Dopamine signalling in mushroom bodies regulates temperature-preference behaviour in Drosophila

The ability to respond to environmental temperature variation is essential for survival in animals. Flies show robust temperature-preference behaviour (TPB) to find optimal temperatures. Recently, we have shown that Drosophila mushroom body (MB) functions as a center controlling TPB. However, neuromodulators that control the TPB in MB remain unknown. To identify the functions of dopamine in TPB, we have conducted various genetic studies in Drosophila. Inhibition of dopamine biosynthesis by genetic mutations or treatment with chemical inhibitors caused flies to prefer temperatures colder than normal. We also found that dopaminergic neurons are involved in TPB regulation, as the targeted inactivation of dopaminergic neurons by expression of a potassium channel (Kir2.1) induced flies with the loss of cold avoidance. Consistently, the mutant flies for dopamine receptor gene (DopR) also showed a cold temperature preference, which was rescued by MB-specific expression of DopR. Based on these results, we concluded that dopamine in MB is a key component in the homeostatic temperature control of Drosophila. The current findings will provide important bases to understand the logic of thermosensation and temperature preference decision in Drosophila.     

High calorie diet augments age-associated sleep impairment in Drosophila

The fruit fly, Drosophila melanogaster is an established model used for aging and longevity studies and more recently for sleep studies. Mammals and Drosophila share various physiological, pathological, pharmacological and genetic similarities in these processes. In particular, sleep is essential for survival in both species and both have age-associated sleep quality alterations. Here we report that a high calorie diet, which accelerates the aging process and reduces lifespan across species, also accelerates age-associated sleep changes in Drosophila. These changes are more evident in the dopamine transporter mutant, fumin, that displays a short sleep phenotype due to enhanced dopaminergic signaling. With normal food, fumin mutants sleep for only one third of the time that the control flies do, but still show equivalent longevity. However, when on a mildly high calorie diet, their sleep length shows a marked decrease and they have a reduced longevity. These data indicate that the age-associated change in sleep in Drosophila is a physiologically regulated aging process that is tightly linked to calorie intake and that the dopamine level plays an important role. In addition, this provides another evidence that sleep is essential for the longevity of Drosophila.     

Ethanol preference in C. elegans

Caenorhabditis elegans senses multiple environmental stimuli through sensory systems and rapidly changes its behaviors for survival. With a simple and well-characterized nervous system, C. elegans is a suitable animal model for studying behavioral plasticity.
Previous studies have shown acute neurodepressive effects of ethanol on multiple behaviors of C. elegans similar to the effect of ethanol on other organisms. Caenorhabditis elegans also develops ethanol tolerance during continuous exposure to ethanol. In mammals, chronic ethanol exposure leads to ethanol tolerance as well as increased ethanol consumption. Ethanol preference is associated with the development of tolerance and may lead to the development of ethanol dependence.
In this study, we show that C. elegans is a useful model organism for studying chronic effects of ethanol, including the development of ethanol preference. We designed a behavioral assay for testing ethanol preference after prolonged ethanol exposure. Despite baseline aversive responses to ethanol, animals show ethanol preference after 4 h of pre-exposure to ethanol and exhibit significantly enhanced preference for ethanol after a lifetime of ethanol exposure. The cat-2 and tph-1 mutant animals have defects in the synthetic enzymes for dopamine and serotonin, respectively. These mutants are deficient in the development of ethanol preference, indicating that dopamine and serotonin are required for this form of behavioral plasticity.     

Developmental and adult acclimation effects of ambient temperature on temperature regulation of mice selected for high and low levels of nest-building

