Francesca Zoratto

Early-stress regulates resilience, vulnerability and experimental validity in laboratory rodents through mother–offspring hormonal transfer

The role of early-life stressors in the calibration of individual responses to future challenges has long been investigated in laboratory rodents. Specifically, countless studies show that exposure to early-life stressors - in the form of various periods of maternal separation, administration of exogenous corticosterone and variable feeding conditions - modulate the regulation of defensive responses (e.g. behavioral fearfulness/anxiety and endocrine stress reactivity) in adulthood. Yet, the link between early-life stress and adult defensive responses is not linear. Specifically, while neonatal moderate stress is generally associated with adult subjects characterized by reduced stress reactivity, neonatal elevated stress is often reported to relate to opposite responses. Not only are these findings relevant to the understanding of individual plasticity to contextual features, but also they can have direct implications in the development of rodent models of human disorders. Specifically, these studies demonstrate that the experimental individual responds to early environmental cues with the consequence of adjusting its adaptation to the future environment. If neglected, this aspect may have detrimental consequences in laboratory animal experimentation. For example, neonatal conditions increasing adult responses to moderate stress may result in experimental subjects showing abnormal hypothalamic-pituitary-adrenocortical (HPA) activation to routine husbandry conditions, test environment and general laboratory procedures. The aim of the present review is threefold: (i) propose that neonatal circulating levels of corticosteroids may constitute a potential mediator connecting early and adult defensive systems; (ii) propose that the link between early and adult stress follows a U-shaped curve, with low levels down-regulating individual reactivity to external stressors and high levels exerting opposite effects; (iii) discuss the methodological implications of these considerations in the development of rodent models of human disorders.

Aerial flocking patterns of wintering starlings, Sturnus vulgaris, under different predation risk

To test the hypothesis that variation in aerial flocking behaviour is adaptively related to predation risk, we described and quantified the flocking patterns of starlings, approaching two urban roosts, which differed in predation pressure (by peregrine falcons, Falco peregrinus). We predicted that the higher predation pressure in one of the roosts would be reflected in larger and more compact flocks, thought to be less vulnerable to predation than small flocks. Incoming flocks, not under direct attack, were observed during winter for 53 days. We identified 12 flocking shapes. Significantly higher frequencies of compact and large flocks were observed in the roost with high predation pressure, while small flocks and singletons were more frequent at the roost with low predation pressure. Similar patterns were observed in both roosts when other flocks displayed antipredator behaviour, even when far away and in the absence of the predator at the focal roost. This may indicate that social information passed between flocks affects flocking decisions. Predation success was higher at the roost with low predation. These results suggest that aerial flocking patterns are affected by predation risk and possibly by the behaviour of other flocks in response to direct attacks.

Propagating waves in starling, Sturnus vulgaris, flocks under predation

The formation of waves is a vivid example of collective behaviour occurring in insects, birds, fish and mammals, which has been interpreted as an antipredator response. In birds a quantitative characterization of this phenomenon, involving thousands of individuals, is missing and its link with predation remains elusive. We studied waves in flocks of starlings, a highly gregarious species, by both direct observation and quantitative computer vision analysis of HD video recordings, under predation by peregrine falcons, Falco peregrinus. We found that waves originated from the position of the attacking predator and always propagated away from it. We measured their frequency and velocities, the latter often being larger than the velocity of the flock. A high positive correlation was found between the formation of waves and reduced predation success. We suggest that the tendency of a prey to escape, when initiated even by a few individuals in a cohesive group, elicits self-organized density waves. Such evident fluctuations in the local structure of the flocks are efficient in confusing predators.

Neonatal tryptophan depletion and corticosterone supplementation modify emotional responses in adult male mice.

