A subiculum-hypothalamic pathway functions in dynamic threat detection and memory updating.

Animals need to detect threats, initiate defensive responses, and, in parallel, remember where the threat occurred to avoid the possibility of re-encountering it. By probing animals capable of detecting and avoiding a shock-related threatening location, we were able to reveal a septo-hippocampal-hypothalamic circuit that is also engaged in ethological threats, including predatory and social threats. Photometry analysis focusing on the dorsal premammillary nucleus (PMd), a critical interface of this circuit, showed that in freely tested animals, the nucleus appears ideal to work as a threat detector to sense dynamic changes under threatening conditions as the animal approaches and avoids the threatening source. We also found that PMd chemogenetic silencing impaired defensive responses by causing a failure of threat detection rather than a direct influence on any behavioral responses and, at the same time, updated fear memory to a low-threat condition. Optogenetic silencing of the main PMd targets, namely the periaqueductal gray and anterior medial thalamus, showed that the projection to the periaqueductal gray influences both defensive responses and, to a lesser degree, contextual memory, whereas the projection to the anterior medial thalamus has a stronger influence on memory processes. Our results are important for understanding how animals deal with the threat imminence continuum, revealing a circuit that is engaged in threat detection and that, at the same time, serves to update the memory process to accommodate changes under threatening conditions.

Disentangling developmental effects of play aspects in rat rough-and-tumble play.

Animal play encompasses a variety of aspects, with kinematic and social aspects being particularly prevalent in mammalian play behaviour. While the developmental effects of play have been increasingly documented in recent decades, understanding the specific contributions of different play aspects remains crucial to understand the function and evolutionary benefit of animal play. In our study, developing male rats were exposed to rough-and-tumble play selectively reduced in either the kinematic or the social aspect. We then assessed the developmental effects of reduced play on their appraisal of standardized human-rat play ('tickling') by examining their emission of 50 kHz ultrasonic vocalizations (USVs). Using a deep learning framework, we efficiently classified five subtypes of these USVs across six behavioural states. Our results revealed that rats lacking the kinematic aspect in play emitted fewer USVs during tactile contacts by human and generally produced fewer USVs of positive valence compared with control rats. Rats lacking the social aspect did not differ from the control and the kinematically reduced group. These results indicate aspects of play have different developmental effects, underscoring the need for researchers to further disentangle how each aspect affects animals.

Bibliometric analysis on cannibalism/infanticide and maternal aggression towards pups in laboratory rodents.

Animal welfare has evolved during the past decades to improve not only the quality of life of laboratory rodents but also the quality and reproducibility of scientific investigations. Bibliometric analysis has become an important tool to complete the current knowledge with academic databases. Our objective was to investigate whether scientific research on cannibalism/infanticide is connected with maternal aggression towards the offspring in laboratory rodents. To carry out our research, we performed a specific search for published articles on each concept. Results were analyzed in the open-source environment RStudio with the package Bibliometrix. We obtained 253 and 134 articles for the first search (cannibalism/infanticide) and the second search (maternal aggression towards the pups) respectively. We observed that the interest in infanticide/cannibalism started in the 1950s, while researchers started showing interest in maternal aggression towards the pups 30 years later. Our analyses indicated that maternal aggression had better citations in scientific literature. In addition, although our results showed some common features (e.g. oxytocin or medial preoptic area in the brain), we observed a gap between cannibalism/infanticide and maternal aggression towards the pups with only 14 published articles in common for both the searches. Therefore, we recommend researchers to combine both concepts in further investigations in the context of cannibalism for better dissemination and higher impact in laboratory rodents' welfare research.

Behavioral analysis in laboratory rats: Challenges and usefulness of 50-kHz ultrasonic vocalizations.

Many rodent species emit and detect vocalizations in the ultrasonic range. Rats use three classes of ultrasonic vocalizations depending on developmental stage, experience and the behavioral situation. Calls from one class emitted by juvenile and adult rats, the so-called 50-kHz calls, are typical for appetitive and social situations. This review provides a brief historical account on the introduction of 50-kHz calls in behavioral research followed by a survey of their scientific applications focusing on the last five years, where 50-kHz publications reached a climax. Then, specific methodological challenges will be addressed, like how to measure and report 50-kHz USV, the problem of assignment of acoustic signals to a specific sender in a social situation, and individual variability in call propensity. Finally, the intricacy of interpreting 50-kHz results will be discussed focusing on the most prevalent ones, namely as communicative signals and/or readouts of the sender's emotional status.