Summary Genetic and environmental components of adaptation to cold inMus musculus were assessed in a study of the effects of selective breeding for behavioral temperature regulation (indexed by high and low levels of nest-building), rearing mice from birth in the cold, and cold acclimation of adult animals, on thermoregulatory traits. Mice from the eleventh selected generation of a high-nesting line maintained higher resting metabolic rates and body temperatures, while at the same time consuming less food when compared with mice from the low-nesting line (Table 1). High-nesting mice were also more discriminating in their temperature preference when placed on a thermal gradient. Thus, common genetic loci must influence a variety of energy conservation measures important for survival in the cold, including insulative nest-building, metabolic efficiency, and optimum microhabitat selection.Rearing mice at 5°C from birth until 70 days of age resulted in permanent increases in nonshivering thermogenesis, weight of interscapular brown adipose tissue, and core body temperature when compared to mice raised at 22°C (Table 1). These greater heat production capacities were accompanied by consumption of more food. Cold acclimation of adults at 5°C for 3 weeks similarly increased measures of thermogenic capacity (nonshivering thermogenesis and interscapular brown adipose tissue) as well as food consumption, when compared to the effects of warm acclimation, but differed from the effects of cold-rearing in that while resting metabolic rates were elevated, no significant differences in body temperature were found (Table 1).Sex differences were also noted for most of the thermoregulatory measures, with the lighter females scoring higher on thermal preference, resting metabolic rate, nonshivering thermogenesis, brown fat, and food consumption.In general, these results suggest that a more precise partitioning of the genetic and environmental factors which influence thermoregulatory traits in mammals could eventually result in a better understanding of the differences which exist between acclimated and acclimatized animals.     

Ih current is necessary to maintain normal dopamine fluctuations and sleep consolidation in Drosophila

HCN channels are becoming pharmacological targets mainly in cardiac diseases. But apart from their well-known role in heart pacemaking, these channels are widely expressed in the nervous system where they contribute to the neuron firing pattern. Consequently, abolishing Ih current might have detrimental consequences in a big repertoire of behavioral traits. Several studies in mammals have identified the Ih current as an important determinant of the firing activity of dopaminergic neurons, and recent evidences link alterations in this current to various dopamine-related disorders. We used the model organism Drosophila melanogaster to investigate how lack of Ih current affects dopamine levels and the behavioral consequences in the sleep:activity pattern. Unlike mammals, in Drosophila there is only one gene encoding HCN channels. We generated a deficiency of the DmIh core gene region and measured, by HPLC, levels of dopamine. Our data demonstrate daily variations of dopamine in wild-type fly heads. Lack of Ih current dramatically alters dopamine pattern, but different mechanisms seem to operate during light and dark conditions. Behaviorally, DmIh mutant flies display alterations in the rest:activity pattern, and altered circadian rhythms. Our data strongly suggest that Ih current is necessary to prevent dopamine overproduction at dark, while light input allows cycling of dopamine in an Ih current dependent manner. Moreover, lack of Ih current results in behavioral defects that are consistent with altered dopamine levels.     

Research progress on <Emphasis Type="Italic">Drosophila</Emphasis> visual cognition in China

Visual cognition,as one of the fundamental aspects of cognitive neuroscience,is generally associated with high-order brain functions in animals and human.Drosophila,as a model organism,shares certain features of visual cognition in common with mammals at the genetic,molecular,cellular,and even higher behavioral levels.From learning and memory to decision making,Drosophila covers a broad spectrum of higher cognitive behaviors beyond what we had expected.Armed with powerful tools of genetic manipulation in Dr...     

Dopamine Receptors Antagonistically Regulate Behavioral Choice between Conflicting Alternatives in C. elegans

Caenorhabditis elegans is a useful model to study the neuronal or molecular basis for behavioral choice, a specific form of decision-making. Although it has been implied that both D1-like and D2-like dopamine receptors may contribute to the control of decision-making in mammals, the genetic interactions between D1-like and D2-like dopamine receptors in regulating decision-making are still largely unclear. In the present study, we investigated the molecular control of behavioral choice between conflicting alternatives (diacetyl and Cu²⁺) by D1-like and D2-like dopamine receptors and their possible genetic interactions with C. elegans as the assay system. In the behavioral choice assay system, mutation of dop-1 gene encoding D1-like dopamine receptor resulted in the enhanced tendency to cross the Cu²⁺ barrier compared with wild-type. In contrast, mutations of dop-2 or dop-3 gene encoding D2-like dopamine receptor caused the weak tendency to cross the Cu²⁺ barrier compared with wild-type. During the control of behavioral choice, DOP-3 antagonistically regulated the function of DOP-1. The behavioral choice phenotype of dop-2; dop-1dop-3 triple mutant further confirmed the possible antagonistic function of D2-like dopamine receptor on D1-like dopamine receptor in regulating behavioral choice. The genetic assays further demonstrate that DOP-3 might act through Gα_o signaling pathway encoded by GOA-1 and EGL-10, and DOP-1 might act through Gα_q signaling pathway encoded by EGL-30 and EAT-16 to regulate the behavioral choice. DOP-1 might function in cholinergic neurons to regulate the behavioral choice, whereas DOP-3 might function in GABAergic neurons, RIC, and SIA neurons to regulate the behavioral choice. In this study, we provide the genetic evidence to indicate the antagonistic relationship between D1-like dopamine receptor and D2-like dopamine receptor in regulating the decision-making of animals. Our data will be useful for understanding the complex functions of dopamine receptors in regulating decision-making in animals.     

