When night becomes day: Artificial light at night alters insect behavior under semi-natural conditions.

Light is the most important Zeitgeber for temporal synchronization in nature. Artificial light at night (ALAN) disrupts the natural light-dark rhythmicity and thus negatively affects animal behavior. However, to date, ALAN research has been mostly conducted under laboratory conditions in this context. Here, we used the field cricket, Gryllus bimaculatus, to investigate the effect of ALAN on insect behavior under semi-natural conditions, i.e., under shaded natural lighting conditions, natural temperature and soundscape. Male crickets were placed individually in outdoor enclosures and exposed to ALAN conditions ranging from <0.01 to 1500 lx intensity. The crickets' stridulation behavior was recorded for 14 consecutive days and nights and their daily activity patterns were analysed. ALAN impaired the crickets' stridulation rhythm, evoking a change in the crickets' naturally synchronized daily activity period. This was manifested by a light-intensity-dependent increase in the proportion of insects demonstrating an intrinsic circadian rhythm (free-run behavior). This also resulted in a change in the population's median activity cycle period. These ALAN-induced effects occurred despite the crickets' exposure to almost natural conditions. Our findings provide further validity to our previous studies on ALAN conducted under lab conditions and establish the deleterious impacts of ALAN on animal behavioral patterns. TEASER: Artificial light at night alters cricket behavior and desynchronizes their stridulation even under near-natural conditions.

Crickets in the spotlight: exploring the impact of light on circadian behavior.

Crickets serve as a well-established model organism in biological research spanning various fields, such as behavior, physiology, neurobiology, and ecology. Cricket circadian behavior was first reported over a century ago and prompted a wealth of studies delving into their chronobiology. Circadian rhythms have been described in relation to fundamental cricket behaviors, encompassing stridulation and locomotion, but also in hormonal secretion and gene expression. Here we review how changes in illumination patterns and light intensity differentially impact the different cricket behaviors as well as circadian gene expression. We further describe the cricket's circadian pacemaker. Ample anatomical manipulations support the location of a major circadian pacemaker in the cricket optic lobes and another in the central brain, possibly interconnected via signaling of the neuropeptide PDF. The cricket circadian machinery comprises a molecular cascade based on two major transcriptional/translational negative feedback loops, deviating somewhat from the canonical model of Drosophila and emphasizing the significance of exploring alternative models. Finally, the nocturnal nature of crickets has provided a unique avenue for investigating the repercussions of artificial light at night on cricket behavior and ecology, underscoring the critical role played by natural light cycles in synchronizing cricket behaviors and populations, further supporting the use of the cricket model in the study of the effects of light on insects. Some gaps in our knowledge and challenges for future studies are discussed.

Exposure to a nocturnal light pulse simultaneously and differentially affects stridulation and locomotion behaviors in crickets.

It is crucial for living organisms to be in synchrony with their environment and to anticipate circadian and annual changes. The circadian clock is responsible for entraining organisms' activity to the day-night rhythmicity. Artificial light at night (ALAN) was shown to obstruct the natural light cycle, leading to desynchronized behavioral patterns. Our knowledge of the mechanisms behind these adverse effects of ALAN, however, is far from complete. Here we monitored the stridulation and locomotion behavior of male field crickets (Gryllus bimaculatus), raised under light:dark conditions, before, during, and after exposure to a nocturnal 3-h pulse of different ALAN intensities. The experimental insects were then placed under a constant light regime (of different intensities); their behavior was continuously monitored; and the period of their daily activity rhythms was calculated. The light pulse treatment induced a simultaneous negative (suppressing stridulation) and positive (inducing locomotion) effect, manifested in significant changes in the average level of the specific activity on the night of the pulse compared to the preceding and the following nights. The transition to constant light conditions led to significant changes in the period of the circadian rhythms. Both effects were light-intensity-dependent, indicating the importance of dark nights for both individual and population synchronization.

Transcriptional Response of Circadian Clock Genes to an 'Artificial Light at Night' Pulse in the Cricket Gryllus bimaculatus.

