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.

Changes in stress hormones and metabolism during a 105-day simulated Mars mission.

BACKGROUND: The Mars-105 project was aimed at simulating crew's activities, workload, and communication during a mission to Mars, evaluating the homeostatic adaptations to prolonged confinement and cohabitation. METHODS: Fasting plasma glucose (FPG) and insulin, C-peptide, leptin, cortisol, and NGF and BDNF plasma levels were monitored in six healthy nonsmoking male subjects taking part in a 105-d Mars mission simulation. Samples were collected from each subject before (0 wk), during (2.5 wk; 5 wk; 10 wk; 15 wk), and after confinement (+1 wk). RESULTS: Confinement resulted in impaired glucometabolic parameters, since FPG increased during the first 5 wk (baseline: 85.2 ± 10.8 mg · dl⁻¹; 2.5 wk: 98.4 ± 4.7 mg · dl⁻¹; 5 wk: 92.5 ± 6.0 mg · dl⁻¹) and insulin dropped at 2.5 wk (baseline: 14.4 ± 4.8 mU · L⁻¹; 2.5 wk: 7.7 ± 2.1 mU · L⁻¹), subsequently returning to baseline values. HOMA-IR paralleled plasma insulin, dropping to 1.8 ± 0.5 at 2.5 wk (baseline: 3.0 ± 1.2). At all time-points tested, plasma leptin levels were decreased (baseline: 4.4 ± 3.3 ng · dl⁻¹; 2.5 wk: 1.6 ± 1.2 ng · dl⁻¹; 5 wk: 1.3 ± 0.8 ng · dl⁻¹; 10 wk: 1.5 ± 1.1 ng · dl⁻¹; 15 wk:1.7 ± 0.8 ng · dl⁻¹), whereas cortisol levels were increased (baseline: 10.8 ± 4.9 ng · dl⁻¹; 2.5 wk: 16.8 ± 3.5 ng · dl⁻¹; 5 wk: 18.1 ± 7.6 ng · dl⁻¹; 10 wk: 18.1 ± 8.3 ng · dl⁻¹; 15 wk:14.2 ± 4.4 ng · dl⁻¹), resulting in a negative correlation between these hormones. BDNF levels increased only at 5 and 10 wk (baseline: 67.1 ± 36.0 pg · ml⁻¹; 5 wk: 164 ± 54 pg · ml⁻¹; and 10 wk: 110.2 ± 28.9 pg · ml⁻¹). DISCUSSION: The data obtained with the Mars-105 experiment suggest that environmental stress has a strong impact upon metabolic and stress response, indicating the need for further studies and the implementation of specific countermeasures.

How to obtain an integrated picture of the molecular networks involved in adaptation to microgravity in different biological systems?

Periodically, the European Space Agency (ESA) updates scientific roadmaps in consultation with the scientific community. The ESA SciSpacE Science Community White Paper (SSCWP) 9, "Biology in Space and Analogue Environments", focusses in 5 main topic areas, aiming to address key community-identified knowledge gaps in Space Biology. Here we present one of the identified topic areas, which is also an unanswered question of life science research in Space: "How to Obtain an Integrated Picture of the Molecular Networks Involved in Adaptation to Microgravity in Different Biological Systems?" The manuscript reports the main gaps of knowledge which have been identified by the community in the above topic area as well as the approach the community indicates to address the gaps not yet bridged. Moreover, the relevance that these research activities might have for the space exploration programs and also for application in industrial and technological fields on Earth is briefly discussed.

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.

Circulating and Salivary NGF and BDNF Levels in SARS-CoV-2 Infection: Potential Predictor Biomarkers of COVID-19 Disease-Preliminary Data.

COVID-19 continues to afflict the global population, causing several pathological diseases and exacerbating co-morbidities due to SARS-CoV-2's high mutation. Recent interest has been devoted to some neuronal manifestations and to increased levels of Nerve Growth Factor (NGF) and Brain-derived Neurotrophic Factor (BDNF) in the bloodstream during SARS-CoV-2 infection, neurotrophins that are well-known for their multifactorial actions on neuro-immune-endocrine and visual functions. Nineteen (19) patients were enrolled in this monocentric prospective study and subjected to anamnesis and biosamples collection (saliva and blood) at hospitalization (acute phase) and 6 months later (remission phase). NGF and BDNF were quantified by ELISA, and biochemical data were related to biostrumental measurements. Increased NGF and BDNF levels were quantified in saliva and serum during the acute phase of SARS-CoV-2 infection (hospitalized patients), and reduced levels were observed in the next 6 months (remission phase), never matching the baseline values. Salivary and circulating data would suggest the possibility of considering sera and saliva as useful matrices for quickly screening neurotrophins, in addition to SARS-CoV2 antigens and RNA. Overall, the findings described herein highlight the importance of NGF and BDNF as dynamic biomarkers for monitoring disease and reinforces the possibility of using saliva and sera for quick, non-invasive COVID-19 screening.