哺乳动物毛被传热性能及其影响因素

毛被能够加强或减弱动物向周围环境的热量散失,毛被的形态结构和颜色是传热性能的决定因素,其传热过程往往是传导、对流和辐射3个过程的耦合。以往研究发现环境因子中,风可增加机体向环境中的散热速率,且散失量与风速正相关,且动物通过调节在风场中的姿态来适应不同风向。动物体与环境间的温差是影响散热速率的另一因素,不同环境中的动物通过改变毛被结构来适应温差变化。毛被含水率上升会引起导热和蒸发冷却作用加强,动物通过行为或毛被结构变化来调节毛被含水率。毛色决定毛被吸收和反射热辐射的能力。毛被传热性能直接把动物的生理特点与环境因子关联起来,这对揭示动物的适应、进化都具有重要意义。同时提出,毛被结构和传热性能的研究还有助于仿生学意义的挖掘。因此,今后应重点在毛被结构和物理性能、研究技术与方法以及毛被生物学和仿生学意义等方面开展研究。     

NMDA receptors‐dependent plasticity in the phototaxis preference behavior induced by visual deprivation in young and adult flies

Adult mammals have experience‐dependent plasticity in visual system, but it is unclear whether adult insects also have this plasticity after the critical period of visual development. Here, we have established a modified Y‐maze apparatus for investigating experience‐dependent plasticity in Drosophila. Using this setup we demonstrate that flies after the critical period have bidirectional modifications of the phototaxis preference behavior (PPB) induced by visual deprivation and experience: Visual deprivation decreases the preference of flies for visible light, while visual experience exerts the opposite effect. We also found an age‐dependent PPB plasticity induced by visual deprivation. Molecular and cellular studies suggest that the N‐methyl‐ d ‐aspartate receptors (NMDARs) mediate ocular dominance plasticity in visual cortex in mammals, but direct behavioral evidence is lacking. Here, we used the genetic approaches to demonstrate that NMDAR1, which is NMDARs subunit in Drosophila, can mediate PPB plasticity in young and adult flies. These findings provide direct behavioral evidence that NMDAR1 mediates PPB plasticity in Drosophila. Our results suggest that mammals and insects have analogous mechanisms for experience‐dependent plasticity and its regulation by NMDAR signaling.     

Dopamine modulates acute responses to cocaine, nicotine and ethanol in Drosophila

BACKGROUND: Drugs of abuse have a common property in mammals, which is their ability to facilitate the release of the neurotransmitter and neuromodulator dopamine in specific brain regions involved in reward and motivation. This increase in synaptic dopamine levels is believed to act as a positive reinforcer and to mediate some of the acute responses to drugs. The mechanisms by which dopamine regulates acute drug responses and addiction remain unknown. RESULTS: We present evidence that dopamine plays a role in the responses of Drosophila to cocaine, nicotine or ethanol. We used a startle-induced negative geotaxis assay and a locomotor tracking system to measure the effect of psychostimulants on fly behavior. Using these assays, we show that acute responses to cocaine and nicotine are blunted by pharmacologically induced reductions in dopamine levels. Cocaine and nicotine showed a high degree of synergy in their effects, which is consistent with an action through convergent pathways. In addition, we found that dopamine is involved in the acute locomotor-activating effect, but not the sedating effect, of ethanol. CONCLUSIONS: We show that in Drosophila, as in mammals, dopaminergic pathways play a role in modulating specific behavioral responses to cocaine, nicotine or ethanol. We therefore suggest that Drosophila can be used as a genetically tractable model system in which to study the mechanisms underlying behavioral responses to multiple drugs of abuse.     