Light is the major signal entraining the circadian clock that regulates physiological and behavioral rhythms in most organisms, including insects. Artificial light at night (ALAN) disrupts the natural light-dark cycle and negatively impacts animals at various levels. We simulated ALAN using dim light stimuli and tested their impact on gene expression in the cricket Gryllus bimaculatus, a model of insect physiology and chronobiology. At night, adult light-dark-regime-raised crickets were exposed for 30 min to a light pulse of 2-40 lx. The relative expression of five circadian-clock-associated genes was compared using qPCR. A dim ALAN pulse elicited tissue-dependent differential expression in some of these genes. The strongest effect was observed in the brain and in the optic lobe, the cricket's circadian pacemaker. The expression of opsin-Long Wave (opLW) was upregulated, as well as cryptochrome1-2 (cry) and period (per). Our findings demonstrate that even a dim ALAN exposure may affect insects at the molecular level, underscoring the impact of ALAN on the circadian clock system.

GSK-3ß Inhibition in Birds Affects Social Behavior and Increases Motor Activity.

Glycogen synthase kinase-3 (GSK-3) is a highly conserved serine/threonine protein kinase that plays a central role in a wide variety of cellular processes, cognition and behaviour. In a previous study we showed that its α and ß isozymes are highly conserved in vertebrates, however the α gene is missing in birds. This selective loss offers a unique opportunity to study the role of GSK-3ß independently. Accordingly, in the present study we aimed to investigate the role of GSK-3ß in social behaviour, motivation, and motor activity in zebra finches (Taeniopygia guttata). We did that by selective inhibition of GSK-3ß and by using tests that were specifically designed in our laboratory. Our results show that GSK-3ß inhibition: 1) Affected social recognition, because the treated birds tended to move closer towards a stranger, unlike the control birds that stood closer to a familiar bird. 2) Caused the treated birds to spend more time in the more middle parts of the cage compared to controls, a behaviour that might indicate anxiety. 3) As the experiment progressed, the treated birds took less time to make a decision where to stand in the cage compared to controls, suggesting an effect on decision-making. 4) Increased in the motor activity of the treated birds compared to the controls, which can be regarded as hyperactivity. 5) Caused the treated birds to pass through a barrier in order to join their flock members faster compared to controls, and regardless of the increase in the level of difficulty, possibly suggesting increased motivation. Our study calls for further investigation, because GSK-3 is well acknowledged as a central player in regulating mood behaviour, cognitive functions, and neuronal viability. Therefore, studying its impact on normal behaviour as we did in the current study, unlike most studies that were done in diseases models, can advance our understanding regarding GSK-3 various roles and can contribute to the discovery and development of effective treatments to repair cognition and behaviour.

Lifelong exposure to artificial light at night impacts stridulation and locomotion activity patterns in the cricket Gryllus bimaculatus.

Living organisms experience a worldwide continuous increase in artificial light at night (ALAN), negatively affecting their behaviour. The field cricket, an established model in physiology and behaviour, can provide insights into the effect of ALAN on insect behaviour. The stridulation and locomotion patterns of adult male crickets reared under different lifelong ALAN intensities were monitored simultaneously for five consecutive days in custom-made anechoic chambers. Daily activity periods and acrophases were compared between the experimental groups. Control crickets exhibited a robust rhythm, stridulating at night and demonstrating locomotor activity during the day. By contrast, ALAN affected both the relative level and timing of the crickets' nocturnal and diurnal activity. ALAN induced free-running patterns, manifested in significant changes in the median and variance of the activity periods, and even arrhythmic behaviour. The magnitude of disruption was light intensity dependent, revealing an increase in the difference between the activity periods calculated for stridulation and locomotion in the same individual. This finding may indicate the existence of two peripheral clocks. Our results demonstrate that ecologically relevant ALAN intensities affect crickets' behavioural patterns, and may lead to decoupling of locomotion and stridulation behaviours at the individual level, and to loss of synchronization at the population level.

