Socialization causes long-lasting behavioral changes

Socialization causes long-lasting behavioral changes | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Socialization causes long-lasting behavioral changes Beatriz Gil-Martí , Julia Isidro-Mézcua , Adriana Poza-Rodriguez , Gerson S. Asti Tello , Gaia Treves , View ORCID Profile Enrique Turiégano , View ORCID Profile Esteban J. Beckwith , View ORCID Profile Francisco A Martin doi: https://doi.org/10.1101/2024.04.25.591071 Beatriz Gil-Martí 1 Cajal Institute, Spanish National Research Council (CSIC) , Av Dr Arce 37, 28002 Madrid, Spain 2 Department of Biology, Autonomous University of Madrid , Madrid, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site Julia Isidro-Mézcua 1 Cajal Institute, Spanish National Research Council (CSIC) , Av Dr Arce 37, 28002 Madrid, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site Adriana Poza-Rodriguez 1 Cajal Institute, Spanish National Research Council (CSIC) , Av Dr Arce 37, 28002 Madrid, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site Gerson S. Asti Tello 3 Instituto de Fisiología, Biología Molecular y Neurociencias (IFIBYNE), UBA-CONICET , Buenos Aires, Argentina Find this author on Google Scholar Find this author on PubMed Search for this author on this site Gaia Treves 1 Cajal Institute, Spanish National Research Council (CSIC) , Av Dr Arce 37, 28002 Madrid, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site Enrique Turiégano 2 Department of Biology, Autonomous University of Madrid , Madrid, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Enrique Turiégano Esteban J. Beckwith 3 Instituto de Fisiología, Biología Molecular y Neurociencias (IFIBYNE), UBA-CONICET , Buenos Aires, Argentina Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Esteban J. Beckwith For correspondence: ebeckwith{at}fbmc.fcen.uba.ar famartin{at}cajal.csic.es Francisco A Martin 1 Cajal Institute, Spanish National Research Council (CSIC) , Av Dr Arce 37, 28002 Madrid, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Francisco A Martin For correspondence: ebeckwith{at}fbmc.fcen.uba.ar famartin{at}cajal.csic.es Abstract Full Text Info/History Metrics Supplementary material Preview PDF SUMMARY In modern human societies, social isolation acts as a negative factor for health and life quality. On the other hand, social interaction also has profound effects on animal and human behaviors, reducing aggressiveness, feeding and loss of sleep. Here, we observe that in the fly Drosophila melanogaster these behavioral changes long-last even after social interaction has ceased, suggesting that the socialization experience triggers behavioral plasticity. We find that impairing long-term memory mechanisms either genetically or by anesthesia abolishes the expected behavioral changes in response to social interaction. Furthermore, we show that socialization increases CREB-dependent neuronal activity and synaptic plasticity in the mushroom body, the main insect memory center analogous to mammalian hippocampus. We propose that social interaction triggers socialization awareness, understood as long-lasting changes in behavior caused by experience with mechanistic similarities to long-term memory formation. INTRODUCTION Most animals live in social contexts. In our modern human society, the feeling of loneliness is increasing despite the technological advances in social media and communication 1 . The prolonged absence of social interaction has detrimental effects on quality of life, lifespan and several health problems 2 , 3 . In Drosophila melanogaster , social interaction strongly modulates several behaviors, diminishing male-to-male aggression, decreasing food consumption and, depending on the context, increasing or decreasing sleep, among others 4 . Socialization impacts several parallel modulatory systems 5 . In particular, activity-regulated genes in dopaminergic neurons modulate aggression and sleep in response to social enrichment 6 – 8 . Key clusters of dopaminergic neurons are also essential components of learning and memory circuits 9 , since they innervate the main Drosophila memory structure, the mushroom body (MB) 10 . At the molecular level, long-term memory (LTM) formation in the MB requires rutabaga (rut-adenylate cyclase) and d unce ( dnc - cAMP phosphodiesterase) gene functions, in order to adequately regulate cAMP levels and ensure neuronal plasticity 11 . cAMP signaling mediates CREB (c-AMP response binding element) phosphorylation. CREB is a conserved