nils-herrmann/clean_scirepeval huggingface dataset:
Estradiol interacts with an opioidergic network to achieve rapid modulation of a vocal pattern generator
Venue: Journal of Comparative Physiology A

Estrogens rapidly regulate neuronal activity within seconds-to-minutes, yet it is unclear how estrogens interact with neural circuits to rapidly coordinate behavior. This study examines whether 17-beta-estradiol interacts with an opioidergic network to achieve rapid modulation of a vocal control circuit. Adult plainfin midshipman fish emit vocalizations that mainly differ in duration, and rhythmic activity of a hindbrain–spinal vocal pattern generator (VPG) directly establishes the temporal features of midshipman vocalizations. VPG activity is therefore predictive of natural calls, and ‘fictive calls’ can be elicited by electrical microstimulation of the VPG. Prior studies show that intramuscular estradiol injection rapidly (within 5 min) increases fictive call duration in midshipman. Here, we delivered opioid antagonists near the VPG prior to estradiol injection. Rapid estradiol actions on fictive calling were completely suppressed by the broad-spectrum opioid antagonist naloxone and the mu-opioid antagonist beta-funaltrexamine, but were unaffected by the kappa-opioid antagonist nor-binaltorphimine. Unexpectedly, prior to estradiol administration, all three opioid antagonists caused immediate, transient reductions in fictive call duration. Together, our results indicate that: (1) vocal activity is modulated by opioidergic networks, confirming hypotheses from birds and mammals, and (2) the rapid actions of estradiol on vocal patterning depend on interactions with a mu-opioid modulatory network.
Functional and molecular evolution of olfactory neurons and receptors for aliphatic esters across the Drosophila genus
Venue: Journal of Comparative Physiology A

Insect olfactory receptor (Or) genes are large, rapidly evolving gene families of considerable interest for evolutionary studies. They determine the responses of sensory neurons which mediate critical behaviours and ecological adaptations. We investigated the evolution across the genus Drosophila of a subfamily of Or genes largely responsible for the perception of ecologically relevant aliphatic esters; products of yeast fermentation and fruits. Odour responses were recorded from eight classes of olfactory receptor neurons known to express this Or subfamily in D. melanogaster and from homologous sensilla in seven other species. Despite the fact that these species have diverged over an estimated 40 million years, we find that odour specificity is largely maintained in seven of the eight species. In contrast, we observe extensive changes in most neurons of the outgroup species D. virilis, and in two neurons across the entire genus. Some neurons show small shifts in specificity, whilst some dramatic changes correlate with gene duplication or loss. An olfactory receptor neuron response similarity tree did not match an Or sequence similarity tree, but by aligning Or proteins of likely functional equivalence we identify residues that may be relevant for odour specificity. This will inform future structure–function studies of Drosophila Ors.
Analysis of impulse adaptation in motoneurons
Venue: Journal of Comparative Physiology A

Animal locomotion results from muscle contraction and relaxation cycles that are generated within the central nervous system and then are relayed to the periphery by motoneurons. Thus, motoneuron function is an essential element for understanding control of animal locomotion. This paper presents motoneuron input–output relationships, including impulse adaptation, in the medicinal leech. We found that although frequency-current graphs generated by passing 1-s current pulses in neuron somata were non-linear, peak and steady-state graphs of frequency against membrane potential were linear, with slopes of 5.2 and 2.9 Hz/mV, respectively. Systems analysis of impulse frequency adaptation revealed a static threshold nonlinearity at −43 mV (impulse threshold) and a single time constant (τ = 88 ms). This simple model accurately predicts motoneuron impulse frequency when tested by intracellular injection of sinusoidal current. We investigated electrical coupling within motoneurons by modeling these as three-compartment structures. This model, combined with the membrane potential–impulse frequency relationship, accurately predicted motoneuron impulse frequency from intracellular records of soma potentials obtained during fictive swimming. A corollary result was that the product of soma-to-neurite and neurite-to-soma coupling coefficients in leech motoneurons is large, 0.85, implying that the soma and neurite are electrically compact.
Identification of qRT-PCR reference genes for analysis of opioid gene expression in a hibernator
Venue: Journal of Comparative Physiology B

