Brain Proteome and Behavioural Analysis in Wild Type, BDNF+/- and BDNF-/- Adult Zebrafish (Danio rerio) Exposed to Two Different Temperatures.

Experimental evidence suggests that environmental stress conditions can alter the expression of BDNF and that the expression of this neurotrophin influences behavioural responses in mammalian models. It has been recently demonstrated that exposure to 34 °C for 21 days alters the brain proteome and behaviour in zebrafish. The aim of this work was to investigate the role of BDNF in the nervous system of adult zebrafish under control and heat treatment conditions. For this purpose, zebrafish from three different genotypes (wild type, heterozygous BDNF+/- and knock out BDNF-/-) were kept for 21 days at 26 °C or 34 °C and then euthanized for brain molecular analyses or subjected to behavioural tests (Y-maze test, novel tank test, light and dark test, social preference test, mirror biting test) for assessing behavioural aspects such as boldness, anxiety, social preference, aggressive behaviour, interest for the novel environment and exploration. qRT-PCR analysis showed the reduction of gene expression of BDNF and its receptors after heat treatment in wild type zebrafish. Moreover, proteomic analysis and behavioural tests showed genotype- and temperature-dependent effects on brain proteome and behavioural responding. Overall, the absent expression of BDNF in KO alters (1) the brain proteome by reducing the expression of proteins involved in synapse functioning and neurotransmitter-mediated transduction; (2) the behaviour, which can be interpreted as bolder and less anxious and (3) the cellular and behavioural response to thermal treatment.

When Nerve Growth Factor Met Behavior.

Since its first characterization in the early 1950s, the role of the polypeptidic nerve growth factor (NGF) in controlling behavior remained elusive. Since the mid-1980s, we undertook a series of experiments aimed at elucidating the biological role(s) played by neurotrophins, particularly NGF, in adult rodents. At the beginning, we concentrated on the submandibular salivary gland of the male mouse, which was known to store massive amount of NGF. We found that under specific stress conditions, the salivary NGF is released in the bloodstream: intermale fighting between isolated males was the first reported context in which salivary NGF was released, thus providing a physiological significance for its presence in the adult, territorial males. We also found that dominant males release less NGF than subordinates and provided a loop-type model which includes intermale social confrontation, adrenal gland size, and functional status, corticosterone release, a model resulting in likelihood to be stabilized in a "dominant" or a "subordinate" social status. A variety of social anxiety contexts of mammals, humans included, has been described since then, and further studies carried out on humans showed that NGF is released in the bloodstream of parachutists at their first skydiving experience and in the case of ranking high on the Passionate Love Scale (amour fou). Ethological data from lab rodents helped in understanding NGF function in subtly controlling social "status" of male mice: the considerations about the interplay among neurobiological, physiological, and behavioral factors in structuring the dominant vs subordinate phenotypes may well apply to other vertebrate species, specifically addressing the underlying role of neurotrophins in relating behavior and brain neuroplasticity.

Own or dam's genotype? Classical colony breeding may bias spontaneous and stress-challenged activity in DAT-mutant rats.

There is considerable interest in understanding what makes an individual vulnerable or resilient to the deleterious effects of stressful events. From candidate genes, dopamine (DA) and dopamine transporter (DAT) have been linked to anxiety, depression, and post-traumatic stress disorder. We investigated role of DAT using the new DAT heterozygous (DAT-HET) and homozygous mutant (DAT-KO) rat models of hyperdopaminergia. We studied the impact of two breeding conditions in spontaneous locomotor behavior of female rats. The classical colony, through mating DAT-HET males × DAT-HET females (breeding HET-HET), was used. A second WT colony was derived and maintained (breeding WT-WT). Additionally, a subgroup of rats was bred through mating DAT-KO males × WT females (atypical HET, breeding KO-WT). We studied the effects of genotype and its interaction with maternal care (depending by breeding condition). HET-HET breeding led to reduced activity in HET females compared to WT rats (from WT-WT breeding). However, HET females from KO-WT breeding did not differ so much from WT rats (WT-WT breeding). The maternal-care impact was then confirmed: HET mothers (breeding HET-HET) showed reduced liking/grooming of pups and increased digging away from nest, compared to WT mothers (breeding WT-WT). In their female offspring (HET, breeding HET-HET vs. WT, breeding WT-WT), isolation plus wet bedding induced higher and more persistent impact on activity of HET rats, even when the stressor was removed. Our results highlight the importance of epigenetic factors (e.g., maternal care) in responses to stress expressed by offspring at adulthood, quite independently of genotype. DAT hypofunction could determinate vulnerability to stressful agents via altered maternal care.

