Journal of Experimental Biology - Latest Issue
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A primary neuron culture system for functional studies of anoxia tolerance in turtles
ABSTRACTCultured neuronal models for non-mammalian vertebrates are uncommon but could prove useful for investigating mechanisms of exceptional physiological performance, such as anoxia tolerance. We describe a procedure to isolate, culture and characterize cerebrocortical neurons of extremely anoxia-tolerant painted turtles; we then imposed anoxia while recording reactive oxygen species (ROS) using fluorescence photometry. Cerebrocortical sheets from hatchlings were dissociated enzymatically and mechanically, and cultured for ≤7 days. Within 24 h, most cells possessed classic neuronal morphology with identifiable soma and fine neurites. Immunocytochemistry showed MAP2-positive cells accounted for 90.9% of DAPI-positive cells. Ca2+ recordings (Fura-2) demonstrated neurons were immediately excitable with KCl and glutamate, but not acetylcholine. ROS recordings (CM-H2DCFDA) showed they avoided excessive ROS production post-anoxia, like other turtle brain preparations, but with higher signal and temporal resolution. These approaches should extend previous work in other brain preparations to isolated cells that possess the morphological and functional features of neurons. -
ECR Spotlight – Natalia Schneider
ECR Spotlight is a series of interviews with early-career authors from a selection of papers published in Journal of Experimental Biology and aims to promote not only the diversity of early-career researchers (ECRs) working in experimental biology but also the huge variety of animals and physiological systems that are essential for the ‘comparative’ approach. Natalia Schneider is an author on ‘ A primary neuron culture system for functional studies of anoxia tolerance in turtles’, published in JEB. Natalia is a Graduate student in the lab of Dr Daniel E. Warren at Department of Biology, Saint Louis University, USA, investigating how animals function in, adapt to and survive extreme conditions in the ecosystem. -
Effects of hypoxia and hyperoxia on exercise-induced metabolomic and transcriptomic profiles in equine skeletal muscle
ABSTRACTTo explore the molecular mechanisms underlying oxygen-dependent regulation of skeletal muscle adaptations, eight Thoroughbred horses performed 2 min of exercise at a velocity corresponding to 95% maximal O2 uptake under a normoxic condition, while using inspired O2 levels of 0.21 (normoxia), 0.26 (hyperoxia) or 0.16 (hypoxia). At the end of the exercise, arterial O2 saturation was significantly higher with hyperoxia and lower with hypoxia than with normoxia. However, no significant difference in plasma lactate or muscle glycogen concentrations was observed across the O2 conditions. A metabolomic analysis showed that muscle metabolite concentrations involved in glycolysis and the tricarboxylic acid cycle significantly changed in response to exercise but did not significantly differ across the O2 conditions. RNA-sequencing data showed that fewer genes were significantly altered by acute exercise in hyperoxia (upregulated: 523; downregulated: 116) and hypoxia (upregulated: 857; downregulated: 320) compared with normoxia (upregulated: 1628, downregulated: 924). Among them, numerous genes, including well-known exercise-responsive genes, such as NR4A3, PPARGC1A, PDK4 and VEGFA, were altered following exercise, irrespective of the O2 environment. Hyperoxic exercise induced responses of genes related to lysosomal activity, such as M6PR and CTNS, whereas hypoxic exercise triggered hypoxia-responsive gene expression, including PIK3R1, THPO and AKAP1. These findings suggest that arterial O2 availability does not necessarily alter global metabolic or transcriptomic response following a single exercise bout in horses. However, inspired O2 fraction-specific gene responses may play roles in long-term skeletal muscle adaptations and could contribute to the development of optimized training strategies for improved well-being and performance. -
Combined climate stressors constrain various mechanisms for thermal tolerance in the scallop Pecten maximus
ABSTRACTUnfavourable climatic conditions challenge an animal's performance and fitness. We investigated how cellular homeostasis relates to whole-animal physiology in the marine bivalve Pecten maximus under warming (W), warming plus hypercapnia (WHc) or hypoxia (WHo), and the combination of all three drivers (deadly trio, DT). Starting at 14°C, temperatures were increased stepwise by 2°C per 48 h while gill tissue was sampled from experimental exposures to test for indicators of intracellular stress, including lipid peroxidation, protein damage and degradation, apoptosis and heat shock responses. Whole-animal water filtration rate, routine metabolic rate (RMR), haemolymph PO2, pseudofaeces ejection, mantle tissue intracellular acidosis and gill tissue antioxidative capacity were measured in W and DT exposures. Filtration peaked at a lower temperature under DT, when high pseudofaeces ejection suggested that ventilation was prioritized over feeding. Warming alone doubled RMR by 22°C, whereas DT increased RMR even further, reaching higher maxima by 20°C. Haemolymph PO2 was consistently lower under DT, implying that supply was less able to meet increasing demand. Warming to 26°C stimulated a gill tissue heat-shock response, accumulated ubiquitin conjugates and apoptosis, whereas adding hypoxia or hypercapnia suppressed apoptosis. DT suppressed both heat shock and apoptotic responses, with ubiquitin conjugates and branched-chain amino acids accumulating, and gills showing visible damage. Our findings indicate that climate drivers cumulatively block protection mechanisms, increase protein damage and block protein synthesis, thereby substantially reducing passive thermal tolerance and survival under extremes. The thermal tolerance of scallops is critically reduced under DT conditions, when mechanisms defending passive tolerance are exhausted at lower temperatures. -
Heat increment of feeding in the common bottlenose dolphin ( Tursiops truncatus ) contributes moderately to field metabolic rate estimates
ABSTRACTDigestion elevates metabolism through the heat increment of feeding (HIF) – the energy expended on mechanical and biochemical processes after eating. Quantifying this cost is essential for bioenergetic models that predict energy flow and prey requirements in populations. Using breath-by-breath respirometry, we measured oxygen consumption (V̇O2) in eight common bottlenose dolphins (Tursiops truncatus) before and after feeding standardized meals (1659–2658 kcal of capelin and herring). Metabolic rate rose by ∼37% above resting levels, peaking 60 min after feeding before returning to baseline within 2 h. When scaled across the day, digestion increased daily metabolic needs by ∼8.2% of basal metabolism, similar to values reported for Steller sea lions (Eumetopias jubatus) and harbour seals (Phoca vitulina), where HIF contributes 4–10% of daily energy expenditure. This study provides the first multi-individual estimate of HIF in dolphins and suggests that the energetic cost of digestion is a moderate contribution to overall daily metabolism, refining energetic models and improving prey requirement estimates for cetaceans in the wild.