An open-source tool for automated analysis of breathing behaviors in common marmosets and rodents.

The respiratory system maintains homeostatic levels of oxygen (O2) and carbon dioxide (CO2) in the body through rapid and efficient regulation of breathing frequency and depth (tidal volume). The commonly used methods of analyzing breathing data in behaving experimental animals are usually subjective, laborious, and time-consuming. To overcome these hurdles, we optimized an analysis toolkit for the unsupervised study of respiratory activities in animal subjects. Using this tool, we analyzed breathing behaviors of the common marmoset (Callithrix jacchus), a New World non-human primate model. Using whole-body plethysmography in room air as well as acute hypoxic (10% O2) and hypercapnic (6% CO2) conditions, we describe breathing behaviors in awake, freely behaving marmosets. Our data indicate that marmosets' exposure to acute hypoxia decreased metabolic rate and increased sigh rate. However, the hypoxic condition did not augment ventilation. Hypercapnia, on the other hand, increased both the frequency and depth (i.e., tidal volume) of breathing.

Differences in exercise capacity and physiological responses in Wistar rats among breeders.

In animal experiments aimed at extrapolation to humans, it is essential to ensure the reproducibility of experiments and universality between animals and humans. However, among animals with the same generic name but from different breeders, which is to say different stocks, even resting physiological conditions, such as genetics, do not coincide, and, therefore, exercise capacity and physiological responses may also vary. To address this issue, we examined the differences in exercise capacity and exercise-induced metabolic and endocrine responses among stocks of Wistar rats using an established treadmill running model for rodents, which mimics physiological responses in humans. Wistar rats from four breeders were acclimated to treadmill running and then had a catheter inserted into their external jugular veins. Subsequently, the rats were subjected to an incremental treadmill running test (IRT). We found that there were significant differences in the exercise capacity among Wistar rats from different breeders. Additionally, the dynamics of blood lactate, glucose, and adrenocorticotropic hormone levels during the IRT were found to vary among the Wistar rats from different breeders; only one stock showed human-type exercise-induced physiological responses. These results indicate that Wistar rats could have different capacities for and physiological responses to the same exercise depending on their stocks. Thus, the selection of the stock of experimental animals may affect the validity of the results when verifying exercise effects.

Self-renewal and differentiation of rat epididymal basal cells using a novel in vitro organoid model†.

The epididymis is composed of a pseudostratified epithelium that is comprised of various cell types. Studies have shown that rat basal cells share common properties with adult stem cells and begin to differentiate in vitro in response to fibroblast growth factor and 5α-dihydrotestosterone. The characterization of rat basal cells is therefore necessary to fully understand the role of these cells. The objectives of this study were to assess the ability of single basal cells to develop organoids and to assess their ability to self-renew and differentiate in vitro. We isolated basal cells from the rat epididymis and established three-dimensional cell cultures from the basal and nonbasal cell fractions. Organoids were formed by single adult epididymal basal cells. Organoids were dissociated into single basal cells, which were able to reform new organoids, and were maintained over 10 generations. Long-term culture of organoids revealed that these cells could be differentiated into cells expressing the principal cell markers aquaporin 9 and cystic fibrosis transmembrane conductance regulator. Electron microscopy demonstrated that organoids were composed of several polarized cell types displaying microvilli and the ability to form tight junctions. Additionally, organoids could be formed by basal cells from either the proximal or distal region of the epididymis and are able to secrete clusterin, a protein implicated in the maturation of spermatozoa. These data indicate that rat basal cells can be used to derive epididymal organoids and further support that notion that these may represent a stem cell population in the epididymis.

High-frequency burst spiking in layer 5 thick-tufted pyramids of rat primary somatosensory cortex encodes exploratory touch.

Diversity of cell-types that collectively shape the cortical microcircuit ensures the necessary computational richness to orchestrate a wide variety of behaviors. The information content embedded in spiking activity of identified cell-types remain unclear to a large extent. Here, we recorded spike responses upon whisker touch of anatomically identified excitatory cell-types in primary somatosensory cortex in naive, untrained rats. We find major differences across layers and cell-types. The temporal structure of spontaneous spiking contains high-frequency bursts (≥100 Hz) in all morphological cell-types but a significant increase upon whisker touch is restricted to layer L5 thick-tufted pyramids (L5tts) and thus provides a distinct neurophysiological signature. We find that whisker touch can also be decoded from L5tt bursting, but not from other cell-types. We observed high-frequency bursts in L5tts projecting to different subcortical regions, including thalamus, midbrain and brainstem. We conclude that bursts in L5tts allow accurate coding and decoding of exploratory whisker touch.