The serotonergic system and the hypothalamic-pituitary-adrenal (HPA) axis are crucially involved in the regulation of emotions. Specifically, spontaneous and/or environmentally mediated modulations of the functionality of these systems early in development may favour the onset of depressive- and anxiety-related phenotypes. While the independent contribution of each of these systems to the emergence of abnormal phenotypes has been detailed in clinical and experimental studies, only rarely has their interaction been systematically investigated. Here, we addressed the effects of reduced serotonin and environmental stress during the early stages of postnatal life on emotional regulations in mice. To this aim, we administered, to outbred CD1 mouse dams, during their first week of lactation, a tryptophan deficient diet (T) and corticosterone via drinking water (C; 80μg/ml). Four groups of dams (animal facility rearing, AFR; T treated, T; C treated, C; T and C treated, TC) and their male offspring were used in the study. Maternal care was scored throughout treatment and adult offspring were tested for: anhedonia (progressive ratio schedule); anxiety-related behaviour (approach-avoidance conflict paradigm); BDNF, dopamine and serotonin concentrations in selected brain areas. T, C and TC treatments reduced active maternal care compared to AFR. Adult TC offspring showed significantly increased anxiety- and anhedonia-related behaviours, reduced striatal and increased hypothalamic BDNF and reduced dopamine and serotonin in the prefrontal cortex and their turnover in the hippocampus. Thus, present findings support the view that neonatal variations in the functionality of the serotonergic system and of HPA axis may jointly contribute to induce emotional disturbances in adulthood.

Effects of maternal L-tryptophan depletion and corticosterone administration on neurobehavioral adjustments in mouse dams and their adolescent and adult daughters.

Major depressive disorder (MDD), a pathology characterized by mood and neurovegetative disturbances, depends on a multi-factorial contribution of individual predisposition (e.g., diminished serotonergic transmission) and environmental factors (e.g., neonatal abuse or neglect). Despite its female-biased prevalence, MDD basic research has mainly focused on male rodents. Most of present models of depression are also devalued due to the fact that they typically address only one of the aforementioned pathogenetic factors. In this paper we first describe the basic principles behind mouse model development and evaluation and then articulate that current models of depression are intrinsically devalued due to poor construct and/or external validity. We then report a first attempt to overcome this limitation through the design of a mouse model in which the genetic and the environmental components of early risk factors for depression are mimicked together. Environmental stress is mimicked through the supplementation of corticosterone in the maternal drinking water while biological predisposition is mimicked through maternal access to an L-tryptophan (the serotonin precursor) deficient diet during the first week of lactation. CD1 dams and their offspring exposed to the L-tryptophan deficient diet (T) and to corticosterone (80mg/l; C) were compared to animal facility reared (AFR) subjects. T and C mice served as intermediate reference groups. Adolescent TC offspring, compared to AFR mice, showed decreased time spent floating in the forced-swim test and increased time spent in the open sectors of an elevated 0-maze. Adult TC offspring showed reduced preference for novelty, decreased breakpoints in the progressive ratio operant procedure and major alterations in central BDNF levels and altered HPA regulation. The route of administration and the possibility to control the independent variables predisposing to depressive-like symptoms disclose novel avenues towards the development of animal models with increased external and construct validity. Furthermore, the observation that, compared to adult subjects, adolescent mice display an opposite profile suggests that peri-pubertal developmental processes may interact with neonatal predispositions to calibrate the adult abnormal phenotype.

Behavioural response of European starlings exposed to video playback of conspecific flocks: Effect of social context and predator threat

We studied the behavioural response of European starlings to a socially mediated predation event. Adult starlings were exposed to either a video of a flock attacked by a peregrine falcon or a video of a flock not under attack. We investigated whether the social condition affected the anti-predator response under the hypothesis that in such a gregarious species singletons would increase their anti-predator behaviour more than individuals in groups, to compensate for potential increased risk. The video of the flock under attack caused an increase in immobility and vigilance, more marked in singletons, both during and after the exposure. The video of the non-threatened flock caused an increase in activity levels, especially during the exposure. Furthermore, we observed a marked increase in comfort activities in singletons as well as in social interactions and vocalizations in mini-flocks. Only birds in mini-flocks vocalized, which may be explained by an audience effect, a process of social cognition mediated by the social context, and not only by the stimulus. The results are in line with previous field studies, which showed that isolated starlings are exposed to a higher risk of predation compared to individuals in flocks.