The C. elegans D2-Like Dopamine Receptor DOP-3 Decreases Behavioral Sensitivity to the Olfactory Stimulus 1-Octanol

We previously found that dopamine signaling modulates the sensitivity of wild-type C. elegans to the aversive odorant 1-octanol. C. elegans lacking the CAT-2 tyrosine hydroxylase enzyme, which is required for dopamine biosynthesis, are hypersensitive in their behavioral avoidance of dilute concentrations of octanol. Dopamine can also modulate the context-dependent response of C. elegans lacking RGS-3 function, a negative regulator of Gα signaling. rgs-3 mutant animals are defective in their avoidance of 100% octanol when they are assayed in the absence of food (E. coli bacterial lawn), but their response is restored when they are assayed in the presence of food or exogenous dopamine. However, it is not known which receptor might be mediating dopamine''s effects on octanol avoidance. Herein we describe a role for the C. elegans D2-like receptor DOP-3 in the regulation of olfactory sensitivity. We show that DOP-3 is required for the ability of food and exogenous dopamine to rescue the octanol avoidance defect of rgs-3 mutant animals. In addition, otherwise wild-type animals lacking DOP-3 function are hypersensitive to dilute octanol, reminiscent of cat-2 mutants. Furthermore, we demonstrate that DOP-3 function in the ASH sensory neurons is sufficient to rescue the hypersensitivity of dop-3 mutant animals, while dop-3 RNAi knockdown in ASH results in octanol hypersensitivity. Taken together, our data suggest that dopaminergic signaling through DOP-3 normally acts to dampen ASH signaling and behavioral sensitivity to octanol.     

Pelota controls self-renewal of germline stem cells by repressing a Bam-independent differentiation pathway

In the Drosophila ovary, germline stem cell (GSC) self-renewal is controlled by both extrinsic and intrinsic factors. The Bmp signal from niche cells controls GSC self-renewal by directly repressing a Bam-dependent differentiation pathway in GSCs. pelota (pelo), which has been previously shown to be required for Drosophila male meiosis, was identified in our genetic screen as a dominant suppressor of the dpp overexpression-induced GSC tumor phenotype. In this study, we reveal the unexpected new role of Pelo in controlling GSC self-renewal by repressing a Bam-independent differentiation pathway. In pelo mutant ovaries, GSCs are lost rapidly owing to differentiation. Results from genetic mosaic analysis and germ cell-specific rescue show that it functions as an intrinsic factor to control GSC self-renewal. In pelo mutant GSCs, Bmp signaling activity detected by Dad-lacZ expression is downregulated, but bam expression is still repressed. Furthermore, bam mutant germ cells are still able to differentiate into cystocytes without pelo function, indicating that Pelo is involved in repressing a Bam-independent differentiation pathway. Consistent with its homology to the eukaryotic translation release factor 1alpha, we show that Pelo is localized to the cytoplasm of the GSC. Therefore, Pelo controls GSC self-renewal by repressing a Bam-independent differentiation pathway possibly through regulating translation. As Pelo is highly conserved from Drosophila to mammals, it may also be involved in the regulation of adult stem cell self-renewal in mammals, including humans.     

Higher brain functions of PACAP and a homologous Drosophila memory gene amnesiac: insights from knockouts and mutants

Neuropeptides usually exert a long-lived modulatory effect on the small-molecule neurotransmitters with which they colocalize via regulation of the response times of second messenger systems. Pituitary adenylate cyclase-activating polypeptide (PACAP) functions as a neuromodulator and neurotransmitter and regulates a variety of physiological processes. PACAP is structurally highly conserved during evolution, implying its vital importance. In Drosophila, loss-of-function mutations in a PACAP-like neuropeptide gene, amnesiac (amn), affect both memory retention and ethanol sensitivity. The amnesiac gene is expressed in neurons innervating the mushroom body lobes, the olfactory associative learning center. Conditional genetic ablation of neurotransmitter release from these neurons mimics the amnesiac memory phenotypes, suggesting an acute role for amnesiac in memory. However, genetic rescue experiments also suggest developmental defects in amnesiac mutants, implying a role in neuronal development. There is a parallel between memory formation in Drosophila and mammals. PACAP-specific (PAC(1)) receptor-deficient mice show a deficit in hippocampus-dependent associative learning and mossy fiber long-term potentiation (LTP). Meanwhile, PACAP-deficient mice display a high early mortality rate and additional CNS phenotypes including behavioral and psychological phenotypes (e.g., hyperlocomotion, intense novelty-seeking behavior, and explosive jumping). A functional comparison between PACAP and amnesiac underlines phylogenetically conserved functions across phyla and may provide insights into the possible mechanisms of action and evolution of this neuropeptidergic system.     