Evidence That Artificial Light at Night Induces Structure-Specific Changes in Brain Plasticity in a Diurnal Bird.

We recently reported that artificial light at night (ALAN), at ecologically relevant intensities (1.5, 5 lux), increases cell proliferation in the ventricular zone and recruitment of new neurons in several forebrain regions of female zebra finches (Taeniopygia guttata), along with a decrease of total neuronal densities in some of these regions (indicating possible neuronal death). In the present study, we exposed male zebra finches to the same ALAN intensities, treated them with 5'-bromo-2'-deoxyuridine, quantified cell proliferation and neuronal recruitment in several forebrain regions, and compared them to controls that were kept under dark nights. ALAN increased cell proliferation in the ventricular zone, similar to our previous findings in females. We also found, for the first time, that ALAN increased new neuronal recruitment in HVC and Area X, which are part of the song system in the brain and are male-specific. In other brain regions, such as the medial striatum, nidopallium caudale, and hippocampus, we recorded an increased neuronal recruitment only in the medial striatum (unlike our previous findings in females), and relative to the controls this increase was less prominent than in females. Moreover, the effect of ALAN duration on total neuronal densities in the studied regions varied between the sexes, supporting the suggestion that males are more resilient to ALAN than females. Suppression of nocturnal melatonin levels after ALAN exhibited a light intensity-dependent decrease in males in contrast to females, another indication that males might be less affected by ALAN. Taken together, our study emphasizes the importance of studying both sexes when considering ALAN effects on brain plasticity.

Artificial Light at Night Increases Recruitment of New Neurons and Differentially Affects Various Brain Regions in Female Zebra Finches.

Despite growing evidence that demonstrate adverse effects of artificial light at night (ALAN) on many species, relatively little is known regarding its effects on brain plasticity in birds. We recently showed that although ALAN increases cell proliferation in brains of birds, neuronal densities in two brain regions decreased, indicating neuronal death, which might be due to mortality of newly produced neurons or of existing ones. Therefore, in the present study we studied the effect of long-term ALAN on the recruitment of newborn neurons into their target regions in the brain. Accordingly, we exposed zebra finches (Taeniopygia guttata) to 5 lux ALAN, and analysed new neuronal recruitment and total neuronal densities in several brain regions. We found that ALAN increased neuronal recruitment, possibly as a compensatory response to ALAN-induced neuronal death, and/or due to increased nocturnal locomotor activity caused by sleep disruption. Moreover, ALAN also had a differential temporal effect on neuronal densities, because hippocampus was more sensitive to ALAN and its neuronal densities were more affected than in other brain regions. Nocturnal melatonin levels under ALAN were significantly lower compared to controls, indicating that very low ALAN intensities suppress melatonin not only in nocturnal, but also in diurnal species.

Artificial light at night affects brain plasticity and melatonin in birds.

Artificial light at night (ALAN), which disrupts the daily cycle of light, has vast biological impacts on all organisms, and is also associated with several health problems. The few existing studies on neuronal plasticity and cognitive functions in mammals indicate that a disruption of the circadian cycle impairs learning and memory and suppresses neurogenesis. However, nothing is known about the effect of ALAN on neuronal plasticity in birds. To this end, zebra finches (Taeniopygia guttata) were exposed to ecologically relevant ALAN intensities (0.5, 1.5 and 5 lx), treated with BrdU to quantify cell proliferation in their ventricular zone (VZ), and compared to controls that were kept under dark nights. We found, in our diurnal birds, that ALAN significantly increased cell proliferation in the VZ. However, neuronal densities in two brain regions decreased under ALAN, suggesting neuronal death. In addition, ALAN suppressed nocturnal melatonin production in a dose-dependent manner, and might also increase body mass. Taken together, our findings add to the notion of the deleterious effect of ALAN.

Questioning Seasonality of Neuronal Plasticity in the Adult Avian Brain.

