Evidence That Artificial Light at Night Induces Structure-Specific Changes

biomolecules

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

Stan Moaraf 1,2,*, Rachel Heiblum 2, Monika Okuliarová 3 , Abraham Hefetz 1, Inon Scharf 1 , Michal Zeman 3 and Anat Barnea 2

1 Faculty of Life Sciences, School of Zoology, Tel-Aviv University, Tel-Aviv 6997801, Israel; [email protected] (A.H.); scharﬁ@tauex.tau.ac.il (I.S.) 2 Department of Natural and Life Sciences, The Open University of Israel, Ra’anana 43710, Israel; [email protected] (R.H.); [email protected] (A.B.) 3 Department of Animal Physiology and Ethology, Faculty of Natural Sciences, Comenius University, 84215 Bratislava, Slovakia; [email protected] (M.O.); [email protected] (M.Z.) * Correspondence: [email protected]; Tel.: +97-25-8590-3936

Abstract: 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 50-bromo-20- 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 Citation: Moaraf, S.; Heiblum, R.; ALAN increased new neuronal recruitment in HVC and Area X, which are part of the song system Okuliarová, M.; Hefetz, A.; Scharf, I.; in the brain and are male-specific. In other brain regions, such as the medial striatum, nidopallium Zeman, M.; Barnea, A. Evidence That caudale, and hippocampus, we recorded an increased neuronal recruitment only in the medial Artificial Light at Night Induces Structure-Specific Changes in Brain striatum (unlike our previous findings in females), and relative to the controls this increase was less Plasticity in a Diurnal Bird. prominent than in females. Moreover, the effect of ALAN duration on total neuronal densities in the Biomolecules 2021, 11, 1069. https:// studied regions varied between the sexes, supporting the suggestion that males are more resilient doi.org/10.3390/biom11081069 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 Academic Editors: Randy J. Nelson less affected by ALAN. Taken together, our study emphasizes the importance of studying both sexes and James C. Walton when considering ALAN effects on brain plasticity.

Received: 14 May 2021 Keywords: artiﬁcial light at night (ALAN); brain plasticity; cell proliferation; new neuronal recruit- Accepted: 12 July 2021 ment; melatonin; sex-differences; birds; zebra ﬁnches (Taeniopygia guttata) Published: 21 July 2021

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional affil- iations. Artificial light at night (ALAN) disrupts the daily light–dark cycle and exposes animals and humans to higher levels of nocturnal light. It has vast biological impacts on many species, and is also associated with several health problems in humans [1,2]. For example, ALAN has been shown to induce earlier reproductive maturity [3,4], compromise immune homeostasis [5], and decrease melatonin (MEL) production [6,7]. Nevertheless, Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. little is known regarding the effects of ALAN on brain plasticity processes such as cell This article is an open access article proliferation (CP) and new neuronal recruitment (NR), and most of the existing studies distributed under the terms and have been conducted on nocturnal mammals, e.g., [8–11]. Therefore, in a previous study we conditions of the Creative Commons investigated this question in diurnal female zebra finches (Taeniopygia guttata), by exposing Attribution (CC BY) license (https:// them to three weeks of ecologically relevant ALAN intensities (0.5, 1.5, and 5 lux), and creativecommons.org/licenses/by/ found that ALAN increased CP in the ventricular zone (VZ) in the brain as compared 4.0/). with controls that were kept under fully dark nights [6]. In birds, many of the new cells

Biomolecules 2021, 11, 1069. https://doi.org/10.3390/biom11081069 https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, 1069 2 of 17

that are born in the VZ migrate into the telencephalon, differentiate into neurons, and settle in various brain regions, where they replace older ones [12,13] and are recruited into functional circuits [14,15]. Therefore, in that study we also recorded neuronal densities in several forebrain regions, and found that despite the increased proliferation, total neuronal densities decreased in some regions, suggesting net neuronal death. In a later study [7], in which we exposed female zebra finches to a longer ALAN exposure (six weeks), we recorded NR in these brain regions and found that ALAN increased recruitment compared to controls, possibly as a compensatory response to ALAN-induced neuronal death, and/or due to increased nocturnal locomotor activity caused by sleep disruption. In addition, the decrease in total neuronal densities, which we observed in some brain regions under short-term ALAN exposure [6], was not found under the longer exposure [7], indicating a temporal effect of ALAN. Our previous studies were conducted on females. Therefore, the aim of the present study was to test whether the observed effects of ALAN on neuronal plasticity occur also in males. Moreover, a major advantage of studying males is their unique song system, which does not exist in females [16] and is known to contain MEL receptors, e.g., [17]. This enabled us to specifically test the effects of ALAN on the song system. Two nuclei from this system were included in our study: (1) HVC, which is involved in the control of vocal behavior, and incorporates new neurons into functional circuits [14,18], and (2) Area X, which exhibits large-scale neuronal addition throughout adulthood, is critical for the acquisition of song in juveniles [16,19], and plays a role in song maintenance in adults (reviewed in [20]). It is well established that female mate choice in many songbird species is influenced by the male singing behavior, which can serve as an indicator of his quality [21]. Therefore, an effect of ALAN on the neuronal composition of the song nuclei could result in the production of an impaired or mistimed song, which in turn might have male-specific fitness consequences by decreasing the males’ ability to attract or retain a mate and defend a territory, and hence might have ecological implications. In addition, our present study, which focused on males, enabled us to add a comparative dimension, specifically in regard to looking for possible sex-differences in the response of the brain to ALAN. Testing this question is important because at least some aspects of neuronal plasticity, such as CP, might differ between sexes, e.g., [22,23]. To achieve these aims, male zebra finches were exposed to ALAN intensities of 1.5- and 5 lux for three and six weeks and compared to controls that were kept under dark nights. We recorded CP in the VZ, as well as NR and total neuronal densities in two song nuclei HVC and Area X. In addition, we recorded the same neuronal parameters in three other forebrain regions, which were also tested in our previous studies with females: the nidopallium caudale (NC), which contains auditory relays [24] and is involved in vocal communication and the integration of auditory information [25,26]; the medial striatum (MSt), which is a part of the avian somatomotor basal ganglia [27] and is linked to visual perception and associative learning [28–30]; and the hippocampus (HC), which processes spatial information [31,32] and plays a role in stress response [33].