Variability in the "stereotyped" prey capture sequence of male cuttlefish (Sepia officinalis) could relate to personality differences.

Studies of animal personality have shown consistent between-individual variation in behaviour in many social and non-social contexts, but hunting behaviour has been overlooked. Prey capture sequences, especially in invertebrates, are supposed to be quite invariant. In cuttlefish, the attack includes three components: attention, positioning, and seizure. The previous studies indicated some variability in these components and we quantified it under the hypothesis that it could relate to personality differences. We, therefore, analysed predation sequences of adult cuttlefish to test their association with personality traits in different contexts. Nineteen subjects were first exposed to an "alert" and a "threat" test and then given a live prey, for 10 days. Predation sequences were scored for components of the attack, locomotor and postural elements, body patterns, and number of successful tentacle ejections (i.e. seizure). PCA analysis of predatory patterns identified three dimensions accounting for 53.1%, 15.9%, and 9.6% of the variance and discriminating individuals based on "speed in catching prey", "duration of attack behaviour", and "attention to prey". Predation rate, success rate, and hunting time were significantly correlated with the first, second, and third PCA factors, respectively. Significant correlations between capture patterns and responsiveness in the alert and threat tests were found, highlighting a consistency of prey capture patterns with measures of personality in other contexts. Personality may permeate even those behaviour patterns that appear relatively invariant.

A Behavioural Case Study of Early Social Isolation of a Subadult White-Handed Gibbon (Hylobates lar).

Our aim in this study was to analyse the effects of early social isolation on the behaviour of a white-handed gibbon (Hylobates lar) and at the same time to improve his level of welfare. The subject was a 6-year-old male, isolated from conspecific as well as other non-human primates since he was 3 months old. We presented the gibbon with a series of species-specific vocalisations, and we then introduced a 23-year-old conspecific female into his cage. Our subject did not respond to playbacks, whereas he immediately interacted positively with the conspecific female. After 2 days of presentation, the pair started to spend time in proximity to each other and initiated grooming through the wire-mesh dividing the cages. Four days later we recorded vocal duets. No obvious ste-reotypic behaviours were observed, and the prolonged isolation did not seem to compromise the ability of the young gibbon to socialise with the female conspecific. It appears that prolonged isolation does not always compromise the possibility of recovering socially in a satisfactory manner.

Not all stressors are equal: early social enrichment favors resilience to social but not physical stress in male mice.

Early experiences profoundly affect the adult coping response to stress and, consequently, adult vulnerability to psychopathologies triggered by stressing conditions, such as major depression. Though studies in animal models have demonstrated that individuals reared in different conditions are differently vulnerable to a stressor of a specific quality, no information is available as to whether such vulnerability differs when facing stressors of different qualities. To this purpose, we reared C57BL/6 male mice either in standard laboratory rearing condition (SN) or in Communal Nest (CN) condition, the latter consisting of a single nest where three mothers keep their pups together and share care-giving behavior until weaning. We scored the amount of interactions with the mother and with peers and found that CN is a form of social enrichment because both these components are significantly increased. At adulthood, we exposed SN and CN mice, for 4 weeks, to either a physical (forced swim) or a social stress (social instability). Immediately before, at week 1 and at week 4 of the stress procedure, corticosterone levels and the hedonic profile were measured. The results show that CN mice are more resilient to social stress than SN mice since they displayed no anhedonia and lower corticosterone levels. By contrast, both experimental groups were similarly vulnerable to physical stress. Overall, our results show that, in male mice, the adult vulnerability to stress changes according to the quality of the stressor, as a function of early experiences. In addition, the stressor to which CN mice are resilient is qualitatively similar to the stimuli they have experienced early on, both concerning the social domain.

Quality and timing of stressors differentially impact on brain plasticity and neuroendocrine-immune function in mice.

