Chronic defeat stress induces monoamine level dysregulation in the prefrontal cortex but not in the hippocampus of OF1 male mice.

Chronic social stress can increase susceptibility to chronic diseases such as depression. One of the most used models to study the physiological mechanisms and behavioral outcomes of this type of stress is chronic defeat stress (CDS) in male mice. OF1 male mice were subjected to a stress period lasting 18 days. During that time, non-stressed animals were housed in groups. The cluster analysis of the behavioral profile displayed during the first social interaction divided subjects into two groups: active/aggressive (AA) and passive/reactive (PR). The day after the end of the stress period, the following behavioral analyses were performed: the sucrose preference test (SPT) on day 19, the open field test (OFT) on day 20, and the forced swim test (FST) on day 21. Immediately after completing the last test, animals were weighed, and blood samples were obtained. Then, they were sacrificed, and their prefrontal cortices and hippocampi were removed and stored to analyze monoamine levels. Stressed animals displayed anhedonia, and solely the PR mice continued to show higher levels of immobility in the OFT and FST. All stressed animals, regardless of the coping strategy, presented higher plasma corticosterone levels. In addition, stressed mice showed lower levels of tyrosine, dopamine, DOPAC, MHPG, kynurenine, kynurenic acid, and 5-HIAA levels but higher serotonin levels in the prefrontal cortex, not in the hippocampus. In conclusion, our results show that CSD induces differences in monoamine levels between brain areas, and these differences did not respond to the coping strategy adopted.

The amygdala NT3-TrkC pathway underlies inter-individual differences in fear extinction and related synaptic plasticity.

Fear-related pathologies are among the most prevalent psychiatric conditions, having inappropriate learned fear and resistance to extinction as cardinal features. Exposure therapy represents a promising therapeutic approach, the efficiency of which depends on inter-individual variation in fear extinction learning, which neurobiological basis is unknown. We characterized a model of extinction learning, whereby fear-conditioned mice were categorized as extinction (EXT)-success or EXT-failure, according to their inherent ability to extinguish fear. In the lateral amygdala, GluN2A-containing NMDAR are required for LTP and stabilization of fear memories, while GluN2B-containing NMDAR are required for LTD and fear extinction. EXT-success mice showed attenuated LTP, strong LTD and higher levels of synaptic GluN2B, while EXT-failure mice showed strong LTP, no LTD and higher levels of synaptic GluN2A. Neurotrophin 3 (NT3) infusion in the lateral amygdala was sufficient to rescue extinction deficits in EXT-failure mice. Mechanistically, activation of tropomyosin receptor kinase C (TrkC) with NT3 in EXT-failure slices attenuated lateral amygdala LTP, in a GluN2B-dependent manner. Conversely, blocking endogenous NT3-TrkC signaling with TrkC-Fc chimera in EXT-success slices strengthened lateral amygdala LTP. Our data support a key role for the NT3-TrkC system in inter-individual differences in fear extinction in rodents, through modulation of amygdalar NMDAR composition and synaptic plasticity.

Perceived professional quality of life and mental well-being among animal facility personnel in Spain.

Animal facility personnel provide the husbandry and care of laboratory animals. We aimed to investigate their work-related quality of life, empathy and mental well-being. Participants living in Spain were contacted by email and asked to complete an anonymous online questionnaire, in which they answered the Professional Quality of Life scale, the Cognitive and affective empathy test, the Warwick-Edinburgh Mental Well-Being Scale, and their perceived human-animal interaction. Participants were asked whether they were receiving psychological therapy or were taking anxiolytics, hypnotics or antidepressant medication. The study comprised 80 participants. No differences were observed related to personal or professional variables. Participants working with small carnivores reported higher total empathy, and those working with non-human primates reported higher emotional comprehension. Higher human-animal interaction was reported by participants working with small carnivores, farm animals and non-human primates. More than half of the participants reported high levels of mental well-being, positively correlated with emotional comprehension, emphatic joy and compassion satisfaction. Participants working with farm animals reported higher levels of secondary traumatic stress that was positively correlated with human-animal interaction and negatively with mental well-being. Most participants reported low-average levels of burnout, which was negatively correlated with mental well-being. The percentage of animal facility personnel in psychotherapy was higher than in the general population, and the consumption of anxiolytics was a little lower and antidepressants higher. Overall, our results indicate that animal-facility personnel who felt stress or worse mental well-being were in therapy and took medication to improve their condition.