2. Materials and Methods 2.1. Experimental Design Our study consisted of two experiments, a cell proliferation (CP) experiment, which included three groups with six male birds per group (Figure1A), and a neuronal recruitment experiment (NR), which included two groups with eight male birds per group (Figure1B). All of the following experimental conditions and procedures were identical to those in our previous studies with females [6,7]. Zebra finches were hatched and reared in our outdoors breeding colonies, at the I. Meier Segals Garden for Zoological Research at Tel-Aviv University, Israel. Sexually mature males (ranging between three to five months of age, out of a lifespan of four to five years [34] for all groups) were transferred, one group at a time, from their native colonies to a room, in which day length and temperature were set to 14:10 L:D and 27 ◦C. Zebra finches are very social birds, and in order to avoid isolation Biomolecules 2021, 11, x 3 of 18

Biomolecules 2021, 11, 1069 3 of 17 one group at a time, from their native colonies to a room, in which day length and temperature were set to 14:10 L:D and 27 °C. Zebra finches are very social birds, and in order tostress, avoid birds isolation of each stress, group birds were of kepteach togethergroup were in onekept cage together (67 × in33 one× 33cage cm). (67 The× 33 cage× 33 wascm). placedThe cage in thewas same placed location in the withinsame location one room, within which one was room, used which for all was the used groups for forall the groups entire duration for the entire of the duration experiment. of the In expe orderriment. to ensure In order constant to ensure and identical constant conditions and iden- withintical conditions and between within groups, and between temperature, groups, moisture temperature, and light moisture intensities and werelight monitoredintensities wereevery monitored 10 days at theevery same 10 locationsdays at the around same andlocations in the around cage. After and the in experimentalthe cage. After birds the wereexperimental transferred birds from were their transferred native colonies from their to indoor native conditions, colonies to they indoor were conditions, all given threethey weekswere all of given acclimation, three weeks a period of acclimation, that ensures a thermogenicperiod that ensures acclimation thermogenic [35] and acclimation is accepted [35]for physiological and is accepted acclimation for physiological in birds, acclimation e.g., [36]. During in birds, the acclimatione.g., [36]. During periods, the theacclima- birds weretion periods, exposed the to abirds fullspectrum were exposed daylight to a ﬂuorescent full spectrum light daylight source (1200 fluorescent lux; CFL, light Hyundai, source (1200Macedonia) lux; CFL, during Hyundai, the day, Macedonia) and were during kept in the complete day, and darkness were kept during in complete the night. darkness Follow- duringing the the acclimation night. Following period, allthe birds acclimation were still period, exposed all birds to the were same still light exposed source to during the same the lightday, assource described during above. the day, During as described the night, ab forove. the During ALAN groups,the night, we for used the the ALAN same groups, type of welight used source the thatsame was type partially of light shaded, source that so that was birds partially in the shaded,CP experiment so that birdswere in exposed the CP experimenteither to 1.5- were or 5lux exposedALAN either intensities to 1.5- foror 5lux three ALAN weeks intensities (Figure1A) for and three birds weeks in the (FigureNR 1A)experiment and birdswere in the exposed NR experiment to 5lux ALAN were for exposed six weeks to (Figure 5lux ALAN1B). The for cage six containedweeks (Figure four perches1B). The on cage which contained the birds four could perches stand on and wh sleep.ich the ALAN birdsintensities could stand were and measured sleep. ALAN at all intensitiesthese perches were in ordermeasured to ensure at all that these the perches birds were in order exposed to ensure to constant that the ALAN birds intensities, were ex- posedregardless to constant of their locationALAN intensities, in the cage. regardlessControl groups of their were location exposed in the to dark cage. nights Control for groupsthe duration were exposed of the respective to dark nights experiment. for the Spectraduration of of all the the respective light sources experiment. were measured Spectra ofby all a spectrometerthe light sources (Jaz were spectrometer, measured Ocean by a spectrometer Optics, Largo, (Jaz FL, spectrometer, USA) and intensities Ocean Optics, by a Largo,digital lightFL, USA) meter and (TES-1337, intensities TES, by Taipei, a digital Taiwan), light meter to ensure (TES-1337, full and TES, uniform Taipei, spectra Taiwan), and tointensities ensure full (Figure and uniform2). For the spectra entire and duration intensit ofies the (Figure experiment, 2). For the food entire (millet duration seeds) of and the water were provided ad libitum three times per week. As an indirect indication of the experiment, food (millet seeds) and water were provided◦ ad libitum three times per week. Asbirds’ an health,indirect each indication bird was of weighedthe birds’ (Precisa, health, each BJ 120 birdC was± 0.01 weighed g) seven (Precisa, times (three BJ 120 times °C ± 0.01before g) andseven four times times (three during times ALAN before exposure), and four every times week, during four ALAN to six hoursexposure), after lightsevery were turned on. We also measured MEL levels in the birds’ plasma, and after three weeks week, four to six hours after lights were 0turned on.0 We also measured MEL levels in the birds’of the ALANplasma, period and after treated three them weeks with of 5the-bromo-2 ALAN period-deoxyuridine treated them (BrdU), with which 5′-bromo-2 is a cell′- birth-date marker (Figure1 and see below). deoxyuridine (BrdU), which is a cell birth-date marker (Figure 1 and see below).

Figure 1. A schematic descriptiondescription ofof the the experimental experimental design. design. (A (A) Timeline) Timeline of of the the cell cell proliferation proliferation experiment. experiment. (B )(B Timeline) Time- forline the for neuronal the neuronal recruitment recruitment experiment. experi Redment. arrows Red indicatearrows indicate blood sampling; blood sampling; syringes indicatesyringes BrdU indicate treatment BrdU times;treatment and times; and the numbers in brackets indicate the number of injections. See text for details. the numbers in brackets indicate the number of injections. See text for details. Biomolecules 2021, 11, 1069 4 of 17 Biomolecules 2021, 11, x 4 of 18

Figure 2. The spectrumFigure 2. andThe intensity spectrum of and each intensity light source of each that light was source used in that the was experiment used in thefor experimentday and night-time for day illumination. and night-time illumination.