transcription factor that is key to form long-term memory and synaptic plasticity, among many other processes 11 , 12 . Social interaction causes structural changes in the MB, an effect that is abolished in mutant flies for memory-related genes like rut and dnc 13 , 14 . Furthermore, the function of such genes is necessary for immediate sleep changes triggered by social interaction 15 , 16 . In this work, we inquired if socialization was able to generate long-lasting changes on behavior, and addressed how these changes are associated with synaptic plasticity. We showed that underlying mechanisms have similarities with LTM: an altered behavior in response to experience lasted for 8 or more hours after the training; it depended on cAMP levels and was blocked by anesthesia; and ultimately, it correlated with changes in number of CREB-responsive neurons and synapses. In summary, we propose that socialization awareness modifies long-term behavior sharing some underlying mechanisms that are characteristic of long-term memory processes. RESULTS LONG-TERM SOCIALIZATION-INDUCED FEEDING BEHAVIOR REQUIRES cAMP SIGNALING Flies that experienced social interaction show reduced food consumption when compared with flies that were socially reared and posteriorly isolated 17 . We used single-fly CApillary FEeding -sCAFE-assay (modified from 18 ) to extend these findings. We compared grouped flies with animals singly reared since eclosion, meaning that they were socially naive. As expected, there was a significant decrease in food uptake of 5-day socialized flies when compared to individual flies in the immediate 24 hours (0 h-24 h time window) ( fig 1A ). Next, to determine if such feeding effect is maintained even in the absence of social interaction, we slightly modified the socially-enriched paradigm: flies were group- or single-reared for 5 days and then animals from both experimental groups were kept isolated for additional 24 hours previous to assessing feeding ( fig 1B ). Using this protocol, we also detected a decreased food consumption of grouped flies in the 24 h-48 h time window, confirming a long-lasting effect of social interaction on feeding behavior ( fig 1C ). We reasoned that the most plausible candidate genes to play a role for such long-lasting effect would be memory-related genes, such as rutabaga ( rut ) 19 . Despite their past experience, isolated rut mutant flies in the 24 h-48 h period after socialization showed no differences in food intake with solitaire animals since eclosion ( fig 1C ). Besides, rut mutant flies do not change their feeding behavior during the first 24 hours (0 h-24 h), suggesting a requirement of cAMP for this response ( fig 1A ). To confirm the involvement of cAMP signaling we repeated the sCAFE assay in animals mutant for dunce ( dnc ). Results were comparable to rut mutant: dnc mutant flies fail to modify their food consumption not only during the first 24 hours after socialization (0 h-24 h) but also according to previous experience, in the 24 h-48 h period ( fig 1A, C ). Strikingly, feeding behavior of memory-related mutant animals lies in an intermediate state between socialized and isolated flies, maybe suggesting that their basal food consumption is different from wild type strain (see below). We propose that this long-lasting effect of social interaction on (feeding) behavior indicates a that animals have a socialization awareness that lasts even after social interaction has ceased. Download figure Open in new tab Figure 1. Reduced food consumption induced by socialization depends on memory-related genes. (A) Quantification of food consumption of wt , rut and dnc mutant flies in socialized and isolated conditions (single fly CAFE assay) in the 0-24 h time window (Kruskal-Wallis chi-squared = 75.905, df = 5, p-value = 6.022e-15; post hoc Dunn comparisons: wt social |wt isolated p = 6.24e-13, rut social | rut isolated p = 1.00, dnc social | dnc isolated = 1.00). (B) Scheme of the socialization protocol: recently eclosed animals were either grouped or isolated for five days, and subsequently isolated for additional 24 hours before tested. C) Quantification of food consumption of wt , rut and dnc mutant flies in socialized and isolated conditions (sCAFE) in the 24 h-48 h h time window (Kruskal-Wallis chi-squared = 32.698, df = 5, p-value = 4.32e-06; post hoc Dunn comparisons: wt social |wt isolated p = 1.04e-03, rut social | rut isolated p = 1.00, and, in fig S1, dnc social | dnc isolated = 1.00). LONG-TERM EFFECT OF