To date, studies that reported seasonal patterns of adult neurogenesis and neuronal recruitment have correlated them to seasonal behaviors as the cause or as a consequence of neuronal changes. The aim of our study was to test this correlation, and to investigate whether there is a seasonal pattern of new neuronal recruitment that is not correlated to behavior. To do this, we used adult female zebra finches (songbirds that are not seasonal breeders), kept them under constant social, behavioral, and spatial environments, and compared neuronal recruitment in their brains during two seasons, under natural and laboratory conditions. Under natural conditions, no significant differences were found in the pattern of new neuronal recruitment across seasons. However, under artificial indoor conditions that imitated the natural conditions, higher neuronal recruitment occurred in late summer (August) compared to early spring (February). Moreover, our data indicate that "mixing" temperature and day length significantly reduces new neuronal recruitment, demonstrating the importance of the natural combination of temperature and day length. Taken together, our findings show, for the first time, that neuroplasticity changes under natural vs. artificial conditions, and demonstrate the importance of both laboratory and field experiments when looking at complex biological systems.

Exploring the Relationship between Brain Plasticity, Migratory Lifestyle, and Social Structure in Birds.

Studies in Passerines have found that migrating species recruit more new neurons into brain regions that process spatial information, compared with resident species. This was explained by the greater exposure of migrants to spatial information, indicating that this phenomenon enables enhanced navigational abilities. The aim of the current study was to test this hypothesis in another order-the Columbiformes - using two closely-related dove species-the migrant turtle-dove (Streptopelia turtur) and the resident laughing dove (S. senegalensis), during spring, summer, and autumn. Wild birds were caught, treated with BrdU, and sacrificed 5 weeks later. New neurons were recorded in the hyperpallium apicale, hippocampus and nidopallium caudolaterale regions. We found that in doves, unlike passerines, neuronal recruitment was lower in brains of the migratory species compared with the resident one. This might be due to the high sociality of doves, which forage and migrate in flocks, and therefore can rely on communal spatial knowledge that might enable a reduction in individual navigation efforts. This, in turn, might enable reduced levels of neuronal recruitment. Additionally, we found that unlike in passerines, seasonality does not affect neuronal recruitment in doves. This might be due to their non-territorial and explorative behavior, which exposes them to substantial spatial information all year round. Finally, we discuss the differences in neuronal recruitment between Columbiformes and Passeriformes and their possible evolutionary explanations. Our study emphasizes the need to further investigate this phenomenon in other avian orders and in additional species.

Theta EEG neurofeedback benefits early consolidation of motor sequence learning.

Procedural learning is subject to consolidation processes believed to depend on the modulation of functional connections involved in representing the acquired skill. While sleep provides the most commonly studied framework for such consolidation processes, posttraining modulation of oscillatory brain activity may also impact on plasticity processes. Under the hypothesis that consolidation of motor learning is associated with theta band activity, we used EEG neurofeedback (NFB) to enable participants to selectively increase either theta or beta power in their EEG spectra following the acquisition phase of motor sequence learning. We tested performance on a motor task before and after training, right after the NFB session to assess immediate NFB effects, 1 day after NFB to assess interaction between NFB effects and overnight sleep-dependent stabilization, and 1 week after the initial session, to assess the effects of NFB on long-term stabilization of motor training. We also explored the extent of the influence of single-electrode NFB on EEG recorded across the scalp. Results revealed a significantly greater improvement in performance immediately after NFB in the theta group than in the beta group. This effect continued for testing up to 1 week following training. Across participants, post-NFB improvement correlated positively with theta/beta ratio change achieved during NFB. Additionally, NFB was found to cause widespread band-power modulation beyond the electrode used for feedback. Thus, upregulating postlearning theta power may yield contributions to the immediate performance and subsequent consolidation of an acquired motor skill.

Possible linkage between neuronal recruitment and flight distance in migratory birds.