2.2. Melatonin2.2. Levels Melatonin in Plasma Levels in Plasma Four bloodFour samples blood were samples taken were from taken each birdfrom (Figure each bi1rd): at (Figure midday 1): on at daymidday one ofon day one of week three ofweek acclimation, three of acclimation, at midday on at day midday one of on week day three one ofof theweek ALAN three period; of the andALAN two period; and at midnight,two three at daysmidnight, after each three of days the midday after each samples, of the to midday allow enough samples, time to forallow the birdsenough time for to recover betweenthe birds bleedings. to recover Blood between collection bleedings. at midnight Blood collection was performed at midnight as described was performed as in [7]. Plasmadescribed MEL concentrations in [7]. Plasma were MEL measured concentrations by direct were radioimmunoassay measured by direct [37 ],radioimmunoassay which has been validated[37], which for zebrahas been finches validated [38]. For for detailed zebra finche descriptions [38]. For see [detailed6]. We used description specific see [6]. We MEL antiserumused specific (Stockgrand MEL Ltd.,antiserum G/S/704-8483, (Stockgrand University Ltd., G/S/704-8483, of Surrey, University Guildford, of UK) Surrey, Guild- and [O-methyl-3H]-labelledford, UK) and [O-methyl-3H]-labelled melatonin (specificactivity: melatonin 3.07 (specific TBq/mmol, activity: Perkin 3.07 TBq/mmol, Elmer, Perkin Waltham, MA,Elmer, USA). Waltham, Sample radioactivityMA, USA). Sample was measured radioactivity in the was scintillating measuredβ-counter in the scintillating for β- liquid samplescounter (Packard for liquid Tri-Carb samples 2900 (Packard TR, Packard Tri-Carb Instruments, 2900 TR, Packard Perkin Elmer, Instruments, Waltham, Perkin Elmer, MA, USA). AllWaltham, samples MA, in each USA). experiment All samples were in measured each experiment in a single were assay measured with intra-assay in a single assay variation coefficientswith intra-assay less than variation 10.0% andcoefficients the assay less sensitivity than 10.0% 0.5 and pg/tube. the assay sensitivity 0.5 pg/tube. 2.3. BrdU Administration and Immunohistochemistry 2.3. BrdU Administration and Immunohistochemistry To study the effect of exposure to ALAN on CP in the brain, all birds from the CP To study the effect of exposure to ALAN on CP in the brain, all birds from the CP experiment were treated with BrdU (SigmaUltra) at the end of the three weeks of ALAN experiment were treated with BrdU (SigmaUltra) at the end of the three weeks of ALAN exposure (Figure1). Two intramuscular injections of 130 µL of BrdU (diluted 10 mg/mL exposure (Figure 1). Two intramuscular injections of 130 µL of BrdU (diluted 10 mg/mL in sterile water; for details see [39]) were administered within 24 h, one at 15:00 on the in sterile water; for details see [39]) were administered within 24 h, one at 15:00 on the day day before the perfusion, and the other at 07:00 on the perfusion day. Birds were killed before the perfusion, and the other at 07:00 on the perfusion day. Birds were killed two two hours after the second injection. This protocol enabled labelling of new cells in the hours after the second injection. This protocol enabled labelling of new cells in the VZ VZ before they start their migration [40]. Histology and immunohistochemistry were performed asbefore described they start in ref. their [39]. migration [40]. Histology and immunohistochemistry were per- To studyformed the effect as described of exposure in ref. to [39]. ALAN on NR in the brain, all birds from the NR experiment wereTo treated study withthe effect four injectionsof exposure of BrdU,to ALAN administrated on NR in the within brain, a 48 all h birds period, from the NR two in the morningsexperiment and were two treated in the afternoons,with four injections at three of weeks BrdU, of administrated ALAN exposure. within Birds a 48 h period, were killedtwo three in weeksthe mornings after the and last two injection. in the afternoon This protocols, at three enabled weeks labelling of ALAN of newexposure. Birds neurons withinwere several killed brainthree regions weeks (seeafter below). the last Histology injection. and This immunohistochemistry protocol enabled labelling for of new the NR experimentneurons werewithin performed several brain as described regions (see in [7 below).,41]. This Histology staining protocoland immunohistochemistry yielded neurons thatfor were the NR stained experiment fluorescent were green performed (with anti-Huc/HuD), as described in and[7,41]. nuclei This ofstaining new protocol yielded neurons that were stained fluorescent green (with anti-Huc/HuD), and nuclei of Biomolecules 2021, 11, 1069 5 of 17 Biomolecules 2021, 11, x 5 of 18

newneurons neurons that that were were stained stained fluorescent fluorescent red red (with (with anti-BrdU) anti-BrdU) (Figure (Figure3). 3). Therefore, Therefore, cells cells with colocalization of green cytoplasm and red fluorescent fluorescent nuclei were identified identified as new + neurons. Accordingly, Accordingly, we could record the location of BrdUBrdU+ neurons and count them in eacheach section, as explained below.

Figure 3. New neurons shown in three microphotographs of the same field, field, in the Medial Striatum (MSt; upper row ), and the nidopallium caudale (NC; lower row ) in brains of zebra finches finches ( Taeniopygia guttata). HU-labelled neurons are shown under a FITC filter (A,D), and BrdU-labelled cells are shown under a rhodamine filter (B,E). The combined images were under a FITC filter (A,D), and BrdU-labelled cells are shown under a rhodamine filter (B,E). The combined images were obtained using a FITC-rhodamine filter to show co-localization of the two markers (C,F). Scale: 10 µm. obtained using a FITC-rhodamine filter to show co-localization of the two markers (C,F). Scale: 10 µm.

2.4. Mapping Mapping and and Quantification Quantification InIn the the CP CP experiment, experiment, we we used used a a computer computerizedized brain-mapping brain-mapping system system (Neurolucida; (Neurolucida; Stereo Investigator; Micro-BrightField Ltd; Ltd; Wi Williston,lliston, VT, USA) to determine the number of labelled labelled cells/mm cells/mm in in each each of of the the VZ VZ wall walls,s, and and followed followed the the protocol protocol as asdescribed described in [39].in [39 We]. We mapped mapped six six sections sections along along the the rostral-caudal rostral-caudal axis axis of of the the VZ, VZ, at at 720 720 µmµm intervals (Figure 44).). TheThe firstfirst sectionsection thatthat containedcontained thethe anterioranterior commissurecommissure (CoA;(CoA; correspondingcorresponding toto A1.6 A1.6 in in the atlas of the canary brain) [42] [42] was determined as the fourth section. In this way, we ensured that the VZ location of eacheach sampled section that was mapped along the rostral-caudalrostral-caudal axis axis will will correspond correspond in in all brains. In In each section, the entire lengths of the ventral and the dorsal walls of the VZ were measured (indicated by the red line and the black line respectively in the 1–6 sections in Figure 44),), andand thethe numbernumber ofof BrdU-labelledBrdU-labelled cellscells that were found along each wall was divi dividedded by the length of that wall. This yielded thethe number number of of labelled labelled cells/mm in in each each of of the the VZ VZ walls. walls. In the NR experiment we counted BrdU+ neurons in several brain regions that are known to recruit new neurons and represent various functions: the MSt, which was divided into its two sub regions, the lateral MSt (lMSt) and the medial MSt (mMSt); the NC; the HC; the HVC; and Area X (Figure5). In each brain region we sampled five sections. In the MSt, the most rostral section corresponded to A4.0 in the canary atlas [42], and the fifth and most caudal section corresponded to A2.2. The MSt sub regions, the lMSt and the mMSt, were separated by drawing a straight dorsal-ventral line, at equal distances to the most lateral and medial boundaries of MSt. In the NC, the middle section corresponded to P1.2 in the canary atlas [42], and two more sections were obtained, rostrally and caudally, respectively, at distances of 360- and 600 µm from the middle one. In the HC, the most rostral section corresponded to A4.0 in the canary atlas [42], and the fifth and most caudal Biomolecules 2021, 11, 1069 6 of 17

section corresponded to A0.2. The three other in-between sections were evenly distributed so that, on average, the distance between each mapped section and the next was 480 µm. In the HVC, the most rostral section corresponded to AP0.0 and the ﬁfth and most caudal section corresponded to P0.8 [42]. In Area X, the most rostral section corresponded to A4.5

Biomolecules 2021, 11, x and third most caudal section corresponded to A3.5 [42]. In the MSt, HC, HVC and6 of 18 Area X, the middle sections were selected for measurements of neuronal density (see below).