Implementing the 3Rs in Laboratory Animal Research-From Theory to Practice.

The regulatory framework for the use of animals in research in many countries is based on the 3Rs: replacement, reduction, and refinement [...].

The role of sociability in social instability stress: Behavioral, neuroendocrine and monoaminergic effects.

Extensive literature has reported a link between social stress and mental health. In this complex relationship, individual strategies for coping with social stress are thought to have a possible modulating effect, with sociability being a key factor. Despite the higher incidence of affective disorders in females and sex-related neurochemical differences, female populations have been understudied. The aim of the present study was, therefore, to analyze the behavioral, neuroendocrine, and neurochemical effects of stress in female OF1 mice, paying special attention to social connectedness (female mice with high vs low sociability). To this end, subjects were exposed to the Chronic Social Instability Stress (CSIS) model for four weeks. Although female mice exposed to CSIS had increased arousal, there was no evidence of depressive-like behavior. Neither did exposure to CSIS affect corticosterone levels, although it did increase the MR/GR ratio by decreasing GR expression. Female mice exposed to CSIS had higher noradrenaline and dopamine levels in the hippocampus and striatum respectively, with a lower monoaminergic turnover, resulting in an increased arousal. CSIS increased serotonin levels in both the hippocampus and striatum. Similarly, CSIS was found to reduce kynurenic acid, 3-HK, and IDO and iNOS enzyme levels in the hippocampus. Interestingly, the observed decrease in IDO synthesis and the increased serotonin and dopamine levels in the striatum were only found in subjects with high sociability. These highly sociable female mice also had significantly lower levels of noradrenaline in the striatum after CSIS application. Overall, our model has produced neuroendocrine and neurochemical but not behavioral changes, so it has not allowed us to study sociability in depth. Therefore, a model that induces both molecular and behavioral phenotypes should be applied to determine the role of sociability.

Individualized Housing Modifies the Immune-Endocrine System in CD1 Adult Male Mice.

In the last years, different research groups have made considerable efforts to improve the care and use of animals in research. Mice (Mus musculus) are the most widely used animal species in research in the European Union and are sociable and hierarchical creatures. During experiments, researchers tend to individualize males, but no consideration is given to whether this social isolation causes them stress. The aim of this study was, therefore, to explore whether 4 weeks of social isolation could induce changes in different physiological parameters in adult Crl:CD1(ICR) (CD1) males, which may interfere with experimental results. Body weight, blood cells, and fecal corticosterone metabolites levels were the analyzed parameters. Blood and fecal samples were collected at weeks 1 and 4 of the experimental procedure. Four weeks of single housing produced a significant time-dependent decrease in monocytes and granulocytes. Fecal corticosterone metabolite levels were higher in single-housed mice after 1 week and then normalized after 4 weeks of isolation. Body weight, red blood cells, and platelets remained unchanged in both groups during this period. We can, therefore, conclude that social isolation affects some immune and endocrine parameters, and that this should be taken into account in the interpretation of research data.

Refinement in the European Union: A Systematic Review.