Relaxation Response in Stressed Volunteers: Psychometric Tests and Neurotrophin Changes in Biological Fluids.

Background: To evaluate the beneficial effects of relaxation response (RR) training in adult stressed subjects by evaluating the psychometric response recorded at relaxation session. Cortisol as well as nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) mediators were quantified in both saliva and tears, and their levels were related to each other and to the psychometric response. Methods: Stressed subjects (n = 23; 10M/13F; age range 21-53 years old) were voluntarily enrolled in the study. RR training sessions were carried out for 2 months, 1 day per week, at the same time (3-5 p.m.). Two different psychological questionnaires, the Perceived Stress Scale-10 (PSS-10) and the Beck Depression Inventory - Short Form (BDI-SF) and Ocular Surface Disease Index (OSDI) tests, were administered before each session. Saliva and tears were sampled for cortisol (EIA), NGF (ELISA), and BDNF (ELISA) quantifications. Questionnaires' data were analyzed and compared to biochemical ones. Results: All subjects reported beneficial effects from training. RR significantly reduced the psychological stress indexes (p = 0.039 for PSS-10 and p = 0.001 for BDI-SF). Specifically, RR training lowered the perception of Perceived Helplessness (items 1, 3, 10; p < 0.05) in PSS-10 and increased the Perceived Self-Efficacy (p < 0.05). OSDI score was in the normal range (0-25). Biochemically, a decrease in cortisol, a trend to a decrease in NGF, and an increase in BDNF levels were observed in saliva samples after RR treatment. Furthermore, a trend to a decrease in NGF and an increase in BDNF were quantified in tear samples. A correlation between PSS-10 total score and saliva NGF variation (%) as well as between BDI-SF total score and BDNF tear levels were also observed. Conclusion: RR training appeared useful to lowering psychological, mental, and physical stress, as supported by both psychological total and single scores. The finding on biochemical levels of BDNF in saliva and tears are sustained by previous studies while those of NGF require further investigation. Overall, these data on a small population highlight the potential use of RR training and potential neurotrophic changes in biological fluids, in stressed volunteers.

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.

Birth spacing in the mouse communal nest shapes adult emotional and social behavior.

The interactions with the mother and with peers are among the most relevant early environmental factors shaping adult brain function and behavior. In order to investigate the role of these factors, we exploited a novel early manipulation, the Communal Nest (CN), consisting in a single nest where three mothers give birth, keep their pups and share care-giving behavior from birth to weaning. In particular, we reared CD-1 swiss mice in three different CN conditions, each one characterized by a different interval between the three deliveries (Birth Spacing) of 3, 5 or 7 days (respectively, CN+/-3, CN+/-5, CN+/-7). Length of birth spacing affected maternal behavior, CN+/-7 pups receiving the highest levels. At adulthood, mice reared in the different conditions showed differences in emotional response and social skills. In the plus maze test, short birth spacing was found to be associated with enhanced emotionality, CN+/-3 mice showing highest levels of anxiety-like responses in the plus maze compared to the other two CN groups. In the social interaction test, the strategies to achieve dominance differed among the three groups. While CN+/-3 mice appeared to have a more aggressive strategy, displaying high levels of attack behavior in the first encounter, CN+/-5 and CN+/-7 mice displayed a more affiliative strategy based on social investigation. Overall, these findings show that birth spacing shapes the early mouse social environment and, in turn, affects the development of social skills and emotional responses.

Review: Environmental impact on ocular surface disorders: Possible epigenetic mechanism modulation and potential biomarkers.