Figure 4. Mapping of cell proliferation in the ventricular zone (VZ). (A) Top view of a zebra finch brain (caudal is to the Figure 4. Mapping of cell proliferation in the ventricular zone (VZ). (A) Top view of a zebra ﬁnch brain (caudal is to the left, left, rostral is to the right). The numbered lines indicate the locations of the mapped sections, and frontal schematic illus- rostraltrations is to the of right).these sections The numbered are shown lines in 1–6 indicate (taken the from locations the atlas of of the the mapped canary br sections,ain [42]). andRed frontallines indicate schematic the ventral illustrations of thesewalls sections of the areVZ in shown each mapped in 1–6 (taken section from and the the black atlas lines of the indicate canary the brain dorsal [42 walls.]). Red (B,C lines) Pictures indicate showing the ventral two different walls of the VZ insmall each mappedparts of the section two walls and theof the black VZ, linesto demonstrate indicate the new- dorsalborn walls.cells labelled (B,C) Pictureswith BrdU showing and stained two differentdark brown small (only parts of one of the labelled cells is marked by an arrow). CoA, anterior commissure; HC, hippocampus; LMD, lamina medullaris the two walls of the VZ, to demonstrate new-born cells labelled with BrdU and stained dark brown (only one of the labelled dorsallis; MSt, medial striatum; NC, nidopallium caudale. Scale bar = 20 µm. (image adapted from [6]). cells is marked by an arrow). CoA, anterior commissure; HC, hippocampus; LMD, lamina medullaris dorsallis; MSt, medial striatum; NC, nidopallium caudale.In Scale the NR bar experiment = 20 µm. (Adapted we counted with BrdU permission+ neurons from in Ref.several [6]. brain Copyright regions 2021, that JOHN are WILEY & SONS, INC.). known to recruit new neurons and represent various functions: the MSt, which was divided into its two sub regions, the lateral MSt (lMSt) and the medial MSt (mMSt); the NC; the HC; the HVC; and Area X (Figure 5). In each brain region we sampled five sections. In the MSt, the most rostral section corresponded to A4.0 in the canary atlas [42], and the fifth and most caudal section corresponded to A2.2. The MSt sub regions, the lMSt and Biomolecules 2021, 11, x 7 of 18

the mMSt, were separated by drawing a straight dorsal-ventral line, at equal distances to the most lateral and medial boundaries of MSt. In the NC, the middle section corresponded to P1.2 in the canary atlas [42], and two more sections were obtained, rostrally and caudally, respectively, at distances of 360- and 600 µm from the middle one. In the HC, the most rostral section corresponded to A4.0 in the canary atlas [42], and the fifth and most caudal section corresponded to A0.2. The three other in-between sections were evenly distributed so that, on average, the distance between each mapped section and the next was 480 µm. In the HVC, the most rostral section corresponded to AP0.0 and the fifth and most caudal section corresponded to P0.8 [42]. In Area X, the most rostral section Biomolecules 2021, 11, 1069 7 of 17 corresponded to A4.5 and third most caudal section corresponded to A3.5 [42]. In the MSt, HC, HVC and Area X, the middle sections were selected for measurements of neuronal density (see below).

Figure 5. Mapping of new neuronal recruitment in several brain regions are shown. Illustrations of the mapped sections Figure 5. of medialMapping striatum of new (MSt), neuronal nidopallium recruitment caudale (NC), in several hippocampus brain regions(HC), HVC are and shown. Area X Illustrations are adapted offrom the [41]. mapped Five sections of medialsections striatum of the (MSt),lateral MSt nidopallium (lMSt) and medial caudale MSt (NC), (mMSt) hippocampus (A4.5—A2.4), (HC),five sections HVC of and the AreaNC (P0.8—P1.6), X are adapted five sections with permission from Ref.of the [41 HC]. Copyright(A4.0—A1.6), 2021, five sections Elsevier. of the Five HVC sections (AP0.0—P1.0) of the lateral and three MSt sections (lMSt) of andArea medial X (A4.5—A3.5) MSt (mMSt) are highlighted. (A4.5—A2.4), five sections of the NC (P0.8—P1.6), five sections of the HC (A4.0—A1.6), five sections of the HVC (AP0.0—P1.0) and three We used the above-described brain-mapping system to draw the boundaries of each sections of Area X (A4.5—A3.5) are highlighted. of the sampled brain regions and subregions in each of the five sampled relevant sections, to mark the position of BrdU+ neurons, and to quantify other neuronal parameters (see below).We usedMapping the above-describedwas done by using brain-mappinga 60× objective. Because system a previous to draw study the boundaries from our of each oflab the [43] sampled did not brain reveal regions any hemispheric and subregions differences in in each the ofdensity the five of labelled sampled neurons relevant in sections, + toany mark region the in position males, we of mapped BrdU sectionsneurons, only and from to the quantify right hemisphere. other neuronal In the lMSt parameters and (see below).the mMSt, Mapping and in the was HC, done HVC by and using Area X, a 60which× objective. are relatively Because small regions, a previous we scanned study from our labthe [43 whole] did area not in reveal each mapped any hemispheric section by using differences the meander in the scan density probe in of our labelled mapping neurons in any region in males, we mapped sections only from the right hemisphere. In the lMSt and the mMSt, and in the HC, HVC and Area X, which are relatively small regions, we scanned the whole area in each mapped section by using the meander scan probe in our mapping system. However, since the NC is a large brain region, we scanned about 35% of the whole NC area in each mapped section as previously done in [44]. We also estimated the total neuronal densities (labelled and unlabeled neurons) in each of the sampled brain regions and subregions, following our previous protocol (see [45]). This was done in additional set of slides, with sections from the same locations as the slides in the above sets (6 µm apart), stained with cresyl violet, which reliably identifies neurons, e.g., [46]. This cresyl staining procedure was identical to the staining preformed in the CP experiment (see above), only without the addition of any antibodies. Finally, in each brain, and for each region, we also measured nuclear diameters of 10–15 BrdU+ neurons, as described in [47], and calculated the mean ± SE. This variable was used in the calculation of the Abercrombie stereological correction equation [48] to accurately estimate the number of BrdU+ neurons per mm3. Biomolecules 2021, 11, 1069 8 of 17

2.5. Statistical Analysis All analyses were performed using JMP®, Version 14 software, (SAS Institute Inc., Cary, NC, USA, www.sas.com). CP and NR data were square-root transformed and those of MEL levels were log transformed to achieve normal distribution (Shapiro–Wilk test). Data were analysed using a repeated measures full-factorial mixed model, with ALAN as the between-subjects fixed factor and either section number, VZ wall, or brain region as the within-subject fixed variable. MEL levels for all groups were analysed by repeated measures mixed model analysis with ALAN as the between-subject factor and LD phase (day-before, night-before, day-during, and night-during) as the within-subject fixed variable. Bird identification was used as the random variable and Toeplitz covariance structure was used in the mixed model analyses. Individual one-way analyses followed whenever a significant interaction was found. Tukey’s post-hoc test was conducted to determine specific differences. Comparison of data between sexes were analysed using a mixed model with sex as a the between-subjects fixed factor.