Previous work has suggested that central and peripheral opioid signaling are involved in regulating torpor behavior and tissue protection associated with the hibernation phenotype. We used quantitative real-time PCR (qRT-PCR) to measure mRNA levels of opioid peptide precursors and receptors in the brain and heart of summer ground squirrels (Ictidomys tridecemlineatus) and winter hibernating squirrels in the torpid or interbout arousal states. The use of appropriate reference genes for normalization of qRT-PCR gene expression data can have profound effects on the analysis and interpretation of results. This may be particularly important when experimental subjects, such as hibernating animals, undergo significant morphological and/or functional changes during the study. Therefore, an additional goal of this study was to identify stable reference genes for use in qRT-PCR studies of the 13-lined ground squirrel. Expression levels of 10 potential reference genes were measured in the small intestine, liver, brain, and heart, and the optimal combinations of the most stable reference genes were identified by the GeNorm Excel applet. Based on this analysis, we provide recommendations for reference genes to use in each tissue that would be suitable for comparative studies among different activity states. When appropriate normalization of mRNA levels was used, there were no changes in opioid-related genes in heart among the three activity states; in brain, DOR expression was highest during torpor, lowest in interbout arousal and intermediate in summer. The results support the idea that changes in DOR expression may regulate the level of neuronal activity in brain during the annual hibernation cycle and may contribute to hibernation-associated tissue protection.
Intestinal osmoregulatory acclimation and nitrogen metabolism in juveniles of the freshwater marble goby exposed to seawater
Venue: Journal of Comparative Physiology B

The objective of this study was to elucidate the role of the intestine from juveniles of the marble goby, Oxyeleotris marmorata, during seawater (SW) exposure. It has been reported elsewhere that SW-exposed juvenile O. marmorata exhibits hypoosmotic and hypoionic regulation, with the induction of branchial Na+/K+-ATPase (NKA), Na+:K+:2Cl− cotransporter (NKCC), and cystic fibrosis transmembrane receptor-like chloride channels. Here, we report that SW exposure also led to significant increases in the activity and protein abundance of NKA in, and probably an increase in Na+ uptake through, its intestine. Additionally, there was an increase in apical NKCC immunoreactivity in the intestinal epithelium, indicating that there could be increased Cl− uptake through the intestine. These results suggest that absorption of ions, and hence water, from the intestinal lumen could be an essential part of the osmoregulatory process in juvenile O. marmorata during exposure to SW. Furthermore, there were significant increases in the glutamate content, and the aminating activity and protein abundance of glutamate dehydrogenase (GDH) in the intestine of fish exposed to SW. Since the intestinal glutamine synthetase activity and protein abundance decreased significantly, and the intestinal glutamine content remained unchanged, in the SW-exposed fish, excess glutamate formed via increased GDH activity in the intestine could be channeled to other organs to facilitate the increased synthesis of amino acids. Taken together, our results indicate for the first time that, besides absorbing ions and water during SW exposure, the intestine of juvenile O. marmorata also participated in altered nitrogen metabolism in response to salinity changes.
A field model of learning: 1. Short-term memory in the crab Chasmagnathus granulatus
Venue: Journal of Comparative Physiology A

Learning and memory studies have been performed for more than two decades using the crab Chasmagnathus in our laboratory. Here, our research was aimed at disclosing some instances of learning in field conditions. Three experiments were performed non-simultaneously, all with a 22.5-min pre-training preceding the first visual-danger-stimulus, an opaque rectangle passing overhead. In Experiment 1, crabs received a single stimulus followed by 22.5-min testing without stimulation, where the re-emerging latency was considered the basic latency response. In Experiment 2, training consisted of 15 stimulus 3-min apart, followed by 22.5-min testing without stimulation. Throughout training crabs were underground but re-emerged at testing with latencies longer than the basic latency response. Both at pre-training and testing the usual strategy of exploring was the short-near excursions. In Experiment 3, training included three stimulus 22.5-min apart, followed by 22.5-min testing. Crabs left their burrows before the end of each inter-trial, showing a mean latency like the basic latency response, but a sensitization to the stimulus and a preponderance of the fast-far excursions over the usual slow-near. In brief: through 15-3 training, crabs learn that the stimulus is iteratively presented; through 3-22.5 training, crabs acquire sensitization to the stimulus and a different strategy of exploration.
A field model of learning: 2. Long-term memory in the crab Chasmagnathus granulatus
Venue: Journal of Comparative Physiology A