Refining experiments and housing conditions so as to cause the minimum possible pain and distress is one of the three principles (3Rs) on which Directive 2010/63/EU is based. In this systematic review, we aimed to identify and summarize published advances in the refinement protocols made by European Union-based research groups from 2011 to 2021, and to determine whether or not said research was supported by European or national grants. We included 48 articles, the majority of which were related to improvements in experimental procedures (37/77.1%) for mice (26/54.2%) and were written by research groups belonging to universities (36/57.1%) and from the United Kingdom (21/33.9%). More than two thirds (35/72.9%) of the studies received financial support, 26 (mostly British) at a national level and 8 at a European level. Our results indicated a clear willingness among the scientific community to improve the welfare of laboratory animals, as although funding was not always available or was not specifically granted for this purpose, studies were published nonetheless. However, in addition to institutional support based on legislation, more financial support is needed. We believe that more progress would have been made in refinement during these years if there had been more specific financial support available at both the national and European Union levels since our data suggest that countries investing in refinement have the greatest productivity in successfully publishing refinements.

Professional quality of life among Spanish veterinarians.

Background: In Spain, the perceived professional quality of life among veterinarians has not been explored. Methods: Veterinarians were invited to complete an online questionnaire in which they answered the Professional Quality of Life scale, the Medical Outcomes Study Social Support Survey and the Warwick-Edinburgh Mental Well-Being Scale. Participants were asked whether they were receiving psychological therapy or were taking anxiolytics, hypnotics or antidepressant medication. Alcohol consumption was measured using the Alcohol Use Disorders Identification Test and nicotine dependence was assessed using the Fagerström test; participants were asked whether they took illegal drugs. Results: The study sample comprised a total of 602 veterinarians, most of whom reported average levels of compassion satisfaction, secondary stress trauma and burnout. Emotional support and mental wellbeing influenced participants' professional quality of life. The percentage of veterinarians in psychological therapy and/or taking anxiolytics was higher than in the general population. Conclusions: A considerable number of clinical veterinarians in Spain may be suffering from work-related stress. Our study identifies salary, emotional support and mental wellbeing as important factors that affect the professional quality of life. Interventions to improve veterinary clinicians' professional quality of life should therefore focus on these factors.

Chronic social instability stress down-regulates IL-10 and up-regulates CX3CR1 in tumor-bearing and non-tumor-bearing female mice.

Extensive literature has reported a link between stress and tumor progression, and between both of these factors and mental health. Despite the higher incidence of affective disorders in females and the neurochemical differences according to sex, female populations have been understudied. The aim of this study was therefore to analyze the effect of stress on tumor development in female OF1 mice. For this purpose, subjects were inoculated with B16F10 melanoma cells and exposed to the Chronic Social Instability Stress (CSIS) model. Behavioral, neurochemical and neuroendocrine parameters were analyzed. Female mice exposed to CSIS exhibited reduced body weight and increased arousal, but there was no evidence of depressive behavior or anxiety. Exposure to CSIS did not affect either corticosterone levels or tumor development, although it did provoke an imbalance in cerebral inflammatory cytokines, decreasing IL-10 expression (IL-6/IL-10 and TNF-α/IL-10); chemokines, increasing CX3CR1 expression (CX3CL1/CX3CR1); and glucocorticoid receptors, decreasing GR expression (MR/GR). In contrast, tumor development did not alter body weight and, although it did alter behavior, it did so to a much lesser extent. Tumor inoculation did not affect corticosterone levels, but increased the MR/GR ratio in the hippocampus and provoked an imbalance in cerebral inflammatory cytokines and chemokines, although differently from stress. These results underscore the need for experimental approaches that allow us to take sex differences into account when exploring this issue, since these results appear to indicate that the female response to stress is mediated by mechanisms different from those often proposed in relation to male mice.

Mice in translational neuroscience: What R we doing?

Animal models play a pivotal role in translational neuroscience but recurrent problems in data collection, analyses, and interpretation, lack of biomarkers, and a tendency to over-reliance on mice have marred neuroscience progress, leading to one of the highest attrition rates in drug translation. Global initiatives to improve reproducibility and model selection are being implemented. Notwithstanding, mice are still the preferred animal species to model human brain disorders even when the translation has been shown to be limited. Non-human primates are better positioned to provide relevant translational information because of their higher brain complexity and homology to humans. Among others, lack of resources and formal training, strict legislation, and ethical issues may impede broad access to large animals. We propose that instead of increasingly restrictive legislation, more resources for training, education, husbandry, and data sharing are urgently needed. The creation of multidisciplinary teams, in which veterinarians need to play a key role, would be critical to improve translational efficiency. Furthermore, it is not usually acknowledged by researchers and regulators the value of comparative studies in lower species, that are instrumental in toxicology, target identification, and mechanistic studies. Overall, we highlight here the need for a conceptual shift in neuroscience research and policies to reach the patients.