3. Results 3.1. ALAN Increases Cell Proliferation in the VZ

Exposure to ALAN increased CP in males (F(3,15) = 8.51; p = 0.0009; mixed model analyses of the means of the values for all six sections, and dorsal and ventral VZ as the repeated factor; Figure6A,B). This increase was present in both the ventral (F (3,15) = 6.83, p = 0.0007) and the dorsal walls (F(3,15) = 8.99, p = 0.0014). This increase was signiﬁcant Biomolecules 2021, 11, x 9 of 18 only when exposing the birds to 5 lux: in sections 1–4 of the ventral wall (Figure6A), and in sections 3 and 5 of the dorsal wall (Figure6B).

Figure 6. Cell proliferation (number of cells/mm; mean mean ±± SE)SE) in in ventral ventral (A (A) )and and dorsal dorsal ( (BB)) walls walls of of the the VZ, VZ, in in brains of birds exposed to ALAN (1.5 and 5 lux) and controls that were exposed to complete darkness at night. Six sections were birds exposed to ALAN (1.5 and 5 lux) and controls that were exposed to complete darkness at night. Six sections were sampled along the rostral-caudal axis of the VZ (for details see Figure 4). Total neuronal densities (number of neu- sampled along the rostral-caudal axis of the VZ (for details see Figure4). Total neuronal densities (number of neurons/mm 3; rons/mm3; mean ± SE; (C)) in the brain regions MSt (medial striatum), NC (nidopallium caudale), HC (hippocampus), ± HVC,mean andSE; Area (C)) X, in of the birds brain exposed regions to 1.5- MSt and (medial 5 lux striatum),ALAN, an NCd controls (nidopallium that were caudale), exposed HC to complete (hippocampus), darkness HVC, at night. and GreyArea X,dots of indicate birds exposed individual to 1.5- data and points 5 lux, ALAN, different and letters controls indicate that weresignificant exposed differences to complete (Tukey darkness post hoc at night. tests, following Grey dots one-wayindicate individualANOVAs within data points, each section). different n letters= 6 birds indicate per group. signiﬁcant When no differences letters are (Tukey mentioned, post hocthere tests, is no following difference one-way among groups.ANOVAs within each section). n = 6 birds per group. When no letters are mentioned, there is no difference among groups.

3.2. ALAN Exposure Differentially Affects Total Neuronal Densities in Some Brain Regions after Short Term Exposure In the CP experiment, in which birds were exposed to three weeks of ALAN, total neuronal densities increased in the HVC (F(2,15) = 7.72, p = 0.0245) and decreased in Area X (F(2,15) = 6.46, p = 0.0332) in both the 1.5 lux and the 5 lux groups compared with control, whereas the MSt, NC and the HC were not affected by either of the ALAN intensities (Figure 6C).

3.3. ALAN Increases New Neuronal Recruitment in Some Brain Regions and Subregions

Overall, exposure to 5 lux ALAN increased the recruitment of new neurons (F(1,14) = 8.46, p = 0.0005; mixed model analyses of the combined data for all brain regions; Figure 7A). The increase was significant compared with control in the lMSt (F(1,14) = 4.83, p = 0.0061), mMSt (F(1,14) = 3.80; p = 0.0009), HVC (F(1,14) = 4.54, p = 0.0042) and Area X (F(1,14) = 4.48, p = 0.007), but not in the NC (F(1,14) = 4.07, p = 0.1871) and the HC (F(1,14) = 1.07, p = 0.1442). Biomolecules 2021, 11, 1069 9 of 17

3.3. ALAN Increases New Neuronal Recruitment in Some Brain Regions and Subregions

Overall, exposure to 5 lux ALAN increased the recruitment of new neurons (F(1,14) = 8.46, p = 0.0005; mixed model analyses of the combined data for all brain regions; Figure7A). The increase was signiﬁcant compared with control in the lMSt (F(1,14) = 4.83, p = 0.0061), mMSt (F(1,14) = 3.80; p = 0.0009), HVC (F(1,14) = 4.54, p = 0.0042) and Area X (F(1,14) = 4.48, Biomolecules 2021, 11, x 10 of 18 p = 0.007), but not in the NC (F(1,14) = 4.07, p = 0.1871) and the HC (F(1,14) = 1.07, p = 0.1442).

Figure 7. (A) Neuronal recruitment (number of new neurons mm3; mean ± SE) and (B) Total neuronal densities (number 3 of neurons/mmFigure3 ± 7.SE)( inA )the Neuronal lMSt (lateral recruitment medial striatum), (number mMSt (medial of new medial neurons striatum), mm NC; mean (nidopallium± SE) caudale), and (B ) Total HC (hippocampus),neuronal HVC densities and Area (number X, in brains of neurons/mmof birds exposed 3to± 5 luxSE) ALAN, in the and lMSt controls (lateral that were medial kept under striatum), com- mMSt plete darkness(medial at night. medial Grey striatum), dots indicate NC individual (nidopallium data points; caudale), *, **, ***—indica HC (hippocampus),te significant differences HVC and (p Area < 0.05, X, p in< brains 0.005, and p < 0.0005 respectively; Tukey post hoc tests following one-way ANOVAs for each section separately). n = 8 birds perof group. birds exposed to 5 lux ALAN, and controls that were kept under complete darkness at night. Grey dots indicate individual data points; *, **, ***—indicate signiﬁcant differences (p < 0.05, p < 0.005, and 3.4. ALAN Decreases Total Neuronal Densities but Only in Area X after Long Term Exposure p < 0.0005 respectively; Tukey post hoc tests following one-way ANOVAs for each section separately). Total neuronal density decreased significantly in Area X of birds that were exposed n = 8 birds per group. to ALAN for six weeks (NR experiment) compared with control (F(1,14) = 9.72, p = 0.0121; Figure 7B). No effect of ALAN on total neuronal densities was observed in any of the other four regions.

3.5. Total Neuronal Densities Relative to the Control Decreased in the HVC as a Function of ALAN Exposure Duration The effect of ALAN exposure duration on total neuronal densities was compared in the two regions related to vocalization, the HVC and Area X. When adjusted to controls, Biomolecules 2021, 11, 1069 10 of 17

3.4. ALAN Decreases Total Neuronal Densities But Only in Area X after Long Term Exposure Total neuronal density decreased signiﬁcantly in Area X of birds that were exposed to ALAN for six weeks (NR experiment) compared with control (F(1,14) = 9.72, p = 0.0121; Figure7B). No effect of ALAN on total neuronal densities was observed in any of the other four regions.