Throughout life, external and the internal environments interact in normal development and tissue homeostasis as well as in onset of disease. Epigenetic modifications occur in response to environmental changes and play a fundamental role in controlling gene expression without modification of the DNA base sequence. Aging, inflammation, drugs, infections and ultraviolet exposure may have profound effects on epigenetic modifications and trigger susceptibility to diseases. Increasing evidence has demonstrated that epigenetic mechanisms play a key role in regulating the physiopathology of the ocular surface. The evaluation of epigenetic factors in ocular disease would lead to further investigation regarding the potential use of therapeutic targets and diagnostic biomarkers. This review examines specific epigenetic and biochemical mechanisms that may occur in the ocular surface microenvironment. Potential correlation between epigenetic factors and targets should be considered in future approaches to prevention and/or treatment of specific ocular surface disorders.

Prolonged prenatal exposure to low-level ozone affects aggressive behaviour as well as NGF and BDNF levels in the central nervous system of CD-1 mice.

The long-term effects on isolation-induced aggressive behaviour and central NGF and BDNF levels of gestational exposures to ozone (O(3)) were evaluated in adult CD-1 mice. Females were exposed to O(3), at the dose of 0.0, 0.3 or 0.6 ppm from 30 days prior the formation of breeding pairs until gestational day 17. Litters were fostered at birth to untreated dams and, at adulthood, male offspring underwent five successive daily encounters (15 min each) with a standard opponent of the same strain, sex, weight and age. The encounters on day 1, 3 and 5 were videotaped and agonistic and non-agonistic behavioural items finely scored. O(3)-exposed mice showed a significant increase in freezing and defensive postures, a decrease in nose-sniffing behaviour and reduced progressively the aggressive behavioural profile displayed on day 1. Reduced NGF levels in the hippocampus and increased BDNF in the striatum were also found upon O(3) exposure.

Repeated acute exposures to hypergravity during early development subtly affect CD-1 mouse neurobehavioural profile.

Exposure to altered gravitational environment, especially during critical ontogenetic phases, may induce persistent nervous system modifications and behavioural anomalies. This study evaluated the effects of hypergravity exposure on the development of the nervous system and assessed the relevance of parity in the mother's responses to altered gravitational stimuli. CD-1 mouse pups of both sexes delivered by primiparous and biparous dams were exposed to 1h of 2 G rotationally induced hypergravity from PND2 to PND9. Sensorimotor responses and somatic growth were daily measured (PND2-PND15), ultrasonic vocalisations recorded on PNDs 2, 5 and 9, and homing behaviour evaluated on PND12. In addition, spatial orientation ability was assessed in a T-maze on PND18, while mice exploratory behaviour and locomotor activity were evaluated in an open-field test (PND21). Long-term effects of hypergravity exposure on both spatial learning (Morris water-maze test) and brain levels of NGF and BDNF were also investigated at adulthood. Rotation per se induced a delay in somatic growth, sensorimotor responses and ultrasonic vocalisation profile, while hypergravity highlighted sex differences in open-field behaviour. Strategies to solve a spatial learning task, rather than learning per se, were affected by early exposure to rotation, while hypergravity selectively altered behavioural profile in the reversal phase of the test. Early exposure to rotation per se also decreased hypothalamic BDNF levels, while hypergravity reduced NGF levels in the frontal cortex. Previous maternal experience did not interact with hypergravity exposure, while differences between offspring of primiparous and biparous dams were observed in sensorimotor development and exploratory behaviour.

Early developmental exposure to BDE 99 or Aroclor 1254 affects neurobehavioural profile: interference from the administration route.

Among the most persistent and bio-accumulative environmental pollutants are the polybrominated diphenyl ethers (PBDEs), a class of chemicals widely used as flame retardants in plastics and textile coating, and the polychlorinated biphenyls (PCBs), previously used as coolants and lubricants in electrical equipment. Monitoring programs revealed high levels of both these classes of compounds in human breast milk, raising concerns for their potential noxious effects on infants. The aim of the present study was to investigate the neurotoxic effects of 2,2',4,4',5-penta BDE (BDE 99: 18mg/kg/day) or Aroclor 1254 (A1254, a PCB mixture: 10mg/kg/day) administration, from gestational day (GD) 6 to postnatal day (PND) 21, on neurobehavioral development in the CD-1 Swiss mouse. In addition, we investigated whether the administration route affects the emergence or the magnitude of the toxic effects of BDE 99 or A1254. In particular, we compared self-administration, consisting in letting the mouse drink spontaneously the compound dissolved in oil from a syringe, with gavage, consisting in force-feeding a substance by a tube inserted in the mouth and then into the stomach, a procedure reported to be stress-inducing. Both compounds induced hyperactivity, though BDE 99 affected activity profile only during adolescence and A1254 mainly at adulthood. Levels of total circulating thyroxine were decreased by both BDE 99 and A1254 administration, though only in the latter group the decrease was statistically significant. These findings suggest a different neurotoxic action exerted by PBDEs and PCBs. An effect of the administration route, independent from the compound administered, was found on thigmotactic behavior and gavage administration affected pup body weight gain only in the A1254 group, suggesting that the stress induced by gavage procedure may either affect results per se or modulate the detrimental action of selected compounds.