SOCIALIZATION ON SLEEP IN ABSENCE OF INTERACTION Sleep (particularly daytime sleep) is also regulated after social interaction. When flies are kept in mixed-sex groups immediately at eclosion, sleep increases 15 . However, courtship experience inhibits sleep in male flies 20 , 21 , which lasts for several hours, proving a complex regulation of sleep by social cues and experiences. Most published sleep studies employ Drosophila Activity Monitors (DAMs), which only detect movement when the fly crosses a midpoint sensor in the housing tube 22 , overestimating actual sleep time 17 . The ethoscope was developed to unequivocally identify immobility periods and assess sleep 23 . We confirmed that social interaction also induced animals to sleep more 15 ( fig 2A ). To ensure consistency with previous works 17 , we focused on the first four hours after lights ON, where the effect is unambiguous and reproducible (i.e. 24 h-28 h time window, ZT0-ZT4, fig 2A ). Sleep quantification shows a significant difference between social-enriched and lonely animals in this 24 h-28 h period ( fig 2B ), in line with previous publications 15 , 17 . Furthermore, rut mutant animals showed no significant difference in 24 h-28 h sleep time ( fig 2A, B ), although socialized dnc mutants did exhibit a significant sleep difference in the 24 h-28 h, ( fig 2C ). Intriguingly, memory-related mutant flies sleep considerably more than their wt counterparts, suggesting additional levels of sleep regulation related to cAMP signaling ( fig 2A-C ). Download figure Open in new tab Figure 2. Sleep behavior after socialization is long-term and relies on rut . (A) Sleep profile and (B) sleep quantification of the 24 h-28 h time window for wt and rut mutant background (Kruskal-Wallis chi-squared = 94.165, df = 3, p < 2.2e-16; post hoc Dunn comparisons: wt social |wt isolated p = 1.07e-02, rut social | rut isolated p = 0.438). (C) Sleep quantification of the 24 h-28 h time window for wt and dnc mutant background (Kruskal-Wallis chi-squared = 36.476, df = 3, p-value = 5.94e-08; post hoc Dunn comparisons: wt social |wt isolated p = 3.43e-02, dnc social |dnc isolated p = 3.48e-03). (D) Scheme of the sleep time course: flies were either isolated or grouped after eclosion for 7, 6 or 4 days and subsequently isolated for 0, 1 or 3 days (named as socialized, 6+1, 4+3 and constant isolation); after introducing them in ethoscopes, sleep behavior was recorded for 3 days. (E) Sleep profile of animals isolated for 1 to 4 days, using isolated flies as control. Total number of days in isolation for E-I is depicted in the panel. (F) Quantification of sleep from ZT0 to ZT4 for day 1-4 and flies under constant isolation (CI). (G-I) Analysis of bout length (G), total number of bouts (H) and latency to first bout (I) from ZT0 to ZT12 for day 1-4 and animals under CI. Our results with dnc and rut mutant flies were apparently contradictory with those previously described using DAMs 15 . However, the ethoscope offers the possibility of analyzing data as they were extracted from DAMs, thus depicting comparable results to published DAM data. This virtual DAM analysis did render a significant difference between rut mutant grouped and single-reared flies, whereas no sleep changes were apparent in dnc mutant animals, in agreement with previous studies (fig S1) 15 . The differing results obtained depending on the type of analysis (regular or virtual DAM) stem from the higher sensitivity of ethoscopes to movement. It also explains why the increased sleep behavior of memory-mutant flies remained unnoticed until now, given that DAMs cannot detect such changes ( 24 and fig S2). Nevertheless, in either case, our data and previous work support the idea that cAMP regulation, necessary for synaptic plasticity, is needed to sustain long-lasting changes in sleep triggered by socialization awareness. We wondered how long animals that experienced a socially-enriched environment maintained such sleep behavior in absence of social interaction. To compare animals of the same age, we socialized flies for 7, 6 or 4 days (which is enough socialization time in order to generate a sleep effect 15 ) and subsequently isolated them for 0, 1 or 3 additional days (named as socialized, 6+1 or 4+3, respectively). Continuously isolated animals were used as control. Then, their sleep behavior was recorded for the following 3 days (i.e. depicted in fig 2D ). In the framework of this experimental approach, we could compare continuously isolated flies with animals