Pronounced Hyperactivity, Cognitive Dysfunctions, and BDNF Dysregulation in Dopamine Transporter Knock-out Rats

Dopamine (DA) controls many vital physiological functions and is critically involved in several neuropsychiatric disorders such as schizophrenia and attention deficit hyperactivity disorder. The major function of the plasma membrane dopamine transporter (DAT) is the rapid uptake of released DA into presynaptic nerve terminals leading to control of both the extracellular levels of DA and the intracellular stores of DA. Here, we present a newly developed strain of rats in which the gene encoding DAT knockout Rats (DAT-KO) has been disrupted by using zinc finger nuclease technology. Male and female DAT-KO rats develop normally but weigh less than heterozygote and wild-type rats and demonstrate pronounced spontaneous locomotor hyperactivity. While striatal extracellular DA lifetime and concentrations are significantly increased, the total tissue content of DA is markedly decreased demonstrating the key role of DAT in the control of DA neurotransmission. Hyperactivity of DAT-KO rats can be counteracted by amphetamine, methylphenidate, the partial Trace Amine-Associated Receptor 1 (TAAR1) agonist RO5203648 ((S)-4-(3,4-Dichloro-phenyl)-4,5-dihydro-oxazol-2-ylamine) and haloperidol. DAT-KO rats also demonstrate a deficit in working memory and sensorimotor gating tests, less propensity to develop obsessive behaviors and show strong dysregulation in frontostriatal BDNF function. DAT-KO rats could provide a novel translational model for human diseases involving aberrant DA function and/or mutations affecting DAT or related regulatory mechanisms. SIGNIFICANCE STATEMENT Here, we present a newly developed strain of rats in which the gene encoding the dopamine transporter (DAT) has been disrupted (Dopamine Transporter Knockout rats [DAT-KO rats]). DAT-KO rats display functional hyperdopaminergia accompanied by pronounced spontaneous locomotor hyperactivity. Hyperactivity of DAT-KO rats can be counteracted by amphetamine, methylphenidate, and a few other compounds exerting inhibitory action on dopamine-dependent hyperactivity. DAT-KO rats also demonstrate cognitive deficits in working memory and sensorimotor gating tests, less propensity to develop compulsive behaviors, and strong dysregulation in frontostriatal BDNF function. These observations highlight the key role of DAT in the control of brain dopaminergic transmission. DAT-KO rats could provide a novel translational model for human diseases involving aberrant dopamine functions.

Polymorphism of the 3'-UTR of the dopamine transporter gene (DAT) in New World monkeys.

Genetic polymorphism in the 3′-untranslated region (3′-UTR) of the dopamine transporter (DAT) gene has been reported in both human and nonhuman primates, and the variable number of tandem repeats (VNTR) polymorphism has been related to several neurological and psychiatric disorders. As New World primates have been employed as models in biomedical research in these fields, in the present study we assessed genetic variation in the DAT gene in 25 robust capuchin monkeys (Sapajus spp.) and 39 common marmosets (Callithrix jacchus). Using enzymatic amplification followed by sequencing of amplified fragments, a VNTR polymorphism in the 3′-UTR region of the DAT gene was identified in both robust capuchins and common marmosets. The polymorphic tandem repeat of 40-bp basic units is similar to the human VNTR consensus sequence, with size variants composed of 9, 10, and 11 units in marmosets and 8, 9, 13, and 17 basic units in capuchins. We found behavioral evidence that carrying the 10-repeat DAT allele promotes flexible choice and maximization of foraging in marmosets tested in an operant choice paradigm. Moreover, in an intertemporal choice task, capuchins with longer repeat variants show less self-controlled choices than capuchins with at least one short repeat variant. Future research should focus on the relationship between these DAT polymorphisms, dopamine reuptake via the dopamine transporter, and behavioral and cognitive variation across New World monkey individuals.