Probing the decision-making mechanisms underlying choice between drug and nondrug rewards in rats.

Delineating the decision-making mechanisms underlying choice between drug and nondrug rewards remains a challenge. This study adopts an original approach to probe these mechanisms by comparing response latencies during sampling versus choice trials. While lengthening of latencies during choice is predicted in a deliberative choice model (DCM), the race-like response competition mechanism postulated by the Sequential choice model (SCM) predicts a shortening of latencies during choice compared to sampling. Here, we tested these predictions by conducting a retrospective analysis of cocaine-versus-saccharin choice experiments conducted in our laboratory. We found that rats engage deliberative decision-making mechanisms after limited training, but adopt a SCM-like response selection mechanism after more extended training, while their behavior is presumably habitual. Thus, the DCM and SCM may not be general models of choice, as initially formulated, but could be dynamically engaged to control choice behavior across early and extended training.

Zp4 is completely dispensable for fertility in female rats†.

Zona pellucida (ZP), which is composed of at most four extracellular glycoproteins (ZP1, ZP2, ZP3, and ZP4) in mammals, shelters the oocytes and is vital in female fertility. Several studies have identified the indispensable roles of ZP1-3 in maintaining normal female fertility. However, the understanding of ZP4 is still very poor because only one study on ZP4-associated infertility performed in rabbits has been reported up to date. Here we investigated the function of mammalian Zp4 by creating a knockout (KO) rat strain (Zp4-/- rat) using CRISPR-Cas9-mediated DNA-editing method. The influence of Zp4 KO on ZP morphology and some pivotal processes of reproduction, including oogenesis, ovulation, fertilization, and pup production, were studied using periodic acid-Schiff's staining, superovulation, in vitro fertilization, and natural mating. The ZP morphology in Zp4-/- rats was normal, and none of these pivotal processes was affected. This study renewed the knowledge of mammalian Zp4 by suggesting that Zp4 was completely dispensable for female fertility.

Role of zinc in female reproduction.

Zinc is a critical component in a number of conserved processes that regulate female germ cell growth, fertility, and pregnancy. During follicle development, a sufficient intracellular concentration of zinc in the oocyte maintains meiotic arrest at prophase I until the germ cell is ready to undergo maturation. An adequate supply of zinc is necessary for the oocyte to form a fertilization-competent egg as dietary zinc deficiency or chelation of zinc disrupts maturation and reduces the oocyte quality. Following sperm fusion to the egg to initiate the acrosomal reaction, a quick release of zinc, known as the zinc spark, induces egg activation in addition to facilitating zona pellucida hardening and reducing sperm motility to prevent polyspermy. Symmetric division, proliferation, and differentiation of the preimplantation embryo rely on zinc availability, both during the oocyte development and post-fertilization. Further, the fetal contribution to the placenta, fetal limb growth, and neural tube development are hindered in females challenged with zinc deficiency during pregnancy. In this review, we discuss the role of zinc in germ cell development, fertilization, and pregnancy with a focus on recent studies in mammalian females. We further detail the fundamental zinc-mediated reproductive processes that have only been explored in non-mammalian species and speculate on the role of zinc in similar mechanisms of female mammals. The evidence collected over the last decade highlights the necessity of zinc for normal fertility and healthy pregnancy outcomes, which suggests zinc supplementation should be considered for reproductive age women at risk of zinc deficiency.

Rats show direct reciprocity when interacting with multiple partners.

Direct reciprocity, where individuals apply the decision rule 'help someone who has helped you', is believed to be rare in non-human animals due to its high cognitive demands. Especially if previous encounters with several partners need to be correctly remembered, animals might either stop reciprocating favours previously received from an individual, or switch to the simpler generalized reciprocity mechanism. Here we tested the decision rules Norway rats apply when interacting with multiple partners before being able to return received help. In a sequential prisoner's dilemma situation, focal subjects encountered four different partners that were either helpful or not, on four consecutive days. On the fifth day, the focal subject was paired with one of the previous four partners and given the opportunity to provide it with food. The focal rats returned received help by closely matching the quantity of help their partner had previously provided, independently of the time delay between received and given help, and independently of the ultimate interaction preceding the test. This shows that direct reciprocity is not limited to dyadic situations in Norway rats, suggesting that cognitive demands involved in applying the required decision rules can be met by non-human animals even when they interact with multiple partners differing in helping propensity.