isolated for 1 to 4 days after socialization ( fig 2E ). We could observe a progressive reduction of sleep time in the ZT0-ZT4 after isolation, with significant decrease at day 4 that was comparable to continuous isolation ( fig 2F ). Thus, 4 days of isolation are enough to modify sleep reaching similarly sleep levels than socially naive flies, in contrast to the need of 5 days described previously using DAMs 17 . The ethoscope also allows a detailed sleep analysis regarding bout length, the total number of bouts and the latency to first bout in a 12-h analysis (ZT0-ZT12). There were no differences in the sleep bout length amongst experimental groups. In contrast, isolated flies for 4 days reduced the number of sleep bouts to similar levels than the ones from socially naive animals, despite we noticed a progressive reduction but still statistically significative ( fig 2H ). Intriguingly, the latency to the first bout in grouped flies remained similar up to day 3, where it raised sharply, similar to the latency of isolated flies ( fig 2I ). We conclude that the effect of socialization lasts at least for 3 days, and indeed, it can be considered as long-term. SOCIALIZATION-INDUCED DIMINISHED AGGRESSIVENESS DEMANDS cAMP SIGNALING In isolated flies that previously experienced social interaction, isolation signals starvation and, as a consequence, increases feeding and decreases sleep, meaning that both behavioral changes are reciprocally related 17 . We wondered if socialization awareness was also evident in a different social behavior. Previous data showed that 5-day grouped male flies since eclosion were less aggressive than their single-reared counterparts when tested immediately after the treatment 25 . This behavior also allowed us to determine the progression of long-lasting effects, in order to compare the temporal requirements of social interaction with those of classical learning and memory assays. Thus, we employed animals from both experimental conditions and then evaluated aggression after different isolation periods in a well-established paradigm 26 . Socially-experienced flies showed reduced aggression (i.e. measured as the proportion of time lunging) at 1, 4, 8 and 24 hours after isolation when compared with single-reared animals ( fig 3 ), evidencing a behavioral change at short- and long-term. Despite social interaction had ceased up to 24 h before, grouped flies still spent considerably less time fighting than lonely flies ( fig 3 ), confirming that socialization awareness is a general feature of socialization. Critically, rut mutant flies did not show any difference according to their previous experience ( fig 3 ), reinforcing the role of memory-related genes. Besides, our data revealed that at short-term (one hour), grouped rut mutant animals were significantly less aggressive than rut single-reared flies, thus differentiating from rut requirements in classical learning assays. However, this rut-independent effect disappeared after four hours, suggesting that social interaction imprinted a temporary effect independently of cAMP, but long-term socialization consequences in aggression depends on rut activity. Overall, rut mutant flies showed decreased levels of aggression, which makes comparison with wt animals difficult, as previously noticed 27 . Download figure Open in new tab Figure 3. Socialized-reduced aggression shows short- and long-term effect. Quantification of proportion of time expend lunging after different times of re-isolation. Flies either wt or in a rut mutant background were grouped or isolated for 5 days and then socialized flies were tested after 1, 4, 8 or 24 hours after isolation (Kruskal-Wallis chi-squared = 139.99, df = 9, p-value < 2.2e-16, post hoc Dunn comparisons: wt 24h_after_social |wt isolated p = 1.08e-07, wt 8h_after_social |wt isolated p = 1.61e-10, wt 4h_after_social |wt isolated p = 1.88e-05, wt 1h_after_social |wt isolated p = 2.24e-09, rut 24h_after_social |rut isolated p = 1.00, rut 8h_after_social |rut isolated p = 0.815, rut 4h_after_social |rut isolated p = 0.598, rut 1h_after_social |rut isolated p = 7.52e-10). ANESTHESIA ABOLISHES SOCIALIZATION EFFECTS Anesthesia blocks long-term memory consolidation in most species 28 , 29 . In Drosophila , a 2-min cold shock acts as anesthetics and is able to impede long-term memory in the classical aversive olfactory conditioning assay 30 . We wondered if anesthesia was also able to block socialization awareness. We exposed adult flies to 3-min cold shock