New neuronal recruitment in an adult animal's brain is presumed to contribute to brain plasticity and increase the animal's ability to contend with new and changing environments. During long-distance migration, birds migrating greater distances are exposed to more diverse spatial information. Thus, we hypothesized that greater migration distance in birds would correlate with the recruitment of new neurons into the brain regions involved with migratory navigation. We tested this hypothesis on two Palearctic migrants - reed warblers (Acrocephalus scirpaceus) and turtle doves (Streptopelia turtur), caught in Israel while returning from Africa in spring and summer. Birds were injected with a neuronal birth marker and later inspected for new neurons in brain regions known to play a role in navigation - the hippocampus and nidopallium caudolateral. We calculated the migration distance of each individual by matching feather isotopic values (δ(2)H and δ(13)C) to winter base-maps of these isotopes in Africa. Our findings suggest a positive correlation between migration distance and new neuronal recruitment in two brain regions - the hippocampus in reed warblers and nidopallium caudolateral in turtle doves. This multidisciplinary approach provides new insights into the ability of the avian brain to adapt to different migration challenges.

Active immunization against vasoactive intestinal polypeptide decreases neuronal recruitment and inhibits reproduction in zebra finches.

Neurogenesis and neuronal recruitment occur in adult brains of many vertebrates, and the hypothesis is that these phenomena contribute to the brain plasticity that enables organisms to adjust to environmental changes. In mammals, vasoactive intestinal polypeptide (VIP) is known to have many neuroprotective properties, but in the avian brain, although widely distributed, its role in neuronal recruitment is not yet understood. In the present study we actively immunized adult zebra finches against VIP conjugated to KLH and compared neuronal recruitment in their brains, with brains of control birds, which were immunized against KLH. We looked at two forebrain regions: the nidopallium caudale (NC), which plays a role in vocal communication, and the hippocampus (HC), which is involved in the processing of spatial information. Our data demonstrate that active immunization against VIP reduces neuronal recruitment, inhibits reproduction, and induces molting, with no change in plasma prolactin levels. Thus, our observations suggest that VIP has a direct positive role in neuronal recruitment and reproduction in birds. J. Comp. Neurol. 524:2516-2528, 2016. © 2016 Wiley Periodicals, Inc.

GSK-3ß Inhibition Affects Singing Behavior and Neurogenesis in Adult Songbirds.

GSK-3 (glycogen synthase kinase-3) is a serine/threonine kinase which is a critical regulator in neuronal signaling, cognition, and behavior. We have previously shown that unlike other vertebrates that harbor both α and ß GSK-3 genes, the α gene is missing in birds. Therefore, birds can be used as a new animal model to study the roles of GSK-3ß in behavior and in regulating adult neurogenesis. In the present study, we inhibited GSK-3ß in brains of adult male zebra finches (Taeniopygia guttata) and accordingly investigated how this inhibition affects behavior and cell proliferation. Our results show that GSK-3 inhibition: (1) affects specific aspects of singing behavior, which might be related to social interactions in birds, and (2) differentially affects cell proliferation in various parts of the ventricular zone. Taken together, our study demonstrates a role of GSK-3ß in regulating singing behavior and neuronal proliferation in birds and highlights the importance of GSK-3ß in modulating cognitive abilities as well as social behavior.

ADNP: A major autism mutated gene is differentially distributed (age and gender) in the songbird brain.

ADNP is a protein necessary for brain development, important for brain plasticity, cognitive and social functioning, characteristics that are all impaired in autism and in the Adnp(+/-) mouse model, in a sex-dependent manner. ADNP was originally discovered as a protein that is secreted from glial cells in response to vasoactive intestinal peptide (VIP). VIP is a major neuroprotective peptide in the CNS and PNS and was also associated with social recognition in rodents and aggression, pair-bonding and parental behaviors in birds. Comparative sequence alignment revealed high evolutionary conservation of ADNP in Chordata. Despite its importance in brain function, ADNP has never been studied in birds. Zebra finches (Taeniopygia guttata) are highly social songbirds that have a sexually dichotomous anatomical brain structure, with males demonstrating a developed song system, presenting a model to study behavior and potential sexually dependent fundamental differences. Here, using quantitative real time polymerase chain reaction (qRT-PCR), we discovered sexually dichotomous and age related differences in ADNP mRNA expression in three different regions of the song bird brain-cerebellum, cerebrum, and brain stem. Higher levels of ADNP mRNA were specifically found in young male compared to the female cerebrum, while aging caused a significant 2 and 3-fold decrease in the female and male cerebrum, respectively. Furthermore, a comparison between the three tested brain regions revealed unique sex-dependent ADNP mRNA distribution patterns, affected by aging. Future studies are aimed at deciphering the function of ADNP in birds, toward a better molecular understanding of sexual dichotomy in singing behavior in birds.