A growing body of evidence suggests that psychological stress is a major risk factor for psychiatric disorders. The basic mechanisms are still under investigation but involve changes in neuroendocrine-immune interactions, ultimately affecting brain plasticity. In this study we characterized central and peripheral effects of different stressors, applied for different time lengths, in adult male C57BL/6J mice. We compared the effects of repeated (7 versus 21 days) restraint stress (RS) and chronic disruption of social hierarchy (SS) on neuroendocrine (corticosterone) and immune function (cytokines and splenic apoptosis) and on a marker of brain plasticity (brain-derived neurotrophic factor, BDNF ). Neuroendocrine activation did not differ between SS and control subjects; by contrast, the RS group showed a strong neuroendocrine response characterized by a specific time-dependent profile. Immune function and hippocampal BDNF levels were inversely related to hypothalamic-pituitary-adrenal axis activation. These data show a fine modulation of the crosstalk between central and peripheral pathways of adaptation and plasticity and suggest that the length of stress exposure is crucial to determine its final outcome on health or disease.

Using videos from social media to study the begging behaviour of peregrine falcon (Falco peregrinus) nestlings.

In 2013, two papers suggested behavioural biologists to use videos available on social media as a tool for investigating animal behaviour, a methodology referred to as video mining. Here, this approach was applied to the study of specific aspects of peregrine falcon (Falco peregrinus) nestlings' behaviour at critical developmental stages. Special attention was given to food begging behaviour and its development. The materials included 254 videos (from 31 nests and 51 different broods) that underwent strict selection procedures to ensure their reliability and quality. Following age estimation of the nestlings, videos were divided into four classes to study age-related differences in begging behaviour. No statistically significant differences emerged among age classes. Video mining may represent a valuable tool for qualitative analyses if wisely and rigorously applied in suitable species and for appropriate research questions. Besides, the video mining approach could also be applied in citizen-science-based studies. Supplementary Information: The online version contains supplementary material available at 10.1007/s12210-022-01129-x.

General and species-specific recommendations for minimal requirements for the use of cephalopods in scientific research.

Here we list species-specific recommendations for housing, care and management of cephalopod molluscs employed for research purposes with the aim of contributing to the standardization of minimum requirements for establishments, care and accommodation of these animals in compliance with the principles stated in Directive 2010/63/EU. Maximizing their psychophysical welfare was our priority. General recommendations on water surface area, water depth and tank shape here reported represent the outcome of the combined action of the analysis of the available literature and an expertise-based consensus reached - under the aegis of the COST Action FA1301 - among researchers working with the most commonly used cephalopod species in Europe. Information on water supply and quality, environmental conditions, stocking density, feeding and handling are also provided. Through this work we wish to set the stage for a more fertile ground of evidence-based approaches on cephalopod laboratory maintenance, thus facilitating standardization and replicability of research outcomes across laboratories, at the same time maximizing the welfare of these animals.

Sustained hippocampal neurogenesis in females is amplified in P66(Shc-/-) mice: An animal model of healthy aging.

Aging is accompanied by poor learning and memory abilities and by decreased hippocampal neurogenesis, a process that is also modulated by oxidative stress (OS). P66(Shc) has recently emerged as a novel mammalian gerontogene able to affect healthspan during aging. Deletion of this gene in mice leads to reduced OS accompanied by decreased incidence of age-related pathologies and reduced signs of behavioral aging. We hypothesized that p66(Shc-/-) mutants might show increased neurogenesis in the hippocampus, a brain region involved in learning and memory processes. To this aim, granule cell number, proliferation, neuronal differentiation, and cell death were assessed in the hippocampus in senescent p66(Shc-/-) [knock out (KO)] and p66(Shc+/+) [wild type (WT)] male and female mice. Spatial learning abilities and spontaneous activity were also investigated in a multifunctional behavioral system-IntelliCages. The behavioral analysis revealed that females in general perform better in spatial learning tasks, with genotype effects being apparent in the activity pattern only. Likewise, all females showed increased neuronal differentiation, whereas increased proliferation was found only in those belonging to the p66(Shc-/-) genotype, indicating that they might be protected from precursor cell loss. The number of dying cells was not affected by genotype or sex; however, all KO mice showed less granule cells than WT. Overall, our data suggest that hippocampal function is protected in the female gender at older age, an effect amplified by reduced OS in the p66(Shc-/-) mutant.