two times per day to single and grouped flies during the training period ( fig 4A ). Both experimental “cold-shocked” groups did not show any significant differences in food consumption in the 24 h-48 h time window after isolation, in contrast to non-shocked control animals ( fig 4B ). Given the reciprocal relationship between feeding and sleep behavior regarding social interaction 17 , we confirmed that sleep between lonely and socialized animals in the 24 h-28 h time window also remained similar after cold shock ( fig 4C, D ). As expected, in non-shocked animals the difference was statistically significant ( fig 4C, D ). In summary, we found that socialization awareness relies on cAMP signaling and is blocked by anesthesia, as it occurs in long-term memory. Download figure Open in new tab Fig 4. Anesthesia abolishes socialization effects on sleep and food consumption. (A) Scheme of the cold-shock protocol (twice per day). (B) Quantification of food consumption using sCAFE (Kruskal-Wallis chi-squared = 15.954, df = 3, p-value = 1.16e-3; post hoc Dunn comparisons: non-shocked social |non-shocked isolated p = 5.26e-3, shocked social |shocked isolated p = 1.00). (C) sleep profile and (D) sleep quantification of the 24 h-28 h time window (Kruskal-Wallis chi-squared = 31.184, df = 3, p-value = 7.78e-07; post hoc Dunn comparisons: non-shocked social |non-shocked isolated p = 3.05e-06, shocked social |shocked isolated p = 0.116) of cold-shocked socialized and isolated wt flies, together with non-shocked control wt flies. SOCIALIZATION CORRELATES WITH INCREASED NEURONAL ACTIVITY AND SYNAPTIC PLASTICITY In Drosophila , LTM increased the number of CREB-activated neurons in the MB 10 , 31 . To evaluate whether or not socialization also correlates with higher levels of CREB activity in the MB, we used the CAMEL reporter tool after 5 days of socialization directly after eclosion. This tool bears a MB-specific transgenic construct that responds to phosphorylated CREB with the production of GFP 31 . We quantified the number of GFP positive soma ( fig 5A ) in adult brains, observing an increase in the number of CREB-positive cells in grouped vs single-reared animals ( fig 5B ). In contrast, this CREB response was lost in rut mutant brains ( fig 5B ). Download figure Open in new tab Figure 5. Socialization correlates with cellular and synaptic plasticity. A) Representative confocal images of CAMEL tool for wt and rut mutant MB, either socialized or isolated. Only one representative MB is shown. B) Number of CREB GFP-positive cells in the MB of socialized or isolated wt and rut mutant animals after 5 days of socialization. Kruskal-Wallis chi-squared = 33.735, df = 3 p-value = 1.93e-05; post hoc Dunn key comparisons: 5 days: wt social |wt isolated p = 6.01e-05, rut social | rut isolated p = 1.00. C) Example of CAMEL tool (MB cells marked by GFP) combined with the pre-sinaptic marker brp-cherry after 5 days of socialization for wt and rut mutant animals, either socialized of isolated. D) Quantification of the number of synapses after either isolation or socialization in both wt and rut mutant flies (see fig S5 for a detail on the quantification). Kruskal-Wallis chi-squared = 9.7691, df = 3, p-value = 0.021; post hoc Dunn key comparisons: wt social |wt isolated p = 2.51 e-02, rut social | rut isolated p = 1.00. LTM formation using an appetitive conditioning paradigm increased the number of MB-input synapses 32 . Thus, to determine if CREB-activated neurons after socialization also showed signals of increased synaptic plasticity, we included in the CAMEL tool a second reporter, the presynaptic marker BRP, fused with the RFP-variant cherry. This reporter combination allowed the visualization of the presynaptic densities ( fig 5C ). We quantified the number of synapses per cell volume in brains of 5-day grouped and single-reared animals (fig S2 shows an example of this quantification technique, see M&M). There was a significant increase in the relative number of pre-synapses in the MB of grouped flies compared to single-reared animals ( fig 5D ), similar to the synaptic plasticity described in mammals after an experience 33 . In contrast, in a rut mutant background we could not detect any difference in the number of MB pre-synapses, which was in agreement with the reduced pre-synapse number in rut MB-input neurons after appetitive conditioning 32 ( fig 5D ). Given that intensity of fluorescence varies greatly depending on the region, for analytical purposes we divided the MB