EMBRYONIC RESISTANCE TO CHEMICAL AND PHYSICAL FACTORS: MANIFESTATION, MECHANISM, ROLE IN REPRODUCTION AND IN ADAPTATION TO ECOLOGY

Chemical and physical factors may adversely affect embryonic development. As an example of chemical factors, the effects of diabetic metabolic factors on embryonic development in mammals was reviewed. The existence of a stage-dependent reaction of embryos was found. At preimplantation stages diabetic metabolic factors are embryotoxic and lethal, and the blastocysts reacted by an "all-or-none" response. Early somite embryos showed a higher resistance to the effects of diabetic metabolic factors resulting in various types of malformations. Both groups of embryos showed a very high sensitivity to the effects of combined diabetic metabolic factors. Congenital defects in term foetuses were lower than those observed during middle phases of pregnancy because some of the severely malformed embryos resorb during gestation. The effects of temperature on embryonic development were presented as an example of physical influences. In man, hyperthermia in pregnancy seems to correlate with defects in the development of the nervous and skeletal systems. In domestic animals, changes in environmental temperature correlated with depressions of reproduction rate. In laboratory animals, hyperthermia caused the development of congenital malformations. Stage-dependent as well as genetic differences in embryonic susceptibility to hyperthermia were found. Critical periods in sensitivity of embryos to hyperthermic influences were also observed. It has been shown that, in spite of similar external manifestations of the reaction of embryos to effects of diabetes and hyperthermia, the mechanism of these reactions was different. High resistance of early reptile and bird embryos to influences of temperature was considered as an example of morphofunctional adaptations in early embryogenesis of vertebrates to their development in terrestrial conditions.     

Thermal nociception in adult Drosophila: behavioral characterization and the role of the painless gene

Nociception, warning of injury that should be avoided, serves an important protective function in animals. In this study, we show that adult Drosophila avoids noxious heat by a jump response. To quantitatively analyze this nociceptive behavior, we developed two assays. In the CO2 laser beam assay, flies exhibit this behavior when a laser beam heats their abdomens. The consistency of the jump latency in this assay meets an important criterion for a good nociceptive assay. In the hot plate assay, flies jump quickly to escape from a hot copper plate (>45 degrees C). Our results demonstrate that, as in mammals, the latency of the jump response is inversely related to stimulus intensity, and innoxious thermosensation does not elicit this nociceptive behavior. To explore the genetic mechanisms of nociception, we examined several mutants in both assays. Abnormal nociceptive behavior of a mutant, painless, indicates that painless, a gene essential for nociception in Drosophila larvae, is also required for thermal nociception in adult flies. painless is expressed in certain neurons of the peripheral nervous system and thoracic ganglia, as well as in the definite brain structures, the mushroom bodies. However, chemical or genetic insults to the mushroom bodies do not influence the nociceptive behavior, suggesting that different painless-expressing neurons play diverse roles in thermal nociception. Additionally, no-bridge(KS49), a mutant that has a structural defect in the protocerebral bridge, shows defective response to noxious heat. Thus, our results validate adult Drosophila as a useful model to study the genetic mechanisms of thermal nociception.     

High sugar-induced insulin resistance in Drosophila relies on the lipocalin Neural Lazarillo

In multicellular organisms, insulin/IGF signaling (IIS) plays a central role in matching energy needs with uptake and storage, participating in functions as diverse as metabolic homeostasis, growth, reproduction and ageing. In mammals, this pleiotropy of action relies in part on a dichotomy of action of insulin, IGF-I and their respective membrane-bound receptors. In organisms with simpler IIS, this functional separation is questionable. In Drosophila IIS consists of several insulin-like peptides called Dilps, activating a unique membrane receptor and its downstream signaling cascade. During larval development, IIS is involved in metabolic homeostasis and growth. We have used feeding conditions (high sugar diet, HSD) that induce an important change in metabolic homeostasis to monitor possible effects on growth. Unexpectedly we observed that HSD-fed animals exhibited severe growth inhibition as a consequence of peripheral Dilp resistance. Dilp-resistant animals present several metabolic disorders similar to those observed in type II diabetes (T2D) patients. By exploring the molecular mechanisms involved in Drosophila Dilp resistance, we found a major role for the lipocalin Neural Lazarillo (NLaz), a target of JNK signaling. NLaz expression is strongly increased upon HSD and animals heterozygous for an NLaz null mutation are fully protected from HSD-induced Dilp resistance. NLaz is a secreted protein homologous to the Retinol-Binding Protein 4 involved in the onset of T2D in human and mice. These results indicate that insulin resistance shares common molecular mechanisms in flies and human and that Drosophila could emerge as a powerful genetic system to study some aspects of this complex syndrome.     