Effects of spatial and cognitive enrichment on activity pattern and learning performance in three strains of mice in the IntelliMaze.

The IntelliMaze allows automated behavioral analysis of group housed laboratory mice while individually assigned protocols can be applied concomitantly for different operant conditioning components. Here we evaluate the effect of additional component availability (enrichment) on behavioral and cognitive performance of mice in the IntelliCage, by focusing on aspects that had previously been found to consistently differ between three strains, in four European laboratories. Enrichment decreased the activity level in the IntelliCages and enhanced spatial learning performance. However, it did not alter strain differences, except for activity during the initial experimental phase. Our results from non-enriched IntelliCages proved consistent between laboratories, but overall laboratory-consistency for data collected using different IntelliCage set-ups, did not hold for activity levels during the initial adaptation phase. Our results suggest that the multiple conditioning in spatially and cognitively enriched environments are feasible without affecting external validity for a specific task, provided animals have adapted to such an IntelliMaze.

NGF, brain and behavioral plasticity.

Nerve Growth Factor (NGF) was initially studied for its role as a key player in the regulation of peripheral innervations. However, the successive finding of its release in the bloodstream of male mice following aggressive encounters and its presence in the central nervous system led to the hypothesis that variations in brain NGF levels, caused by psychosocial stressor, and the related alterations in emotionality, could be functional to the development of proper strategies to cope with the stressor itself and thus to survive. Years later this vision is still relevant, and the body of evidence on the role of NGF has been strengthened and expanded from trophic factor playing a role in brain growth and differentiation to a much more complex messenger, involved in psychoneuroendocrine plasticity.

Developing effective animal-assisted intervention programs involving visiting dogs for institutionalized geriatric patients: a pilot study.

AIM: An ever increasing interest in the therapeutic aspects of the human-animal bond has led to a proliferation of animal-assisted interventions (AAI) involving dogs. However, most of these programs lack a solid methodological structure, and basic evaluative research is needed. The purpose of this study was to test the value of dog-assisted interventions as an innovative tool to increase quality of life in the geriatric population. METHODS: Nineteen patients (men and women) with a mean age of 85 years participated in the study. Interactions between patients and visiting dogs occurred either in a social situation (socialization sessions) or in a therapeutic context (physical therapy sessions). We derived and characterized a specific ethogram of elderly-dog interactions aimed at evaluating the effectiveness of visiting dogs in improving mood, catalyzing social interactions and reducing their everyday apathetic state. Cortisol levels were also measured in the saliva, and depressive state was evaluated. RESULTS: Overall, results show a time-dependent increase in social behaviour and spontaneous interactions with the dogs. Dog-mediated interactions affected the daily increase in cortisol levels, thus having an 'activational effect', in contrast to the apathetic state of institutionalized elderly. CONCLUSIONS: Dog-mediated intervention programs appear to be promising tools to improve the social skills and enrich the daily activities of the institutionalized elderly.

Daily serum and salivary BDNF levels correlate with morning-evening personality type in women and are affected by light therapy.

INTRODUCTION: BDNF is present in human serum and its level changes have been used as a marker of antidepressant efficacy in some psychiatric disorders. In addition, the positive effects of light therapy on major depression suggest that circadian-regulated factors should be taken into account in the management of mood disorders. The aim of the present study was to test ultradian fluctuations in serum and salivary BDNF levels and their interaction with light therapy in a sample of healthy women. METHODS: The study included 16 young women. Psychopathological status and chronotype traits were assessed by SPAQ, BDI, STAI, TAS, and MEQ. Standard light treatment protocol was applied. Serum and saliva were collected at 8.00, 13.00 and 20.00 hrs on the same day and at the end of light therapy. RESULTS: BDNF levels declined over the course of the day both in serum and saliva, and a correlation between diurnal BDNF trend and personality traits and habits characterizing the morning and evening types in healthy women was found. CONCLUSIONS: The present study is one of the first to show measurable BDNF in human saliva and to demonstrate its daily fluctuations in both saliva and serum of healthy young women. The correlation between diurnal changes in BDNF and the personality traits associated with body rhythms corroborates the notion that salivary BDNF may be a useful biomarker for stress-related research and different clinical investigations.