Stimulation of 5-HT7 receptor during adolescence determines its persistent upregulation in adult rat forebrain areas.

Brain serotonin 7 (5‐HT7) receptors play an important functional role in learning and memory, in regulation of mood and motivation, and for circadian rhythms. Recently, we have studied the modulatory effects of a developmental exposure (under subchronic regimen) in rats with LP‐211, a brain‐penetrant and selective 5‐HT7 receptor agonist. We aimed at further deciphering long‐term sequelae into adulthood. LP‐211 (0.250 mg/kg i.p., once/day) was administered for 5 days during the adolescent phase (postnatal days 43–45 to 47–49). When adult (postnatal days >70), forebrain areas were obtained for ex vivo immunohistochemistry, whose results prompted us to reconsider the brain connectivity maps presented in our previous study (Canese et al., Psycho‐Pharmacol 2015;232:75–89.) Significant elevation in levels of 5‐HT7 receptors were evidenced due to adolescent LP‐211 exposure, in dorsal striatum (which also shows an increase of dopaminergic D2 auto‐receptors) and—unexpectedly—in piriform cortex, with no changes in ventral striatum. We observed that functional connectivity from a seed on the right hippocampus was more extended than reported, also including the piriform cortex. As a whole, the cortical loop rearranged by adolescent LP‐211 exposure consisted in a hippocampus receiving connections from piriform cortex and dorsal striatum, the latter both directly and through functional control over the ‘extended amygdala’. Such results represent a starting point to explore neurophysiology of 5‐HT7 receptors. Further investigation is warranted to develop therapies for sleep disorders, for impaired emotional and motivational regulation, for attentive and executive deficit. The 5‐HT7 agonist LP‐211 (0.250 mg/kg i.p., once/day) was administered for 5 days during adolescence (postnatal days 43–45 to 47–49) in rats. When adult (postnatal days >70), a significant elevation in levels of 5‐HT7 receptors were evidenced in dorsal striatum and—unexpectedly—in piriform cortex. Synapse 69:533–542, 2015.

Low empathy-like behaviour in male mice associates with impaired sociability, emotional memory, physiological stress reactivity and variations in neurobiological regulations

Deficits in empathy have been proposed to constitute a hallmark of several psychiatric disturbances like conduct disorder, antisocial and narcissistic personality disorders. Limited sensitivity to punishment, shallow or deficient affect and reduced physiological reactivity to environmental stressors have been often reported to co-occur with limited empathy and contribute to the onset of antisocial phenotypes. Empathy in its simplest form (i.e. emotional contagion) is addressed in preclinical models through the evaluation of the social transmission of emotional states: mice exposed to a painful stimulus display a higher response if in the presence of a familiar individual experiencing a higher degree of discomfort, than in isolation. In the present study, we investigated whether a reduction of emotional contagion can be considered a predictor of reduced sociality, sensitivity to punishment and physiological stress reactivity. To this aim, we first evaluated emotional contagion in a group of Balb/cJ mice and then discretised their values in four quartiles. The upper (i.e. Emotional Contagion Prone, ECP) and the lower (i.e. Emotional Contagion Resistant, ECR) quartiles constituted the experimental groups. Our results indicate that mice in the lower quartile are characterized by reduced sociability, impaired memory of negative events and dampened hypothalamic-pituitary-adrenocortical reactivity to external stressors. Furthermore, in the absence of changes in oxytocin receptor density, we show that these mice exhibit elevated concentrations of oxytocin and vasopressin and reduced density of BDNF receptors in behaviourally-relevant brain areas. Thus, not only do present results translate to the preclinical investigation of psychiatric disturbances, but also they can contribute to the study of emotional contagion in terms of its adaptive significance.