in three areas, alpha, beta and the tip of beta. Interestingly, the former two showed only a marginal increase that did not reach statistical significance, however the tip of the MB concentrated most of the increase (fig S3). In summary, results showed a clear correlation of CREB-activated neurons and increased synaptic plasticity with effective social interaction that is abolished in memory impaired mutants, thus supporting a resemblance between socialization awareness and LTM. DISCUSSION Socialization induces several changes in animal behavior and here we show that such changes are long-lasting, as a result of social interaction experience. Not surprisingly, socialization awareness shows similarities with a long-term memory process: involvement of cAMP signaling and processes of neuronal and synaptic plasticity. However, it presents differences with LTM. For instance, the role of rut in short-term behavioral changes seems dispensable, at least for aggression ( fig 3A, B ). It may indicate that short-term effect is independent of rut . A striking peculiarity is its temporal dynamics since it would be hard to distinguish putative learning and consolidation stages during socialization, while in long-term memory paradigms both phases are clearly distinguishable (as, for example, in appetitive or aversive olfactory conditioning). The classic view on sleep regulation indicates that this behavioral state is regulated by the circadian clock and the internal sleep homeostat 34 , but recent work in many species including Drosophila show that sleep regulation goes beyond these two processes and includes temperature, starvation, sexual arousal, and social context, among others 35 . Our data suggest that recalling a past social experience may also regulate sleep in flies ( fig 1 ), similar to what happens with psychophysiological insomnia in humans 36 . In mammals, social isolation has profound effects on behavior and cognition, which is accompanied by detectable alterations in brain structure and function at several levels 37 . For instance, the hippocampus shows reduced dendritic spine density after either postnatal or juvenile social isolation 38 , 39 . The hippocampus is the main structure related to long-term memory, analog to the insect Mushroom Body 40 . In fact, it was previously described that socialization increases the fiber number in the MB, an increase that is impeded by classic learning mutation such as rutabaga 13 , 14 . In addition, our results revealed that socialization also induces rut-dependent changes in synaptic plasticity of the previously activated MB neurons. The increased synaptic densities in CREB-positive neurons might be explained by the socialization-induced enhanced sleep, given that sleep loss diminishes pre-synaptic densities in cholinergic neurons, including the MB neurons 41 , 42 . This is unlikely because despite rut mutant animals do sleep much more ( fig 2B ), their MB presynaptic densities do not reach levels of socialized wt animals, although they are higher than in isolated flies ( fig 5D ). Besides, rut mutant flies did not reach enough sleep levels as to restore behavioral plasticity 24 , thus suggesting that rut increased synaptic activity might be due to the excess of sleep but it is unable to rescue the effect of social interaction ( fig 1 - 3 ). This reinforces the idea that socialization awareness may induce behavioral plasticity by similar mechanisms to long-term memory. An apparent contradictory result was that memory-mutant animals did not behave as expected, i.e., as wt socialized flies, resembling more to wt isolated flies ( fig 1 - 3 ). There are several reasons to explain this presumed inconsistency. The most obvious one is that the basal behavior of rut and dnc mutant flies are different due to the lack of cAMP signaling, as previously described for aggression 27 . Actually, the reduced basal behavioral response of wt isolated animals correlated with their decreased number of CREB-positive cells and pre-sinapses when compared to rut mutant animals, which showed intermediate levels. We might speculate that isolated animals would also generate an “isolation awareness” that enhanced the effect of isolation by diminishing synaptic plasticity due to a cAMP-related mechanisms and, as a consequence, the behavioral response. Why do isolated flies that were previously socialized behave similar to single-reared flies since eclosion 15 ? Actually, chronic isolation displays starvation-like phenotypes in Drosophila 17 