Dopaminergic modulation of the circadian activity and sociability: Dissecting parental inheritance versus maternal styles as determinants of epigenetic influence.

In mammalians, social life and circadian rhythms find their neurobiological basis in a network that includes the dopaminergic system. The malfunctioning of dopamine pathways can lead to various disorders such as Attention-Deficit/Hyperactivity and Obsessive/compulsive disorders. A useful research approach is to exploit animal models that carry a functional silencing of SLC6A3 gene, encoding the dopamine transporter (DAT). Hyperactivity, working memory deficits, and asocial tendencies are core features in truncated-DAT rats, for example. We investigated how inheritance and maternal caring style influence circadian rhythms and social behaviours in DAT heterozygous (HET) rats, belonging to four groups: Mat-P, Mat-M, Mix-P, and Mix-M (Mat label stands for care from wild-type dam, Mix label stands for care by heterozygous dam; M label stands for maternal wild-DAT and P label stands for paternal wild-DAT). In Experiment 1, we monitored 24/7 the spontaneous locomotor activity of peri-adolescent subjects. Hyperactivity occurred only in P-asset subjects (with maternal-origin truncated-DAT allele) at specific bins of the day. In Experiment 2, we observed social interactions of the same rats. Mix-M subjects (raised by HET dams and/or inheriting the wild-DAT allele from mothers) tend to interact with all rats; Mat-P (cared by WT dams and/or inheriting the truncated-DAT allele from mothers) seem to be ignored, when acting as stimulus subjects. Overall, results confirm complex modulations for circadian cycle and social life: flexible DAT expression in HET subjects depends on epigenetic combinations of parental inheritance and early experiential factors. Once confirmed, these data could shed light on trans-generational contributions to dopaminergic-related disorders.

An evolutionary perspective for contemporary psychiatric research.

Behaviour represents the ultimate output of the brain and is characterized by a high level of inter-individual variability. On the one hand, taking into account evolutionary history and adaptive significance of behavioural responses allows to design experimental protocols that improve both data quality and interpretation. On the other hand, a multilevel approach, which analyses factors ranging from the genetic set up to the socioeconomic status, leads to a more comprehensive and effective investigation of mechanisms underlying brain function. Exploitation of this approach in clinical studies may provide new strategies to more precisely investigate psychiatric disorders.

Use of the Milan Pet Quality of Life Instrument (MPQL) to Measure Pets' Quality of Life during COVID-19.

The COVID-19 pandemic caused lifestyle changes, with unknown effect on pets' quality of life (QoL). Between May and July 2020, we distributed an online survey to investigate the role of several factors on feline and canine QoL, including lockdown-related factors. We used existing scales to measure human and pets' personalities (Reinforcement Sensitivity Theory Personality Questionnaire, RST-PQ; RST-Dog; RST-Cat) and the human-animal relationship (Lexington Attachment to Pets Scale, LAPS) and the Milan Pet Quality of Life instrument (MPQL). Overall, 235 participants reported about 242 adult pets (Ncats = 78, Ndogs = 164). Factor analysis confirmed the structure and internal reliability of the existing scales (RST-PQ, RST-Dog, RST-Cat, LAPS) and suggested a four-factor structure for the MPQL (physical, psychological, social, environmental). The results indicate that the pets' psysical QoL was largely explained by pet-related elements (pets' demographics and life experience, and pets' personality). Conversely, the pets' psychological QoL was explained mostly by owner-related elements, such as the owners' demographics, COVID-19-related changes, and the owners' personality. Predictably, the pets' environmental QoL is mostly explained by environmental factors, such as the outdoor access in the home environment and the country. Finally, the pets' social QoL was explained by the larger combination of models: pets' characteristics and personality, environment and COVID-19-related changes, and the pet-human relationship. These findings can be explained by two non-mutually exclusive mechanisms. The reported changes may be a by-product of the COVID-19 pandemic's psychological and lifestyle effects on the owners, which in turn alter the way the owners interact with their pets and look after them. However, the owners' characteristics and mood may bias their answers regarding their pets.

Altering the development of the dopaminergic system through social play in rats: Implications for anxiety, depression, hyperactivity, and compulsivity.