In the previous companion paper, the possibility of learning by Chasmagnathus in field conditions was demonstrated. Here, we study long-term memory inquiring to what extent an internal representation could be maintained in a complex environment. Two 45-min training sessions, each of 15 visual danger stimulus presentations with 3-min intertrials, were given at a 24-h interval. Throughout the first training session and during the first 22.5 min of re-training on day 2, crabs kept the same hiding response level but then, during the second phase of re-training, the re-emerging increased up to the point that 32% of surface crabs ignored the stimulus. Each session was followed by a 22.5-min testing without stimulation. At testing on day 2 after re-training, crabs showed a change in the usual exploring strategy. Results reveal long persistency in responding despite a rest interval of 24 h followed by a gradual decrease in response until it vanishes. The statistical analysis was performed by comparing performances at day 2 ( 1988) and then confirmed through comparisons between day 1 and day 2. However, it is not possible to identify separate and taxonomically well-defined learnings but rather a tangled collection of processes that influence each other blurring some of the diagnostic features of each learning.
The effects of progressive hypoxia and re-oxygenation on cardiac function, white muscle perfusion and haemoglobin saturation in anaesthetised snapper (Pagrus auratus)
Venue: Journal of Comparative Physiology B

The effects of progressive hypoxia and re-oxygenation on cardiac function, white muscle perfusion and haemoglobin saturation were investigated in anaesthetised snapper (Pagrus auratus). White muscle perfusion and haemoglobin saturation were recorded in real time using fibre optic methodology. A marked fall in heart rate (HR) was evoked when the water bath dissolved oxygen (DO) concentration decreased below 1.5 mg L−1. This bradycardia deepened over the subsequent 20 min of progressive hypoxia and noticeable arrhythmias occurred, suggesting that hypoxia had direct and severe effects on the cardiac myocytes. Perfusion to the white muscle decreased below a DO concentration of 3 mg L−1, and oxyhaemoglobin concentration decreased once the DO fell below ca. 2 mg L−1. During re-oxygenation, heart rate and white muscle perfusion increased as the DO concentration exceeded 1.9 ± 0.1 mg L−1, whereas haemoglobin saturation increased once the external DO concentration reached 2.9 mg L−1. These changes occurred in anaesthetised fish, in which sensory function must be impaired, if not abolished. As white muscle perfusion both fell and increased prior to changes in white muscle oxyhaemoglobin saturation, a local hypoxia is more likely to be the consequence than the cause of the reduced blood delivery, and changes upstream from the tail vasculature must be responsible. HR and tissue haemoglobin concentrations did increase simultaneously on re-oxygenation suggesting an increased cardiac output as the cause.
Energy metabolism of Inuit sled dogs
Venue: Journal of Comparative Physiology B

We explored how seasonal changes in temperature, exercise and food supply affected energy metabolism and heart rate of Inuit dogs in Greenland. Using open flow respirometry, doubly labeled water, and heart rate recording, we measured metabolic rates of the same dogs at two different locations: at one location the dogs were fed with high energy food throughout the year while at the other location they were fed with low energy food during summer. Our key questions were: is resting metabolic rate (RMR) increased during the winter season when dogs are working? Does feeding regime affect RMR during summer? What is the proportion of metabolic rate (MR) devoted to specific dynamic action (SDA), and what is the metabolic scope of working Inuit sled dogs? The Inuit dogs had an extremely wide thermoneutral zone extending down to −25°C. Temperature changes between summer and winter did not affect RMR, thus summer fasting periods were defined as baseline RMR. Relative to this baseline, summer MR was upregulated in the group of dogs receiving low energy food, whereas heart rate was downregulated. However, during food digestion, both MR and HR were twice their respective baseline values. A continuously elevated MR was observed during winter. Because temperature effects were excluded and because there were also no effects of training, we attribute winter elevated MR to SDA because of the continuous food supply. Working MR during winter was 7.9 times the MR of resting dogs in winter, or 12.2 times baseline MR.
Molecular and functional characterization of the cystic fibrosis transmembrane conductance regulator from the Australian common brushtail possum, Trichosurus vulpecula
Venue: Journal of Comparative Physiology B