A survey on the use of mice, pigs, dogs and monkeys as animal models in biomedical research in Spain.

BACKGROUND: The use of animals in biomedical science remains controversial. An individual's level of concern is generally influenced by their culture, previous or current experience with animals, and the specific animal species in question. In this study we aimed to explore what people in Spain who had never or who no longer worked with laboratory animals thought of the use of mice, pigs, dogs and monkeys for biomedical research purposes. We also aimed to determine whether or not people currently involved in biomedical research with the aforementioned species felt their work was justified. RESULTS: The study comprised a total of 807 participants (never worked = 285, used to work = 56, currently working = 466), almost two thirds of whom were women. Our results revealed that the phylogenetic scale is an important factor in people's opinions of the use of certain species in research. The percentage of people who were against the use of dogs or monkeys was higher than that of those who were against the use of mice or pigs. The main reasons given for having stopped working with laboratory animals were change of professional career and change in research project. Participants who were currently working with animals believed that their work was justified, but said they did not talk about it with people outside their immediate social circle. CONCLUSIONS: Our findings suggest that there is a difference in moral status between monkeys and mice, as well as between companion animals (dogs) and farm animals (pigs). Our results support the idea that working with laboratory animals is a sensitive issue in Spain.

Professional Quality of Life in Research Involving Laboratory Animals.

Many workers contribute to the success of animal welfare and study outcomes in biomedical research. However, the professional quality of life (ProQoL) of those who work with laboratory animals has not been explored in Spain. To this end, we adapted the ProQoL scale to the Spanish population working with laboratory animals. Participants were contacted by email and asked to complete an anonymous on-line questionnaire. The study comprised a total of 498 participants, 12.4% welfare officers/veterinarians, 19.5% caretaker/technicians, 13.9% principal investigators, 20.7% investigators, 13.6% research technicians, and 19.9% PhD students. The adapted scale revealed very good reliability and internal validity, providing information about two different subscales, compassion satisfaction and compassion fatigue. Animal-facility personnel showed higher total ProQoL and compassion-satisfaction scores than researchers; PhD students showed the lowest scores. Thus, our results indicate that job category is a contributing factor in perceived professional quality of life. We observed that compassion satisfaction is negatively associated with the perceived animal stress/pain. Participants reporting poorer compassion satisfaction also reported lower social-support scores. Overall, our ProQoL scale is a useful tool for analyzing the professional quality of life in the Spanish population, and may help to design future interventions to improve workplace wellbeing in Spain and other Spanish-speaking populations.

Working with laboratory rodents in Spain: a survey on welfare and wellbeing.