Dopamine is essential to many functions like reward, motivation, and attention; when its neural pathways do not function properly, various disorders (e.g., anxiety, depression, hyperactivity, compulsions) can arise. Truncated-DAT rats display persistent stereotypies and aggressiveness; hence they are a new valuable animal model to study the pathogenesis of these disorders. The focus of research is often on the individual epigenetic determinants and much less on the impact of social experiences. Here, we investigate the developmental impact of the social environment on adolescent wild type (WT) rats. We divided subjects at weaning into three groups: living with another adolescent (WT Peer), with a WT adult, or with a truncated-DAT one, and we observed homecage social behavior of these pairs (play, jump, victory, and "bullying") during whole adolescence. When adult, we observed the same subjects in plus maze, forced swim, and social preference tests to measure levels of anxiety, depression, and quality of social interactions. Compared to the other groups, WT rats that had spent their adolescence with a truncated-DAT adult as companion show more anxious, depressive, hyperactive, impulsive, and compulsive behaviours. Results confirm that social interactions and healthy play (i.e., when play has behavioural, social, and psychomotor rewards that support the cognitive, emotional and physical development of the individual) are essential to neurobehavioral maturation. Conversely, anomalous interactions like poor play and "bullying" in developing rats may impact onto their dopaminergic system. Consequently, an impoverished social play could be one of the factors contributing to the appearance of putative indexes of attention deficit hyperactivity disorder (ADHD) and\or obsessive-compulsive disorder (OCD).

A new "sudden fright paradigm" to explore the role of (epi)genetic modulations of the DAT gene in fear-induced avoidance behavior.

Alterations in dopamine (DA) reuptake are involved in several psychiatric disorders whose symptoms can be investigated in knock out rats for the DA transporter (DAT-KO). Recent studies evidenced the role of epigenetic DAT modulation in depressive-like behavior. Accordingly, we used heterozygous (HET) rats born from both HET parents (termed MIX-HET), compared to HET rats born from WT-mother and KO-father (MAT-HET), implementing the role of maternal care on DAT modulation. We developed a "sudden fright" paradigm (based on dark-light test) to study reaction to fearful inputs in the DAT-KO, MAT-HET, MIX-HET, and WT groups. Rats could freely explore the whole 3-chambers apparatus; then, they were gently confined in one room where they experienced the fright; finally, they could freely move again. As expected, after the fearful stimulus only MAT-HET rats showed a different behavior consisting of avoidance towards the fear-associated chamber, compared to WT rats. Furthermore, ex-vivo immuno-fluorescence reveals higher prefrontal DAT levels in MAT-HET compared to MIX-HET and WT rats. Immuno-fluorescence shows also a different histone deacetylase (HDAC) enzymes concentration. Since HDAC concentration could modulate gene expression, within MAT-HET fore brain, the enhanced expression of DAT could well impair the corticostriatal-thalamic circuit, thus causing aberrant avoidance behavior (observed only in MAT-HET rats). DAT expression seems to be linked to a simply different breeding condition, which points to a reduced care by HET dams for epigenetic regulation. This could imply significant prefronto-cortical influences onto the emotional processes: hence an excessively frightful response, even to mild stressful agents, may draw developmental trajectories toward anxious and depressed-like behavior.

Effects of low-dose methylcyclopentadienyl manganese tricarbonyl-derived manganese on the development of diencephalic dopaminergic neurons in zebrafish.

Fuel additive methylcyclopentadienyl manganese tricarbonyl (MMT) is counted as an organic manganese (Mn)-derived compound. The toxic effects of Mn (alone and complexed) on dopaminergic (DA) neurotransmission have been investigated in both cellular and animal models. However, the impact of environmentally relevant Mn exposure on DA neurodevelopment is rather poorly understood. In the present study, the MMT dose of 100 µM (about 5 mg Mn/L) caused up-regulation of DA-related genes in association with cell body swelling and increase in the number of DA neurons of the ventral diencephalon subpopulation DC2. Furthermore, our analysis identified significant brain Mn bioaccumulation and enhancement of total dopamine levels in association with locomotor hyperactivity. Although DA levels were restored at adulthood, we observed a deficit in the acquisition and consolidation of memory. Collectively, these findings suggest that developmental exposure to low-level MMT-derived Mn is responsible for the selective alteration of diencephalic DA neurons and with long-lasting effects on fish explorative behaviour in adulthood.