3.5. Total Neuronal Densities Relative to the Control Decreased in the HVC as a Function of Biomolecules 2021, 11, x ALAN Exposure Duration 11 of 18

The effect of ALAN exposure duration on total neuronal densities was compared in the two regions related to vocalization, the HVC and Area X. When adjusted to controls, totaltotal neuronal neuronal densities densities in in the the HVC HVC was was lower lower in birdsin birds that that were were exposed exposed to longer to longer duration dura- of ALAN exposure (six weeks) compared with short duration (three weeks; F(3,15) = 4.55, tion of ALAN exposure (six weeks) compared with short duration (three weeks; F(3,15) = p = 0.0001; Figure8). We did not detect an effect of ALAN duration on the total neuronal 4.55, p = 0.0001; Figure 8). We did not detect an effect of ALAN duration on the total neu- densities in Area X (F(1.14) = 11.55, p = 0.0824; Figure8). ronal densities in Area X (F(1.14) = 11.55, p = 0.0824; Figure 8).

3 FigureFigure 8. 8.Total Total neuronal neuronal densitiesdensities (adjusted(adjusted to controls; number number of of neurons/mm neurons/mm mean±SE)3 mean ± inSE) HVC in and Area X, in brains of birds exposed to short (three weeks) and long (six weeks) term 5lux ALAN. HVC and Area X, in brains of birds exposed to short (three weeks) and long (six weeks) term 5lux *—indicates a significant difference of p < 0.0001; Tukey post hoc tests following one-way ANOVAs ALAN. *—indicates a signiﬁcant difference of p < 0.0001; Tukey post hoc tests following one-way for each brain region separately. n = 8 birds/group. ANOVAs for each brain region separately. n = 8 birds/group.

3.6.3.6. ALAN ALAN Reduces Reduces Nocturnal Nocturnal Melatonin Melatonin Levels Levels DiurnalDiurnal and and nocturnal nocturnal MEL MEL plasma plasma concentrations concentrations before before and and during during ALAN ALAN expo- expo- suressures are are presented presented in in Figure Figure9. 9. Overall, Overall, as as expected, expected, diurnal diurnal MEL MEL levels levels were were lower lower than than (2,14) thethe nocturnal nocturnal ones ones inin bothboth thethe CP and the the NR NR experiments experiments (F (F(2,14) = 151.557,= 151.557, p =p 0.0091,= 0.0091, and (1,20) andF F(1,20) = 216.557,= 216.557, p = 0.0042,p = 0.0042, respectively). respectively). Similarly, Similarly, in each in each experiment, experiment, before before ALAN ALAN ex- exposure,posure, when when all all groups groups experienced complete darkdark nights,nights, nocturnalnocturnal MELMEL levels levels were were similarsimilar across across groups.groups. However, However, in in both both experi experiments,ments, when when the theexperimental experimental groups groups were wereexposed exposed to ALAN, to ALAN, nocturnal nocturnal MEL MELlevels levels were werereduced reduced compared compared to the tocontrols the controls (F(2,20) = (F2.618,(2,20) = p 2.618,= 0.0111p = in 0.0111 the CP in theexperiment, CP experiment, FigureFigure 9A; and9A; F and(1,14) = F 3.425,(1,14) = p 3.425, = 0.0223p = 0.0223in the NR in theexperiment; NR experiment; Figure Figure9B). 9B). Biomolecules 2021, 11, 1069 11 of 17 Biomolecules 2021, 11, x 12 of 18

FigureFigure 9. 9.Day Day and and night night plasma plasma melatoninmelatonin levelslevels (pg/mL;(pg/mL; Mean ±± SE)SE) before before and and after after exposu exposurere to to ALAN ALAN (1.5, (1.5, 5 5lux), lux), compared with controls that were kept under dark nights in (A) proliferation (n = six birds per group); and (B) neuronal compared with controls that were kept under dark nights in (A) proliferation (n = six birds per group); and (B) neuronal recruitment (n = eight birds per group) experiments. Different letters indicate significant differences (p < 0.05, Tukey post recruitmenthoc tests following (n = eight one-way birds per ANOVAs group) experiments.between groups). Different Groups letters with indicateno letters signiﬁcant are not significantly differences different. (p < 0.05, Tukey post hoc tests following one-way ANOVAs between groups). Groups with no letters are not signiﬁcantly different.

3.7.3.7. ALANALAN Does Not Affect Body Mass NoNo differencesdifferences were found in the body body mass mass between between the the ALAN ALAN and and control control groups, groups, beforebefore and after exposure exposure to to ALAN ALAN for for either either three three or six or sixweeks. weeks. Body Body weights weights were were13.4 13.4± 0.32± g0.32 in the g in control the control group; group; 13.5 ± 13.5 0.31± g 0.31in the g in1.5 the lux 1.5 ALAN lux ALAN group; group; 13.3 ± 0.33 13.3 g± in0.33 the g 5 lux ALAN group (n = six birds per group) after three weeks exposure (F(1,19) = 1.59, p = in the 5 lux ALAN group (n = six birds per group) after three weeks exposure (F(1,19) = 1.59, p0.8142);= 0.8142); 12.4 12.4 ± 0.31± 0.31g in gthe in control the control group; group; and and13.1 13.1± 0.32± g0.32 in the g in 5 thelux 5ALAN lux ALAN group group (n = eight birds per group) after six weeks (F(1,15) = 2.56, p = 0.7141). (n = eight birds per group) after six weeks (F(1,15) = 2.56, p = 0.7141).