Effects of acute hypergravity exposure and parity on maternal behavior in CD-1 mice.

We assessed the behavioral response to acute hypergravity exposure in lactating mother mice, Mus musculus of the outbred CD-1 strain. Primiparous or terziparous dams were exposed with their litters to a centrifugal force equivalent to 2G hypergravity for 1 h daily from postnatal day 2 (P2) to P9. We made detailed behavioral observations before, during and after the rotation on selected days to identify elements of the maternal behavioral repertoire vulnerable to 2G challenge. Licking and nest building were reduced during rotation while mothers sniffed and snouted their pups more. Nursing and total time in physical contact with pups were relatively stable. The effects of rotation were most pronounced on P2, dams appearing to habituate to the treatment with repeated exposure. Dam parity had a limited effect on the behavioral response to rotation, primiparous mothers tending to spend longer nursing their pups during the rotation and showing a greater tendency to lick and nest-build in post-rotation. Differences between parity groups diminished over days. Body weight was decreased in rotated primiparous dams and their pups gained less weight than stationary controls. Ultrasonic vocalization (USV) rates recorded on P2, P5 and P9 seemed to indicate delayed behavioral development in rotated pups.

The oral health of institutionalized older adults in Malta.

PURPOSE: Studies on the oral health status of institutionalized older adults are less prevalent than those of community-dwelling older adults, as institutionalized older adults tend to be frailer. Poor oral health in older adults has a negative impact on the quality of life and self-confidence of older people and potentially poses a financial burden on both the older adult and society in general. The objective of this study was to assess and compare the oral health status of state institutionalized older adults in Malta with that of their European counterparts. MATERIALS AND METHODS: A total of 278 older adults with an average age of 83.6 ± 6.5 years from nine state institutions in Malta were randomly selected. Participants were clinically examined for caries, periodontal disease, oral mucosal lesions, and prosthetic status. RESULTS: The oral health status of state institutionalized older adults was poor, with a very low routine dental attendance (29.3%) and, consequently, a high level of treatment need (44.4% of dentate individuals required extractions and 42.1% of dentate individuals required restorations). Only 2% of dentate individuals had healthy periodontal tissues with no clinical attachment loss over 4 mm. Edentulism among state institutionalized older adults stood at 41%. CONCLUSIONS: Institutionalized older adults from Malta have a poor oral health status comparable to institutionalized older adults from Europe in general, which poses fiscal and cultural challenges that need to be addressed by the dental community.

The Oral Health-Related Quality of Life in State Institutionalized Older Adults in Malta.

PURPOSE: The objective of this study was to determine the oral health-related quality of life in state institutionalized older adults and correlate it to their oral health status. MATERIALS AND METHODS: A group of 278 older adults (average age: 83.5±6.5 years) from 9 state institutions in Malta was randomly selected. Participants were clinically examined and answered three questionnaires (Oral Health Impact Profile [OHIP-14], Geriatric Oral Health Assessment Index [GOHAI], and Denture Satisfaction). RESULTS: Oral health-related quality of life measurements were significantly associated with pocket depth (OHIP-14 and GOHAI, P<.05); decayed, missing, or filled teeth (OHIP-14 and GOHAI, P<.05); carious teeth (OHIP-14 and GOHAI, P<.05); number of missing teeth (OHIP-14 and GOHAI, P<.05); and maxillary and mandibular dentures (OHIP-14 and GOHAI, P<.0001). Denture satisfaction was associated with denture age (P=.010) and types of prostheses (P<.05). CONCLUSION: Institutionalized older adults have high levels of oral disease. Although oral health-related quality of life is generally good, those participants with a poorer oral health-related quality of life had significant associations with poor oral health. However, the extent varies with the individual's perception of good health.