BACKGROUND: Replacement, reduction and refinement, the 3R principles, provide a framework to minimize the use and suffering of animals in science. In this context, we aimed to determine the actual perception that individuals working with laboratory rodents in biomedical research have on animal welfare and on their interaction with the animals, as well as how they perceive its impact on their social relations. To this end, we designed an anonymous on-line survey for people working with rodents, at three responsibility levels, in Spain. RESULTS: Of the 356 participants, 239 were women (67 %); 263 were researchers (74 %), and 93 animal facility staff (26 %), of which 55 were caretakers/technicians (15 %), and 38 welfare officer/veterinarians (11 %). Animal facility staff indicated environmental enrichment to be a universal practice. About half of the participants reported that, in their opinion, animals suffer "little to none" or "minor" stress and pain. Animal caretakers/technicians and researchers perceived higher levels of stress and pain than welfare officers/veterinarians. Participants judged decapitation the most unpleasant method to kill rodents, whereas anaesthetic overdose was the least one. A sizable proportion - 21 % of animal caretakers/technicians and 11.4 % of researchers - stated that they were never given the choice not to euthanize the rodents they work with. Overall, women reported higher interactions with animals than men. Nevertheless, we could detect a significant correlation between time spent with the animals and interaction scores. Notably, 80 % of animal facility staff and 92 % of researchers rarely talked about their work with laboratory rodents with people outside their inner social circle. CONCLUSIONS: Overall, the participants showed high awareness and sensitivity to rodent wellbeing; animal facility staff reported a similar perception on welfare questions, independently of their category, while researchers, who spent less time with the animals, showed less awareness and manifested lower human-animal interaction and less social support. Regarding the perception on social acceptance of laboratory animal work, all groups were cautious and rarely talked about their job, suggesting that it is considered a sensitive issue in Spain.

Looking backward to move forward: a meta-analysis of stem cell therapy in amyotrophic lateral sclerosis.

Transplantation of several types of stem cells (SC) for the treatment of amyotrophic lateral sclerosis (ALS) has been evaluated in numerous Phase I/II clinical trials with inconclusive results. Here, we conducted a meta-analysis to systematically assess the outcome of SC therapy trials which report the evolution of each patient before and after cell administration. In this way, we aimed to determine the effect of the SC intervention despite individual heterogeneity in disease progression. We identified 670 references by electronic search and 90 full-text studies were evaluated according to the eligibility criteria. Eleven studies were included comprising 220 cell-treated patients who received mesenchymal (M) SC (n = 152), neural (N) SC (n = 57), or mononuclear cells (MNC: CD34, CD117, and CD133 positive cells) (n = 11). Our analyses indicate that whereas intrathecal injection of mesenchymal stromal cells appears to have a transient positive effect on clinical progression, as measured by the ALS functional rating score, there was a worsening of respiratory function measured by forced vital capacity after all interventions. Based on current evidence, we conclude that optimal cell product and route of administration need to be determined in properly controlled preclinical models before further advancing into ALS patients. In addition, in-depth understanding of disease mechanisms in subsets of patients will help tailoring SC therapy to specific targets and increase the likelihood of improving outcomes.

Postweaning Grouping as a Strategy to Reduce Singly Housed Male Mice.

Rearing laboratory mice in groups is important since social isolation after weaning induces brain alterations, which entails behavioral abnormalities in adulthood. Age is an important factor when grouping males of different litters due to inter-male aggressiveness. The aim of this study was to determine whether newly weaned mice could safely be grouped with late juvenile or early and late pubescent mice, and whether cage cleaning, the number of the hosting group members and testosterone plasma levels have any influence. Newly weaned C57BL/6J, CD1, and SCID Beige male mice were systematically grouped with same strain late juvenile, early or late pubescent male mice in clean or dirty cages of 1, 2 or 3 hosting members. We also analyzed plasma testosterone levels at different postnatal days. Our result showed that only strain and hosting male's age influence agonistic behavior toward newly weaned mice. Thus, in order not to house a recently weaned male alone, we would recommend grouping it with late juvenile same strain mice in all studied strains. In the same way, CD1 and SCID Beige pubescent mice will admit a newly weaned mouse in their group. However, we would not recommend grouping newly weaned and pubescent C57BL/6J males.

Chimeras for the twenty-first century.

Recent advances in stem cell biology and molecular engineering have improved and simplified the methodology employed to create experimental chimeras, highlighting their value in basic research and broadening the spectrum of potential applications. Experimental chimeras have been used for decades during the generation of murine genetic models, this being especially relevant in developmental and regeneration studies. Indeed, their value for the research and modeling of human diseases was recognized by the 2007 Nobel Prize to Mario Capecchi, Martin Evans, and Oliver Smithies. More recently, their potential application in regenerative medicine has generated a lot of interest, particularly the enticing possibility to generate human organs for transplantation in livestock animals. In this review, we provide an update on interspecific chimeric organogenesis, its possibilities, current limitations, alternatives, and ethical issues.