4.4. DiscussionDiscussion 4.1.4.1. ALANALAN IncreasesIncreases Cell Proliferation in the VZ Overall,Overall, inin allall three groups (control, 1.5 lux, and 5 5 lux), lux), CP CP in in the the ventral ventral VZ VZ wall wall was was higherhigher thanthan inin thethe dorsaldorsal wall, similar to what was previously previously found found in in canaries canaries (Serinus (Serinus canariacanaria)[) [49],49], andand inin femalefemale zebra finches finches [6]. [6]. TheThe ALANALAN intensities that we used used in in the the present present study, study, 1.5 1.5 and and 5 5 lux, lux, were were chosen chosen becausebecause of their ecological ecological relevance relevance [49]. [49 ].Howe However,ver, exposure exposure of male of male zebra zebra finches finches to 1.5 to 1.5lux lux ALAN ALAN did did not not affect affect CP, CP, unlike unlike the the increase increase that that we we previously previously observed observed in in females females whichwhich werewere keptkept underunder identicalidentical conditions [6 [6].]. Only Only exposure exposure to to 5 5 lux lux ALAN ALAN increased increased CPCP in specific specific locations locations along along the the rostral-caudal rostral-caudal axis axis of the of theVZ VZin males in males in both in bothwalls walls(alt- (althoughhough more more so in so the in ventral). the ventral). This result This echoes result echoesour findings our findings in females in [6]. females Our previous [6]. Our previoussuggestion suggestion that an increase that an increasein cell proliferation in cell proliferation might be might caused be by caused perturbation by perturbation of the ofcircadian the circadian rhythm rhythm under ALAN, under ALAN,and is mediated and is mediated by MEL by[6], MEL might [6 ],hold might here hold as well. here asHowever, well. However, this suggestion this suggestion does not explain does not why explain in males, why unlike in males, females, unlike no females,differences no differenceswere observed were under observed 1.5 lux under ALAN. 1.5 The lux fact ALAN. that in The males fact only that the in higher males ALAN only the intensity higher ALANaffected intensity cell proliferation affected cellmight proliferation indicate that might in this indicate respect, that males in this are respect,more resilient males areto moreALAN resilient than females. to ALAN At this than stage, females. we cannot At this explain stage, wewhy. cannot However, explain if intersexual why. However, differ- if intersexualences occur differencesalso in other occur avian also species, in other it should avian be species, taken itinto should account be takenwhen intoconducting account whenecological conducting studies. ecological studies. InIn thethe ventralventral wall of the VZ, VZ, the the increase increase of of cell cell proliferation proliferation under under exposure exposure to to 55 lux lux ALANALAN waswas significantsignificant in the four more rostral rostral sections. sections. This This seems seems to to be be a ashift shift rostrally, rostrally, comparedcompared toto thethe patternpattern that we previously found in in females females ([6], ([6], upper upper panel panel in in FigureFigure 44 there).there). A rostral-caudal pattern pattern of of cell cell prol proliferationiferation in in the the VZ VZ was was already already reported reported in in songbirdssongbirds (canaries(canaries [[50]),50]), and was explained by by the the need need to to supply supply ne neww neurons neurons to to nearby nearby neurogenicneurogenic brain regions. regions. Accordingly, Accordingly, the the more more rostral rostral increased increased proliferation proliferation in the inven- the ventraltral VZ VZin males in males might might be due bedue to Area to Area X, which X, which exists exists only only in males, in males, and is and located is located in the in therostral rostral part part of the of the brain brain (Figure (Figure 5)5 and) and corre correspondssponds to to our our Section Section 11 (Figure (Figure 44).). However, Biomolecules 2021, 11, 1069 12 of 17

in the dorsal VZ wall, the increased proliferation under ALAN in males seems to occur more caudally compared to that in females ([6], upper panel in Figure4), although the same brain regions (such as the HC) are located dorsally to it in both sexes. Therefore, the explanation that increased proliferation answers the need to supply new neurons to nearby regions might not hold.

4.2. ALAN Increases Neuronal Recruitment in Some Brain Regions Overall, exposure of males to 5 lux ALAN caused an increase in new neuronal recruitment in several brain regions. The two nuclei that are part of the song-system and are male-specific, HVC and Area X, seem to be sensitive to ALAN, as evident by the increase in neuronal recruitment in these brain regions (Figure7A). HVC is involved in vocal learning and song production [51], and Area X is important for song learning and maintenance [52,53]. Therefore, any changes in the homeostasis and functionality of these two regions might affect song production and structure, and that, in turn, might have direct fitness consequences [21]. It could be that the effect of ALAN on neuronal recruitment in these regions is mediated by the observed suppression of nocturnal MEL levels. This is because MEL receptors were found in the song control system in several bird species [54–56], and in European starlings (Sturnus vulgaris) exogenous MEL attenuated the long-day-induced volumetric increase in HVC [57]. Taken together, these evidences suggest a functional role for MEL in brain plasticity. MSt (which includes lMSt and mMSt) exhibited increased recruitment in males. The MSt is part of the somatomotor basal ganglia in birds, plays a role in visual perception, and receives inputs from the substantia nigra pars compacta and from regions involved in somatosensory, visual, auditory, and motor function, as reviewed in [7]. Therefore, we previously suggested [7] that the increased recruitment in this region under ALAN might be due to increased nocturnal locomotor activity, caused by sleep disruption, which has been reported in various avian species including zebra finches, e.g., [48]. Support for this suggestion comes also from our yet-unpublished behavioral data, indicating that zebra finches that are exposed to ALAN are more active during the night than control birds that are kept under dark nights. MSt exists in both sexes, and in a previous study [7] we found a similar effect of increased recruitment of new neurons also in females. However, it is interesting to note that the effect was more robust in females than in males, and this difference is demonstrated in Figure 10A: 110% more than the corresponding control (calculated from Figure1 in [ 7]), vs. 49% (based on Figure7A in the present study), accordingly. This difference might indicate that the MST of males is more resilient to ALAN than that in females. Another indication that some brain regions in males might be more resilient to ALAN than those in females, is that neuronal recruitment in the two other tested brain regions (NC and HC) was not affected by ALAN in males, unlike our previous findings in females [7]. This sex difference which is overall significant (F(1,15) = 15.848; p < 0.0005), is also demonstrated in Figure 10A. Biomolecules 2021, 11, 1069 13 of 17 Biomolecules 2021, 11, x 14 of 18