Neurobehavioral coping to altered gravity: endogenous responses of neurotrophins.

An altered gravitational environment represents a unique challenge for biological systems that have evolved against gravitational background. Ground-based and space research indicates that the developing nervous system is potentially affected by exposure to hyper/microgravity. With the construction of the orbiting International Space Station long-term research on the nervous system will be possible. With this perspective, we started ground-based studies to characterize mouse behavioral responses to rotation-induced 2 g hypergravity, using a custom-made centrifuge device. Brain levels of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) as well as NGF and BDNF expression and mast cell distribution in heart and lung, were evaluated and correlated with the changes in mouse behavior upon hypergravity exposure. Hypergravity strongly affected the spontaneous activity of the animals, selectively modifying mouse behavioral repertoire. Such changes were mainly related to variations in brain levels of NGF, while BDNF was slightly affected, thus confirming a role for these neurotrophins in neuronal plasticity underlying experience-induced neurobehavioral changes. Moreover, gender differences were observed in both behavioral and neurobiological responses to hypergravity. These results indicate that changes in the gravitational environment might represent a useful tool to investigate the neurobiological and behavioral responses to stressors and may provide insights into the mechanisms underlying development and plasticity of nervous system in brain, heart, and lung.

Effects of acute and repeated daily exposure to hypergravity on spatial learning in mice.

Studies in humans have revealed that exposure to altered gravity may lead to impairments in cognitive functions. The objective of this study was to test whether mice exposed to hypergravity using a centrifuge apparatus showed learning impairments in a spatial learning task. Mice rotating at 1G or at 2G acceleration gravity and non-rotating controls were tested for reactivity to a spatial change after either a single 1 h or five repeated 1 h daily rotations in the centrifuge. While no differences among groups were found in the performance after single exposure to altered gravity, 5 days of repeated exposures to 1G or 2G gravity conditions significantly affected mouse ability to discriminate a new spatial arrangement. Additionally, this effect was stronger in the animals repeatedly exposed to 2G rather than to 1G conditions.

Cognitive and emotional alterations in periadolescent mice exposed to 2 g hypergravity field.

The development of the nervous system is a dynamic process where epigenetic factors play a fundamental role. Both ground-based and space research indicate that exposure to an altered gravitational environment affects rodent neurobehavioral profile and stage of development as well as duration of exposure appear to be critical for the observed effects. The behavioral profile of adolescent (28-day-old) male and female CD-1 mice upon acute 2 g exposure was characterized and emotional/anxiety responses (plus-maze), as well as spatial learning performance (Morris water-maze), were assessed respectively 24 and 48 h after hypergravity exposure. Behavioral observation indicated a transient mild sickness associated with hypergravity, with a decrease in spontaneous activity. Rotation per se induced an increase in emotional/anxious responses and a deterioration of spatial learning acquisition, while hypergravity specifically improved flexibility of spatial orientation.

Development of a Maltese version of oral health-associated questionnaires: OHIP-14, GOHAI, and the Denture Satisfaction Questionnaire.

PURPOSE: To show the reliability of the Maltese translations of OHIP-14, GOHAI, and the Denture Satisfaction Questionnaire, define the reliability of the responses, and determine the correlation between OHIP-14 and GOHAI. MATERIALS AND METHODS: The items of the three questionnaires (OHIP-14, GOHAI, and Denture Satisfaction) were translated into Maltese and back into English to compare with the original version. Specific sampling of a population well versed in Maltese and English was carried out to obtain a sample of respondents for each questionnaire. Data were gathered through self-administered questionnaires: first administering the Maltese version and following with the English version 1 week later. RESULTS: Participation rates were high (98%). Cronbach's alpha for all three questionnaires was high (> 0.7), indicating satisfactory test-retest reliability of the instruments. Similarly, the Spearman correlation coefficients for both the English and Maltese versions of OHIP-14 and GOHAI were good (> 0.6). CONCLUSION: The Maltese versions of OHIP-14, GOHAI, and the Denture Satisfaction Questionnaire can be safely used as a valid alternative to the English versions in studies of patients who are limited in linguistic proficiency.

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.