Unlike eutherian mammals, the colon of the Australian common brushtail possum, Trichosurus vulpecula, a metatherian mammal, is incapable of electrogenic Cl− secretion and has elevated levels of electrogenic Na+ absorption, while the ileum secretes HCO3− rather than Cl−. In eutherian mammals, the cystic fibrosis transmembrane conductance regulator (CFTR) is essential for both Cl− and HCO3− secretion and the regulation of Na+ absorption. Therefore, we have sequenced possum (p)CFTR, described its distribution and characterized the properties of cloned pCFTR expressed in Fischer rat thyroid (FRT) cells. pCFTR (GenBank accession No. AY916796) has a 1,478 amino acid open reading frame, which has >90% identity with CFTR from other marsupials and >80% identity with non-rodent eutherian mammals. In pCFTR, there is a high level of conservation of the transmembrane and nucleotide binding domains although, with the exception of other marsupials, there is considerable divergence from other species in the R domain. FRT cells transfected with pCFTR express mature CFTR protein which functions as a small Cl− channel activated by cAMP-dependent phosphorylation. In whole-cell recordings it has a linear, time and voltage-independent conductance, with a selectivity sequence $$ P_{\text{Br}} > P_{\text{Cl}} > P_{\text{I}} > P_{{{\text{HCO}}_ }} } . $ pCFTR transcript is present in a range of epithelia, including the ileum and the colon. The presence of pCFTR in the ileum and its measured HCO3− permeability suggest that it may be involved in ileal HCO3− secretion. Why the possum colon does not secrete Cl− and has elevated electrogenic Na+ absorption, despite the apparent expression of CFTR, remains to be determined.
Calcium transport and homeostasis in gill cells of a freshwater crab Dilocarcinus pagei
Venue: Journal of Comparative Physiology B

Crustaceans present a very interesting model system to study the process of calcification and calcium (Ca2+) transport because of molting-related events and the deposition of CaCO3 in the new exoskeleton. Dilocarcinus pagei, a freshwater crab endemic to Brazil, was studied to understand Ca2+ transport in whole gill cells using a fluorescent probe. Cells were dissociated, all of the gill cell types were loaded with fluo-3 and intracellular Ca2+ change was monitored by adding Ca as CaCl2 (0, 0.1, 0.25, 0.50, 1.0 and 5 mM), with a series of different inhibitors. For control gill cells, Ca2+ transport followed Michaelis–Menten kinetics with Km = 0.42 ± 0.04 mM and Vmax = 0.50 ± 0.02 μM (Ca2+ change × initial intracellular Ca−1 × 180 s−1; N = 14, r2 = 0.99). Verapamil (a Ca2+ channel inhibitor) and amiloride (a Na+/Ca2+ exchanger [NCX] inhibitor) completely reduced intracellular Ca2+ transport, while nifedipine, another Ca2+ channel inhibitor, did not. Vanadate, a plasma membrane Ca2+-ATPase inhibitor (PMCA), increased intracellular Ca2+ in gill cells through a decrease in the efflux of Ca2+. Ouabain increased intracellular Ca2+, similar to the effect of KB-R, a specific NCX inhibitor for Ca2+ in the influx mode. Alterations in extracellular [Na] in the saline did not affect intracellular Ca2+ transport. Caffeine, responsible for inducing Ca release from sarcoplasmic reticulum in vertebrate muscle, increased intracellular Ca2+ compared to control, suggesting an effect of this inhibitor in gill epithelial cells of Dilocarcinus pagei, probably through release of intracellular stores. We also demonstrate here that intracellular Ca2+ in gill cells of Dilocarcinus pagei was kept relatively constant in face of an extracellular Ca concentration of 50-fold, suggesting that crustaceans are able to display Ca2+ homeostasis through various Ca2+ intracellular sequestration mechanisms and/or plasma membrane Ca2+ influx and outflux that are highly regulatory. In summary, studies using whole gill cells are an interesting approach for working with real regulatory Ca2+ mechanisms in intact cells under physiological Ca levels (mM range), compared to earlier work using isolated vesicles of various epithelial cells.
Effects of hypothyroidism on myosin heavy chain composition and fibre types of fast skeletal muscles in a small marsupial, Antechinus flavipes
Venue: Journal of Comparative Physiology B