and starvation disables aversive long-term memory 43 , probably because increased metabolism in the MB and glia are necessary 44 , 45 . It might well be that socialization awareness was prevented as a consequence of the starvation signaling, and this would explain the similar phenotypes achieved by isolation after socialization and isolation since eclosion, despite mechanistically they should be different. Indeed, one might hypothesize that rescuing such starvation-like phenotype would reveal differences between both experimental conditions. Notably, socialization-induced behavioral changes are sexually dimorphic, since grouped and single-reared females behave similarly 46 . In fact, male-specific P1 interneurons act as an internal state regulatory hub for sleep, aggression, sleep and spontaneous locomotion 47 . Together with Diuretic hormone 44 -( DH44 ) and Tachykinin -( TK ) expressing interneurons, P1 neurons form a male-specific neural circuit that regulates spontaneous locomotion in response to social interaction, thus suggesting a possible common mechanism for socially-induced behavioral changes 46 . Interestingly, P1 neurons directly activate a specific subset of dopaminergic neurons that innervate the MB and it drives LTM appetitive olfactory memory formation 48 . The MB is not only a memory regulatory center but also acts as a sleep and feeding regulatory center 49 , 50 . In this work we have shown that social interaction correlates with increased synaptic plasticity in the MB itself ( fig 4 ). Thus, it is tempting to postulate that socialization awareness may use a general neural circuit connecting P1 neurons, dopaminergic neurons and the MB in order to modify several behaviors with long-lasting effects. MATERIALS Stocks and fly husbandry Flies were raised and experiment performed using standard food at 25°C on a 12/12h light/dark cycle. rutabaga 2080 (#9405), dunce 1 (#6020) and Wild Type ( Canton S #64349) stocks were obtained from Bloomington Drosophila Stock Center. The CAMEL tool is composed by 6xCRE-splitGal4 AD , UAS-eGFP and R21B06-splitGal4 DBD , gently donated by Dr Jan Pielage 31 . rut 2080 ; 6xCRE-splitGal4 AD and UAS-cherry-Brutchpilot; R21B06-splitGal4 DBD stocks were combined in our laboratory and are available under request. Isolation/socialization Protocol Male virgin flies were collected under CO 2 anesthesia within 4 hours post-eclosion and isolated in individual glass vials or socialized (25:25 male:female) in a plastic bottle. After 5 days of socialization or isolation, all flies were isolated without using anesthesia for 24 h (except where indicated) and then, behavioral experiments or dissections were performed. In the case of cold shock, flies were ice-cold shocked twice a day (Zeitgeber Time 1 -ZT01- and ZT9) during the five days of isolation/socialization protocol for 2-3 minutes (i.e. until flies fainted). Glass vials were used to allow good cold transfer from ice. Afterwards vials were placed horizontally in a RT surface to let flies recover. single fly Capillary Feeding (sCAFE) The protocol from 18 was used with slight modifications. Males were placed in individual vials with a wet filter paper at the bottom and a 5 μl capillary (Blaubrand, 708707) with 5% sucrose water food. The capillary was introduced through a 5mm cut 200 μl pipet tip that goes through a wet plug and sustained with an additional tip. After 24 h food intake is measured (0 h-24 h time window), the capillary substituted by a new one and plugs are wet again to preserve moisture. 24 h later food intake is measured again. Once the experiment has finished flies are weighted. Additional 3 individual tubes without flies were measured to control the evaporation rate. Sleep For all experiments, flies were sorted into glass tubes [70 mm × 5 mm × 3 mm (length × external diameter × internal diameter)] containing the same food used for rearing under a regime of 12:12 Light:Dark (LD) condition in incubators set at 25°C. Activity recordings were performed using ethoscopes 23 . Behavioural data analysis was performed in RStudio (RStudio Team. RStudio: Integrated Development for r. RSudio, Inc. Boston, MA; 2015. http://www.rstudio.com/ ) employing the Rethomics suite of packages 51 . All sleep assays were repeated at least twice with 20–40 flies/treatment/experiment. Aggression The protocol from 26 with slight modifications was used. Briefly, two flies were placed into each chamber of the arena (4×3 mm grid) with food. One-to-one socialization was achieved by