The cell birth marker BrdU does not affect recruitment of subsequent cell divisions in the adult avian brain.

BrdU is commonly used to quantify neurogenesis but also causes mutation and has mitogenic, transcriptional, and translational effects. In mammalian studies, attention had been given to its dosage, but in birds such examination was not conducted. Our previous study suggested that BrdU might affect subsequent cell divisions and neuronal recruitment in the brain. Furthermore, this effect seemed to increase with time from treatment. Accordingly, we examined whether BrdU might alter neurogenesis in the adult avian brain. We compared recruitment of [(3)H]-thymidine(+) neurons in brains of zebra finches (Taeniopygia guttata) when no BrdU was involved and when BrdU was given 1 or 3 months prior to [(3)H]-thymidine. In nidopallium caudale, HVC, and hippocampus, no differences were found between groups in densities and percentages of [(3)H]-thymidine(+) neurons. The number of silver grains per [(3)H]-thymidine(+) neuronal nucleus and their distribution were similar across groups. Additionally, time did not affect the results. The results indicate that the commonly used dosage of BrdU in birds has no long-term effects on subsequent cell divisions and neuronal recruitment. This conclusion is also important in neuronal replacement experiments, where BrdU and another cell birth marker are given, with relatively long intervals between them.

A possible relation between new neuronal recruitment and migratory behavior in Acrocephalus warblers.

Evidence suggests a possible correlation between learning abilities of adults and new neuronal recruitment into their brains. The hypothesis is that this brain plasticity enables animals to adapt to environmental changes. We examined whether there are differences in neuronal recruitment between resident and migrant birds. We predicted that migrants, which are more exposed to spatial changes than residents, will recruit more new neurons. To test this, we compared neuronal recruitment in two closely related bird species - the migrant reed warbler (Acrocephalus scirpaceus), and the resident Clamorous warbler (A. Stentoreus) - during spring, summer, and autumn. Wild birds were caught, treated with BrdU and sacrificed five weeks later. New neurons were recorded in the Hippocampus and Nidopallium caudolateral. The results support our hypothesis, as more new neurons were found in the migrant species, in both brain regions and all seasons. We suggest that this phenomenon enables enhanced navigational abilities, which are required for the migratory lifestyle. However, in contrast to our hypothesis, in spring we found less new neurons in adults of both species, as compared to other seasons. We suggest that in spring, when birds settle in breeding territories, they require less spatial skills, and this might enable to reduce the cost of neuronal recruitment, as reflected by less new neurons in their brains. We also found age differences, with overall higher neuronal recruitment in juveniles. Finally, we advocate the importance of studying wild populations, for a better understanding of the adaptive significance of neuronal replacement in the vertebrate brain.

Better than sleep: theta neurofeedback training accelerates memory consolidation.

Consistent empirical results showed that both night and day sleep enhanced memory consolidation. In this study we explore processes of consolidation of memory during awake hours. Since theta oscillations have been shown to play a central role in exchange of information, we hypothesized that elevated theta during awake hours will enhance memory consolidation. We used a neurofeedback protocol, to enhance the relative power of theta or beta oscillations. Participants trained on a tapping task, were divided into three groups: neurofeedback theta; neurofeedback beta; control. We found a significant improvement in performance in the theta group, relative to the beta and control groups, immediately after neurofeedback. Performance was further improved after night sleep in all groups, with a significant advantage favoring the theta group. Theta power during training was correlated with the level of improvement, indicating a clear relationship between memory consolidation, and theta neurofeedback.