Effects of drug-induced hypothyroidism on myosin heavy chain (MyHC) content and fibre types of fast skeletal muscles were studied in a small marsupial, Antechinus flavipes. SDS-PAGE of MyHCs from the tibialis anterior and gastrocnemius revealed four isoforms, 2B, 2X, 2A and slow, in that order of decreasing abundance. After 5 weeks treatment with methimazole, the functionally fastest 2B MyHC significantly decreased, while 2X, 2A and slow MyHCs increased. Immunohistochemistry using monospecific antibodies to each of the four MyHCs revealed decreased 2b and 2x fibres, and increased 2a and hybrid fibres co-expressing two or three MyHCs. In the normally homogeneously fast superficial regions of these muscles, evenly distributed slow-staining fibres appeared, resembling the distribution of slow primary myotubes in fast muscles during development. Hybrid fibres containing 2A and slow MyHCs were virtually absent. These results are more detailed but broadly similar to the earlier studies on eutherians. We hypothesize that hypothyroidism essentially reverses the effects of thyroid hormone on MyHC gene expression of muscle fibres during myogenesis, which differ according to the developmental origin of the fibre: it induces slow MyHC expression in 2b fibres derived from fast primary myotubes, and shifts fast MyHC expression in fibres of secondary origin towards 2A, but not slow, MyHC.
Reflex bradycardia does not influence oxygen consumption during hypoxia in the European eel (Anguilla anguilla)
Venue: Journal of Comparative Physiology B

Most teleost fish reduce heart rate when exposed to acute hypoxia. This hypoxic bradycardia has been characterised for many fish species, but it remains uncertain whether this reflex contributes to the maintenance of oxygen uptake in hypoxia. Here we describe the effects of inhibiting the bradycardia on oxygen consumption (MO2), standard metabolic rate (SMR) and the critical oxygen partial pressure for regulation of SMR in hypoxia (Pcrit) in European eels Anguilla anguilla (mean ± SEM mass 528 ± 36 g; n = 14). Eels were instrumented with a Transonic flow probe around the ventral aorta to measure cardiac output (Q) and heart rate (fH). MO2 was then measured by intermittent closed respirometry during sequential exposure to various levels of increasing hypoxia, to determine Pcrit. Each fish was studied before and after abolition of reflex bradycardia by intraperitoneal injection of the muscarinic antagonist atropine (5 mg kg−1). In the untreated eels, fH fell from 39.0 ± 4.3 min−1 in normoxia to 14.8 ± 5.2 min−1 at the deepest level of hypoxia (2 kPa), and this was associated with a decline in Q, from 7.5 ± 0.8 mL min−1 kg−1 to 3.3 ± 0.7 mL min−1 kg−1 in normoxia versus deepest hypoxia, respectively. Atropine had no effect on SMR, which was 16.0 ± 1.8 μmol O2 kg−1 min−1 in control versus 16.8 ± 0.8 μmol O2 kg−1 min−1 following treatment with atropine. Atropine also had no significant effect on normoxic fH or Q in the eel, but completely abolished the bradycardia and associated decline in Q during progressive hypoxia. This pharmacological inhibition of the cardiac responses to hypoxia was, however, without affect on Pcrit, which was 11.7 ± 1.3 versus 12.5 ± 1.5 kPa in control versus atropinised eels, respectively. These results indicate, therefore, that reflex bradycardia does not contribute to maintenance of MO2 and regulation of SMR by the European eel in hypoxia.
The vitellogenin of the bumblebee, Bombus hypocrita: studies on structural analysis of the cDNA and expression of the mRNA
Venue: Journal of Comparative Physiology B

In this present study, the cDNA of Bombus hypocrita vitellogenin (Vg) was cloned and sequenced. It is composed of 5,478 bp and contains an ORF of 1,772 amino acids within a putative signal peptide of 16 residues. The deduced amino acid sequence shows significant similarity with Bombus ignitus (95%) and Apis mellifera (52%) and a high number of conserved motifs. Close to the C terminus there is a GL/ICG motif followed by nine cysteines, and a DGXR motif is located 18 residues upstream from the GL/ICG motif. Moreover, we predicted the 3D structure of B. hypocrita Vg. Furthermore, the Vg mRNA of B. hypocrita was spatio-temporally analyzed in different castes (such as queen, worker and drone) from pupae to adult. The Vg mRNA was found in the white-eyed pupal (Pw) stage in queens, and the expression increased during the entire pupal development and attained its peak in the dark brown pupal stage. It also had a high expression in the adult fat body. In workers, the Vg expression was detected in the Pw stage, and its levels increased with age with the highest in 15 days. Afterward, it decreased progressively. Vg mRNA was also observed in drones, with a higher level of expression shown in only freshly molted adult drones.