Implementing strategies to reduce singly housed male mice.

There are different husbandry situations that lead to social isolation of male mice. Besides legal considerations, single housing has a negative impact on behavioural and molecular studies. We have implemented two strategies, the 'companion mouse' and just-weaned male grouping, to reduce the number of male mice housed singly in our facility. We have achieved a progressive reduction (42% in three years) without an increase in aggression.

Generation of two transgene-free human iPSC lines from CD133+ cord blood cells.

We have generated two human induced pluripotent stem cell (iPSC) lines from CD133+ cells isolated from umbilical cord blood (CB) of a female child using non-integrative Sendai virus. Here we describe the complete characterization of these iPSC lines: PRYDi-CB5 and PRYDi-CB40.

Proteolytic Degradation of Hippocampal STEP61 in LTP and Learning.

Striatal-enriched protein tyrosine phosphatase (STEP) modulates key signaling molecules involved in synaptic plasticity and neuronal function. It is postulated that STEP opposes the development of long-term potentiation (LTP) and that it exerts a restraint on long-term memory (LTM). Here, we examined whether STEP61 levels are regulated during hippocampal LTP and after training in hippocampal-dependent tasks. We found that after inducing LTP by high frequency stimulation or theta-burst stimulation STEP61 levels were significantly reduced, with a concomitant increase of STEP33 levels, a product of calpain cleavage. Importantly, inhibition of STEP with TC-2153 improved LTP in hippocampal slices. Moreover, we observed that after training in the passive avoidance and the T-maze spontaneous alternation task, hippocampal STEP61 levels were significantly reduced, but STEP33 levels were unchanged. Yet, hippocampal BDNF content and TrkB levels were increased in trained mice, and it is known that BDNF promotes STEP degradation through the proteasome. Accordingly, hippocampal pTrkBTyr816, pPLCγTyr783, and protein ubiquitination levels were increased in T-SAT trained mice. Remarkably, injection of the TrkB antagonist ANA-12 (2 mg/Kg, but not 0.5 mg/Kg) elicited LTM deficits and promoted STEP61 accumulation in the hippocampus. Also, STEP knockout mice outperformed wild-type animals in an age- and test-dependent manner. Summarizing, STEP61 undergoes proteolytic degradation in conditions leading to synaptic strengthening and memory formation, thus highlighting its role as a molecular constrain, which is removed to enable the activation of pathways important for plasticity processes.

Pharmacogenetic modulation of STEP improves motor and cognitive function in a mouse model of Huntington's disease.

Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by an expansion of a CAG repeat in the huntingtin (htt) gene, which results in an aberrant form of the protein (mhtt). This leads to motor and cognitive deficits associated with corticostriatal and hippocampal alterations. The levels of STriatal-Enriched protein tyrosine Phosphatase (STEP), a neural-specific tyrosine phosphatase that opposes the development of synaptic strengthening, are decreased in the striatum of HD patients and also in R6/1 mice, thereby contributing to the resistance to excitotoxicity described in this HD mouse model. Here, we aimed to analyze whether STEP inactivation plays a role in the pathophysiology of HD by investigating its effect on motor and cognitive impairment in the R6/1 mouse model of HD. We found that genetic deletion of STEP delayed the onset of motor dysfunction and prevented the appearance of cognitive deficits in R6/1 mice. This phenotype was accompanied by an increase in pERK1/2 levels, a delay in the decrease of striatal DARPP-32 levels and a reduction in the size of mhtt aggregates, both in the striatum and CA1 hippocampal region. We also found that acute pharmacological inhibition of STEP with TC-2153 improved cognitive function in R6/1 mice. In conclusion, our results show that deletion of STEP has a beneficial effect on motor coordination and cognition in a mouse model of HD suggesting that STEP inhibition could be a good therapeutic strategy in HD patients.