Figure 10. (A) Percent change in neuronal recruitment (number of new neurons/mm3; mean ± SE) Figure 10. (A) Percent change in neuronal recruitment (number of new neurons/mm3; mean ± SE) compared to corresponding control, and (B) percent change in total neuronal densities (number of compared to corresponding control, and (B) percent change in total neuronal densities (number of neurons/mm3 ± SE), in MSt, NC and HC, in brains of female and male zebra finches exposed to 5 lux 3 ± neurons/mmALAN for 6 weeks.SE), Female in MSt, data NC taken and HC, from in [7]. brains N = of 8 femalefor each and sex. male **, ***—indicate zebra finches significant exposed todif- 5ferences lux ALAN (p < for 0.005, 6 weeks. and p Female < 0.0005 data respectively; adapted from Tukey Ref. post [7]. hoc N =tests 8 for following each sex. one-way **, ***—indicate ANOVAs significantfor each section differences separately). (p < 0.005, and p < 0.0005 respectively; Tukey post hoc tests following one-way ANOVAs for each section separately). 4.3. Differential Effects of the Duration of Exposure to ALAN on Total Neuronal Densities 4.3. Differential Effects of the Duration of Exposure to ALAN on Total Neuronal Densities In birds, new neurons that are recruited in various brain regions replace older ones In birds, new neurons that are recruited in various brain regions replace older ones that that die [13,58], and the assumption is that the total number of neurons within a region die [13,58], and the assumption is that the total number of neurons within a region remains remains constant. In three of the tested regions (MSt, NC, and HC) we did not observe constant. In three of the tested regions (MSt, NC, and HC) we did not observe any change any change in total neuronal densities compared to controls, both in short (three weeks; in total neuronal densities compared to controls, both in short (three weeks; Figure6C ) Figure 6C) and in long-term (six weeks; Figure 7B) exposure to ALAN, despite the in- and in long-term (six weeks; Figure7B) exposure to ALAN, despite the increased NR in creased NR in MSt, possibly due to higher turnover rate in these regions under ALAN. By MSt, possibly due to higher turnover rate in these regions under ALAN. By contrast, in contrast, in the two male-specific regions (HVC and Area X) ALAN affected total neuronal the two male-specific regions (HVC and Area X) ALAN affected total neuronal densities. Althoughdensities. both Although HVC both and AreaHVC Xand are Area part X of are the part song of the system song in system birds in [31 birds,32], the[31,32], effect the ofeffect ALAN of ALAN on their on total their densities total densities varied. varied. In HVC In thereHVC wasthere a was temporary a temporary increase increase in the in short-termthe short-term exposure exposure (Figure (Figure5), which 5C), disappeared which disappeared in the longer in the term, longer as neuronal term, as densities neuronal weredensities then comparablewere then comparable to control levels to control (Figures levels7B (Figures and8). This 7B and outcome, 8). This which outcome, occurred which despiteoccurred the despite increase the in increase neuronal in recruitment, neuronal recruitment, indicates a indicates possible fastera possible neuronal faster turnover. neuronal Byturnover. contrast, By Area contrast, X was theArea only X was region the that only did region not retain that itsdid total not densitiesretain its undertotal densities ALAN, whichunder decreased ALAN, which compared decreased to controls compared both to in cont therols short- both (Figure in the6C) short-and (Figure the long-term 6C) and (Figuresthe long-term7B and8 (Figures) exposure. 7B Thisand 8) outcome exposure. might This be outcome because apoptosismight be because exceeded apoptosis neuronal ex- recruitment,ceeded neuronal despite recruitment, the fact that despite the latter the increasedfact that the compared latter increased to controls. compared We currently to con- dotrols. not We have currently an explanation do not tohave these an variedexplanatio effectsn to of these ALAN varied on total effects densities of ALAN in the on two total regionsdensities that in are the part two of regions the song-system, that are part and of future the song-system, studies should and record future singing studies behavior should Biomolecules 2021, 11, 1069 14 of 17

under ALAN and its potential effects on mating success. Investigating song production and structure under ALAN conditions might be interesting since, as mentioned above, MEL binding sites were found in song control nuclei of several species, and there is a strong relation between MEL and song [59]. Such information will help to understand the interplay between ALAN, MEL, neuronal plasticity, and song production, and might yield information of ecological relevance regarding conservation of bird species. An intersexual comparison regarding the effect of ALAN on total neuronal densities raises again the possibility that males are more resilient than females (as indicated in Figure 10B;F(1,15) = 4.848; p < 0.02). This is due to the fact that, unlike the situation in males, in which total densities in MSt, NC, and HC remained similar to controls both in the short (Figure6C) and long-term (Figures7B and 10B), in females we observed a decrease in the NC and the MSt in the short-term (Figure 4 in [6]) and an increase in HC in the long-term in females (Figure 3 in [7]). These differential sex effects on total densities, despite the similar positive effects on cell proliferation and neuronal recruitment, might indicate different levels of apoptosis between the sexes.

4.4. Effects of ALAN on Nocturnal Melatonin Levels MEL levels in our control groups were comparable to previously published data for this species [60]. In all groups that were exposed to ALAN, nocturnal MEL levels were lower compared to the controls that were exposed to dark nights, and this is in line with the MEL suppression previously observed by others in great tits [61], and in female zebra ﬁnches [6,7]. Under control (dark nights) conditions, females and males had similar MEL levels (417 ± 22 pg/mL and 407 ± 19.5 pg/mL, respectively; the females’ concentration is calculated from the means of the control groups before ALAN exposure in [6,7], and the males’ concentration is calculated from the respective means in the present study). This indicates that under normal conditions there are no intersexual differences in MEL levels. However, when exposed to ALAN, there seems to be a difference between the sexes in their response to various intensities. Females showed about a 50% decrease in MEL levels between control and both 1.5 and 5 lux ALAN, with no difference between the two latter ones [6]. However, males exhibited a more gradual decrease, of about 30% between control and 1.5 lux ALAN, and then a 45% decrease between 1.5 and 5 lux ALAN (this study). This difference supports our suggestion that males are more resilient to ALAN, at least in low ALAN intensities.

5. Conclusions Our study indicates that ALAN affects the brains of male zebra finches. This is demonstrated by the increased CP in the VZ and increased NR in several brain regions, compared to controls that were kept under dark nights. In addition, we found, for the first time, that song nuclei were highly affected by ALAN, which caused an increased in NR in these regions. In line with our previous observations in females, we can conclude that ALAN similarly affects both sexes in zebra finches. However, there were several indications that males might be more resilient to ALAN exposure than females, for example in regards to the effect of ALAN on nocturnal MEL levels, which were affected less in males than in females by low intensity. The effects of the duration to ALAN exposure on total neuronal densities in the studied regions varied between the sexes and between the song nuclei in males, suggesting different levels of apoptosis. Taken together, these findings emphasize the importance of studying both sexes when considering environmental effects on brain plasticity and lay the ground for the next challenges in our understanding of the underlying mechanisms of ALAN and MEL effects on neuronal plasticity and, in turn, on behavior. We believe that more work should be done in both sexes in order to complete the picture. For example, our results do not distinguish how ALAN is perceived by the birds, whether it is through the eyes or the pineal gland. This question can be tested by experimentally blocking the pineal gland, or by using monochromatic light that is less likely to reach through the skull. Other interesting future directions include recording apoptosis, exposing Biomolecules 2021, 11, 1069 15 of 17

birds to various durations of ALAN (including life-long exposure), and investigating various behavioral patterns such as nocturnal activity and song.

Author Contributions: Conceptualization, A.B. and S.M.; methodology, A.B., S.M., M.O., and M.Z.; validation, A.B., S.M., M.O., and M.Z.; formal analysis, R.H.; investigation, S.M., M.O., and M.Z.; resources, A.B. and M.Z.; writing—original draft, A.B., S.M., and R.H.; writing—review & editing, all co-authors; visualization, S.M., A.B., and R.H.; supervision, A.B., I.S., A.H., and M.Z.; project administration, A.B. and S.M.; Funding acquisition, A.B. and M.Z. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by The Israel Science Foundation, grant number 132/18, The Slovak Research and Development Agency, grant number APVV-17-0178, and The Open University of Israel Research Fund. Institutional Review Board Statement: This study was approved by the Tel-Aviv University Insti- tutional Animal Care and Use Committee (permit No. 04-18-075, issued on 31.12.2018) and was carried out in accordance with its regulations and guidelines regarding the care and use of animals for experimental procedures. Acknowledgments: Thanks are due to Yulia Vistoropsky, and the staff of the I. Meier Segals Garden for Zoological Research at Tel Aviv University for their technical help. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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