recall their original training and remember not to breathe. But, since reawakened memories may be particularly fragile, Sangha and colleagues asked whether the reactivation of this memory meant that it now had to be
THE JOURNAL OF EXPERIMENTAL BIOLOGY 207 (1)
Outside JEB reconsolidated. To do this they reactivated the memories of one half of the snails that were originally trained. They then exposed all the trained animals to one of three memory-disrupting treatments. Some animals were cooled, a commonly used method of memory disruption, whereas other animals were injected with actinomycin D, blocking the synthesis of new RNA and disrupting the production of new proteins involved in memory formation. In the third group of trained animals, the cell body of a neurone within the breathing network, RPeD1, was removed, also preventing new RNA and protein synthesis. Four hours after the treatment the animals were tested to see whether they still remembered to keep their pneumostomes shut when they got breathless. Only those snails that had had their memories reactivated showed impairment in their performance – they started to use their pneumostome to breathe at the air–water interface again. These results suggest that the impaired memory was contingent on memory reactivation and requires new RNA/protein synthesis and the cell body of RPeD1. Combining these results with previous work from Lukowiak’s laboratory, which showed that memory consolidation is also dependent upon RNA/protein synthesis and the cell body of RPeD1, makes us wonder whether similar mechanisms underlie both memory consolidation and reconsolidation. One way to get closer to an answer to this question would be to determine if the same genes are transcribed and translated during memory consolidation and reconsolidation. Another obvious link lies between reconsolidation and forgetting. Is it possible that reconsolidation provides an opportunity to forget previously formed memories and overwrite them with new ones? Lymnaea is proving an excellent model system for studying the mechanisms underlying memory formation in a simple neural network, and the answers to these and other questions may not be too far away. 10.1242/jeb.00751
Sangha, S., Scheibenstock, A. and Lukowiak, K. (2003). Reconsolidation of a long-term memory in Lymnaea requires new protein and RNA synthesis and the soma of right pedal dorsal 1. J. Neurosci. 23, 8034-8040.
Jeremy E. Niven University of Cambridge
[email protected]
TOXIN ACCUMULATION
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FISH BRAINS: NERVOUS ACCUMULATION OF TRIBUTYLTIN The blood–brain barrier is impervious to many toxicants and can protect the brain from accumulation of substances present in the blood stream. In fish, toxicants can enter the blood stream from the diet and across the gills from the external environment. But circumvention of the blood–brain barrier can occur if toxins are taken up via nerves that innervate waterexposed sensory organs. Nerve terminals in the lateral line, olfactory and gustatory systems, have all been highlighted as routes of uptake for waterborne metals. Interference with sensory systems can have severe effects on fish behavior. Direct accumulation of metals in the olfactory, lateral line and gustatory systems can disrupt processes relying on sensory detection such as predator avoidance, social interaction, migration and feeding. Uptake of metals into the brain via sensory neurons could also interfere with the entire nervous system. It is therefore important that we understand this potential route of uptake from the external environment. Previously, the transport of contaminants to the brain of fish via sensory pathways has only been considered for trace metals. Whether the extremely toxic organometals, such as tributyltin, a chemical applied to the underside of boats to prevent growth of marine life, can reach the brain via waterexposed sensory nerves is not known. Neither is much known about the effects of tributyltin on fish behavior. Claude Rouleau and colleagues set out to determine whether tributyltin could penetrate the rainbow trout’s neural system.
exposed to waterborne tributyltin and following an intravenous injection of the compound. In this way, they could distinguish between any differences in uptake patterns of tributyltin due to exposure routes using whole-body autoradiography. The team reasoned that, after injection, tributyltin would accumulate in the brain if it crossed the blood–brain barrier, whereas waterborne tributyltin could either accumulate in the brain by crossing the gills and subsequently the blood–brain barrier or if it was taken up by the sensory nervous system. The autoradiographs showed that tributyltin crossed the blood–brain barrier and was present in the brains of water-exposed fish and those given tributyltin in an intravenous injection. In the water-exposed trout, it was therefore difficult to separate the amount of tributyltin taken up via the gills and via the nervous system. However, this study is particularly interesting as the authors demonstrate hot spots of tributyltin accumulation in brain regions known to receive nerve fibers innervating waterexposed sensory organs, in particular the eminentia granulares, where nerve fibers from the mechanoreceptors in lateral lines terminate. Tributyltin was also seen in the olfactory system. This evidence strongly supports brain uptake of tributyltin via axonal transport from water-exposed sensory organs. In this study, Rouleau and colleagues demonstrate that the uptake of contaminants via sensory systems is not limited to trace metals but is also seen in organometals. Investigating mechanisms of uptake and tissue distribution of waterborne contaminants is vital to our understanding of how toxicants manifest their effects. In particular, discovering how toxins interfere with sensory systems could help explain associated changes in fish behavior. 10.1242/jeb.00752
Rouleau, C., Xiong, Z. H., Pacepavicius, G. and Huang, G. L. (2003). Uptake of waterborne tributyltin in the brain of fish: axonal transport as a proposed mechanism. Environ. Sci. Technol. 37, 3298-3302.