Avoidance behaviour in laboratory house mice (Musmusculus) and Norway rats (Rattus norvegicus) towards predator odours.

Mus musculus and Rattus sp. are considered pest species because they reach high densities in urban areas, crop fields and food storage and productive systems such as breeding farms and orchards. Their control relies mainly on rodenticide application, but the effectiveness of this application is reduced due to behavioural responses and resistance. Novel methods are based on the use of chemical signals as odours that may be attractants, repellents or may reduce the reproductive success of pest species. The aim of this paper is to study the aversive effect of TMT, cat urine and cat body odour on predator-inexperienced Mus musculus and Rattus norvegicus under laboratory conditions. The experimental apparatus comprised three boxes connected by PVC pipes in a linear arrangement. In lateral boxes, odour sources or distilled water were introduced, while animals were placed in the central box at the beginning of the experiment. Rats showed freezing behaviour, reduced visits in the presence of TMT and cat fur. Mice reduced their visits with cat body and cat urine. This study provides evidence of the usefulness of using fear responses as a way to control rodent pests, which must be adapted to the environment and species to be applied.

Relationship between exploratory activity and adrenocortical activity in the black rat (Rattus rattus).

The relationship between physiological and behavioral stress markers is documented in several rodent species. However, there is no information regarding the role of adrenocortical activity in behavior of the black rat (Rattus rattus). Therefore, we hypothesize that the adrenocortical activity of black rats varies between individuals and is related to some of the behaviors in a novel environment. To test this hypothesis, we (i) validated a method for quantifying glucocorticoid metabolites from feces (fGCMs) with an enzyme immunoassay (EIA); (ii) examined variation and diurnal rhythms of feces and GCM production; and (iii) examined the relationship between GCM levels and exploratory behavioral traits. We fulfilled the first aim (i) by successfully performing an ACTH challenge test to validate the use of a 5α-pregnane-3ß,11ß,21-triol-20-one EIA for measuring fGCMs. Second (ii) we detected considerable consistent interindividual variability in production of both feces and glucocorticoids. The peak production of feces occurred in the first hour of the dark cycle, the peak of fGCMs occurred approximately 3 h later. Lastly, (iii) there was no clear relationship between behavior in the hole board test and GCMs. Grooming, a typical behavioral stress marker, was negatively associated with stress reactivity, while head-dipping in the hole-board test (traditionally considered an exploratory behavior independent of stress) was not correlated with the GCMs. This study offers a first look at GCMs in the black rat, successfully validates a method for their measurement and opens possibilities for future research of the relationship between glucocorticoids and exploratory behavior in this species.

Determination of basal bone mineral density in the femur bones of male and female Wistar rats.

Changes in bone mineral content of calcium (Ca), phosphorous (P), magnesium and potassium for male and female Wistar rats during their development from 3 weeks old to adulthood (27 weeks old) were measured. Bone mineral content was related to areal bone mineral density (BMD) which was measured in vivo at the femoral neck using a calibrated X-ray transmission system to obtain basal curves as a function of the age of the specimen. Diagnostic curves were built to determine low BMD (osteopaenia) and osteoporosis in female rats fed a Ca-depleted diet (50%) based on the obtained data and the criteria established by the World Health Organization. Bone mineral content is directly related to sex and age, but P did not change throughout the experimental period. P content did not exhibit significant changes with growing, while Ca was greatest in male rats, producing significant differences in the Ca:P ratio. Male rats reach the Ca:P ratio peak before female rats. However, areal BMD does not follow the same trend. On the other hand, osteoporosis produced a 45% decrease in this parameter for young and mature adults. These results make Z-score values available to diagnose bone-mass losses and hence the possibility of improving the conditions of non-contact measurement of BMD in vivo. This technique can be used for future experiments with Wistar rats.

The smell of cooperation: rats increase helpful behaviour when receiving odour cues of a conspecific performing a cooperative task.