Searching for sleep mutants of Drosophila melanogaster

The functions of sleep are still unknown, but are probably related to cellular and molecular aspects of neural function. To better understand the benefits that sleep may bring at the cellular level, recent studies have employed Drosophila melanogaster as a model system and shown that fruit flies share the fundamental features of mammalian sleep. As in mammals, sleep in Drosophila is characterized by increased arousal threshold and by changes in brain electrical activity. Fly sleep is homeostatically regulated independent of the circadian clock, is modulated by stimulants and hypnotics, and is affected by age. Also, fly sleep is associated with changes in brain gene expression similar to those observed in mammals. While Drosophila neurobiology is sufficiently complex to permit meaningful generalizations to mammals and humans, Drosophila genetics is simple enough to allow a rapid mutagenesis screening. An ongoing mutagenesis study has screened approximately 5000 mutant Drosophila lines and found that sleep amount, sleep pattern, and the homeostatic regulation of sleep are highly conserved phenotypes in flies. So far, this study has identified 10 short sleeper lines and 4 lines that show no sleep rebound after sleep deprivation. Ultimately, the characterization of these lines should help identifying crucial cellular pathways involved in the regulatory mechanisms of sleep and its functional consequences.     

The evolution of endothermy

A theory to account for the evolution of heat production for endothermy in the vertebrates is presented. It is argued that thermoregulatory responses to cold, thyroxine, and the Na⁺ pump are related functionally and phylogenetically.Fish regulate their body temperature behaviorally. For example, if the ambient temperature is too cold, they exhibit appetitive behavior and swim to an area where it is warmer. The incidental heat produced by the increased activity is lost through the gills due to the pattern of circulation. In fish the increased requirement for oxygen during increased activity demands an increased transfer of oxygen and of ions and water across the gill membranes. Thus, any increase in oxygen demand causes an obligatory stimulation of the Na⁺ pump. The evolution of endothermy (that is, of non-shivering thermogenesis) from behavioral thermoregulation of fish can be envisioned as a bypassing of the behavoiral response of fish and a direct stimulation of the Na⁺ pump to produce heat. The attraction of this argument is the ubiquity of Na⁺ transport across membranes.It is also argued that thyroxine was selected for as one type of control. Thyroxine could be selected as a control mechanism because its main function in fish is ion regulation. In addition, thyroxine effects the general level of spontaneous activity, increases chill resistance, alters the ability to sense salinity, and also alters the behavioral response of fish to changes in temperature. The argument is further supported by recent observations which indicate that a major fraction of the thyroxine induced elevated metabolic rate in mammals is due to stimulation of the Na⁺ pump. The above suppositions may also explain why the internal temperature sense of mammals is so sensitive to Na⁺, and may in fact suggest one possible feedback signal in homeotherms.     

Use of helium-oxygen to examine the effect of cold acclimation on the summit metabolism of a marsupial, Dasyuroides byrnei

To determine whether marsupial mammals increase their metabolic capabilities during cold acclimation, the metabolism of both warm and cold acclimated Dasyuroides byrnei was examined by exposure to cold in a helium-oxygen atmosphere. Mean values of heat production and conductance were significantly higher in a helium-oxygen atmosphere than in air. Body temperature did not change until metabolic capacity was exhausted. Both cold and warm acclimated groups could maintain a metabolic scope of 10-11 times the basal or standard level for this species. Such a metabolic scope is much higher than levels recorded for placental mammals. At very low ambient temperatures cold acclimated D. byrnei could sustain a high level of heat production longer than could warm acclimated animals. While there are some similarities between marsupial mammals and placental mammals in their responses to cold acclimation, an increase in maximum metabolism, as reported for placentals, does not seem to occur in marsupials.