The team looked at the body distribution of tributyltin in rainbow trout that had been
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Katherine A. Sloman Brunel University
[email protected]
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RETROGRADE SIGNAL
they played key roles. Furthermore, they were able to show that wit was expressed presynaptically, and gbb post-synaptically, in the neuromuscular junction, suggesting that they might signal from post- to presynaptic cells. Other work had also implicated the calcium/calmodulinsensitive protein kinase, CamKII, in retrograde signalling.
MUSCLE MEMORY The chemical synapse is a uniquely dynamic structure, and the conversation across it is bidirectional. A retrograde signal (from post-synaptic to pre-synaptic cell) is thought to be important for both the initial development of the synapse (synaptogenesis) and its subsequent modulation by activity (synaptic plasticity). In a pair of papers published in Neuron, a group at Berkeley used an elegant combination of Drosophila transgenics, electrophysiology and ultrastructure to delineate a retrograde feedback pathway at the Drosophila neuromuscular junction. The authors’ recent work had dealt them a fortunate hand of cards; they knew that mutating either glass bottom boat (gbb, encoding an orthologue of bone morphogenetic protein) or wishful thinking (wit, encoding its receptor) produced grossly deficient synapses, suggesting that
The team modulated the activity of CamKII in the postsynaptic muscle. The effects were clear: there was a small but significant increase in quantal size (the unit of signalling) when CamKII was inhibited, and a decrease when it was overexpressed, suggesting that CamKII in the postsynaptic cell could alter the scale of neurotransmitter release from the presynaptic cell. Consistent with this, the authors observed remodelling of the ‘active zone’, the region of the presynaptic cell specialised for neurotransmitter release; inhibition of CamKII causes the number of ‘T-bars’ in the active zone to increase, and vice versa. Just as depth charges are lined up on rails on the destroyer’s deck, so Tbars line up neurotransmitter vesicles ready for rapid, efficient release. The changes in morphology are thus consistent with the changes in quantal size. This leads to two further questions: how is the CamKII controlled, and how does this intracellular protein kinase get its message back to a different cell? The first question is easily answered. Camkinases are activated by calcium, and the glutamergic receptor is highly permeable to calcium. But how about the retrograde message? The paper shows that wit
mutants (those lacking the bone morphogenetic protein receptor in presynaptic cells) show defects resembling the post-synaptic inhibition of CamKII; that is, this receptor seems to be involved in receiving the retrograde signal. And, in the accompanying paper, the authors show that interfering with the expression of gbb (the ligand) in the post-synaptic cell has the same effect. Over the years, many retrograde transmitters have been (controversially) proposed, from nitric oxide to cannabinoids; in the insect neuromuscular junction, the retrograde signalling pathway can now be traced rather completely. Calcium entry into the post-synaptic cell activates CamKII and triggers release of gbb into the synaptic cleft; its reception by wit in the presynaptic cell triggers changes in the synaptic architecture that alter the size of future neurotransmission events. 10.1242/jeb.00749
Haghighi, A. P., McCabe, B. D., Fetter, R. D., Palmer, J. E., Hom, S. and Goodman, C. S. (2003). Retrograde control of synaptic transmission by postsynaptic CaMKII at the Drosophila neuromuscular junction. Neuron 39, 255-267. McCabe, B. D., Marques, G., Haghighi, A. P., Fetter, R. D., Crotty, M. L., Haerry, T. E., Goodman, C. S. and O’Connor, M. B. (2003). The BMP homolog Gbb provides a retrograde signal that regulates synaptic growth at the Drosophila neuromuscular junction. Neuron 39, 241-254.
© 2004 The Company of Biologists Limited
THE JOURNAL OF EXPERIMENTAL BIOLOGY 207 (1)
Julian Dow Glasgow University
[email protected]