Reciprocity can explain cooperative behaviour among non-kin, where individuals help others depending on their experience in previous interactions. Norway rats (Rattus norvegicus) cooperate reciprocally according to direct and generalized reciprocity. In a sequence of four consecutive experiments, we show that odour cues from a cooperating conspecific are sufficient to induce the altruistic help of rats in a food-exchange task. When rats were enabled to help a non-cooperative partner while receiving olfactory information from a rat helping a conspecific in a different room, they helped their non-cooperative partner as if it was a cooperative one. We further show that the cues inducing altruistic behaviour are released during the act of cooperation and do not depend on the identity of the cue provider. Remarkably, olfactory cues seem to be more important for cooperation decisions than experiencing a cooperative act per se. This suggests that rats may signal their cooperation propensity to social partners, which increases their chances to receive help in return.

Effects of long-term hydrogen intervention on the physiological function of rats.

The potential therapeutic effects of molecular hydrogen (H2) have now been confirmed in various human and animal-disease models. However, the effects of H2 on the physiological function in a normal state have been largely neglected. Hydrogen-rich water (HRW) intake and hydrogen inhalation (HI) are the most common used methods for hydrogen administration, the difference in the effects between HRW intake and HI remains elusive. In the present study, the body weight and 13 serum biochemical parameters were monitored during the six-month hydrogen intervention, all these parameters were significantly altered by oral intake of HRW or HI. Among the 13 parameters, the most striking alterations induced by hydrogen treatment were observed in serum myocardial enzymes spectrum. The results also showed that the changes in these parameters occurred at different time points, and the alterations in most of the parameters were much more significant in HI than HRW. The results of this study provides the basic data for the mechanism research and application of molecular hydrogen in the future.

Divergent projections of the prelimbic cortex bidirectionally regulate active avoidance.

The prefrontal cortex (PFC) integrates incoming information to guide our actions. When motivation for food-seeking competes with avoidance of danger, the PFC likely plays a role in selecting the optimal choice. In platform-mediated active avoidance, rats avoid a tone-signaled footshock by stepping onto a nearby platform, delaying access to sucrose pellets. This avoidance requires prelimbic (PL) PFC, basolateral amygdala (BLA), and ventral striatum (VS). We previously showed that inhibitory tone responses of PL neurons correlate with avoidability of shock (Diehl et al., 2018). Here, we optogenetically modulated PL terminals in VS and BLA to identify PL outputs regulating avoidance. Photoactivating PL-VS projections reduced avoidance, whereas photoactivating PL-BLA projections increased avoidance. Moreover, photosilencing PL-BLA or BLA-VS projections reduced avoidance, suggesting that VS receives opposing inputs from PL and BLA. Bidirectional modulation of avoidance by PL projections to VS and BLA enables the animal to make appropriate decisions when faced with competing drives.

Gradients in the biophysical properties of neonatal auditory neurons align with synaptic contact position and the intensity coding map of inner hair cells.

Sound intensity is encoded by auditory neuron subgroups that differ in thresholds and spontaneous rates. Whether variations in neuronal biophysics contributes to this functional diversity is unknown. Because intensity thresholds correlate with synaptic position on sensory hair cells, we combined patch clamping with fiber labeling in semi-intact cochlear preparations in neonatal rats from both sexes. The biophysical properties of auditory neurons vary in a striking spatial gradient with synaptic position. Neurons with high thresholds to injected currents contact hair cells at synaptic positions where neurons with high thresholds to sound-intensity are found in vivo. Alignment between in vitro and in vivo thresholds suggests that biophysical variability contributes to intensity coding. Biophysical gradients were evident at all ages examined, indicating that cell diversity emerges in early post-natal development and persists even after continued maturation. This stability enabled a remarkably successful model for predicting synaptic position based solely on biophysical properties.

Different methods of fear reduction are supported by distinct cortical substrates.

Understanding how learned fear can be reduced is at the heart of treatments for anxiety disorders. Tremendous progress has been made in this regard through extinction training in which the aversive outcome is omitted. However, current progress almost entirely rests on this single paradigm, resulting in a very specialized knowledgebase at the behavioural and neural level of analysis. Here, we used a dual-paradigm approach to show that different methods that lead to reduction in learned fear in rats are dissociated in the cortex. We report that the infralimbic cortex has a very specific role in fear reduction that depends on the omission of aversive events but not on overexpectation. The orbitofrontal cortex, a structure generally overlooked in fear, is critical for downregulating fear when novel predictions about upcoming aversive events are generated, such as when fear is inflated or overexpected, but less so when an expected aversive event is omitted.