allowing both flies to interact, whereas isolation was caused by a black divider that allowed physical separation of flies. After 5 days, socialized flies were also separated by the divider for 1, 4, 8 or 24 h. After removing the divider, reunited flies were recorded for 20 minutes and agression analyzed by means of the FlyTracker (MATLAB) software and the platform JAABA ( Janelia Automatic Animal Behavior Annotator ), that identifies when the animal is lunging. The proportion of time fighting is the number of frames in which a particular animal lunges divided by the total number of frames. Immunolabeling, imaging and image analysis Adult brain preparations were stained following the same protocol as in 52 . Dissections were always performed at ZT4-5 to avoid possible circadian-induced changes. For CREB+ cells experiment, primary antibodies used were anti-GFP rabbit (1/200; DSHB???REF) and anti-Fasciclin II mouse (1/50; DSHB). To quantify synapse number, primary antibodies used were anti-GFP goat (1/200; DSHB), anti-RFP rabbit (1/200; DSHB) and anti-Fasciclin II mouse (1/50; DSHB). Secondary antibodies used were Alexia 488, 568 or 680 (1/500; Life Technologies). Images were taken by a Leica SP5 confocal microscopy re-using the same experimental conditions, avoiding saturation. CREB+ cell images were taken using a 40X objective, with slices of 3 um. Synapse quantification confocal images were taken the same day using a 63X objective, slices of 0,8um. Posteriorly images were treated using Imaris 6.3.1 software. Axon volume was rebuilt using the Volume tool and brutchpilot signal was quantified using the Spots tool. To adjust brightness parameters accurately the MB was divided in three parts (alfa, beta and beta tip) (Fig. Supp. 2). Synaptic density for each Mushroom Body is the summatory of spots/volume from each part. Statistical analysis For the behavioral and morphological experiments ( figures 1 , 2 , 3 , 4 , 5 , S1 and S3), the data was analyzed in R (version 3.6.3) through Rstudio (Version 1.0.153), employing the Kruskal-Wallis non-parametric test (library stats ). When appropriate, we performed post hoc Dunn analyses (library FSA) to identify specific differences between treatments. All assays were repeated at least twice with sample sizes as indicated within the figure Author contributions FAM conceptualized and designed the project; FAM and EJB supervised the project; BGM, GT and JIM performed aggression experiments and cellular studies; BGM and APZ performed feeding assays; GSAT and EJB performed and analyzed sleep experiments; EJB and ET analyzed data; FAM and EJB wrote the original draft and made the figures; ET made extensive editing to the manuscript and revised statistics; FAM and EJB were responsible of funding acquisition. Competing interests The authors declare no competing interests. ACKNOWLEDGEMENTS We would like to thank Javier Gil Castillo for his invaluable help and advices in 3D printing. We also appreciate flies and reagents from the Bloomington and VDRC stock centers. Special thanks to our colleagues Prof Alberto Ferrús, Dr Sergio Casas-Tintó, Dr Pablo Méndez, Dr Abhijit Das and Dr JL Trejo-Pérez for their helpful comments and suggestions on this manuscript. Special thanks to Dr Pavan Agrawal and his lab for their critical reading and suggestions on BioRxiv manuscript. We thank the support of the scientific image and microscopy unit (Cajal Institute). FAM was a recipient of a RyC-2014-14961 contract (2016-2022). Grant RyC-2014-14961 funded by MICIU/AEI/10.13039/501100011033 and by ESF Investing in your future. Grant CNS2022-135223 funded by MICIU/AEI/10.13039/501100011033 and by European Union NextGeneration EU/PRTR. BG-M is a recipient of a FPI-UAM predoctoral fellowship, grant number SFPI/2020/00878. JIM was a recipient of a JAE intro fellowship (grant number JAEINT_22_01271) funded by the Spanish National Research Council (CSIC). EJB is a member of the Argentine Research Council (CONICET), and he is funded by Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación , Argentina, through grants PICT-2020-SERIEA-01240, PICT-PRH-2021-00009, and CONICET through grant PIP 11220200102510CO. Footnotes ↵ 4 Lead contact Authorship updated; text (results and discussion) and fig 3 revised. REFERENCES 1. ↵ Donovan , N.J. , and Blazer , D . ( 2020 ). Social Isolation and Loneliness in Older Adults: Review and Commentary of a National Academies Report . American Journal of Geriatric Psychiatry 28 , 1233 – 1244 . doi: 10.1016/j.jagp.2020.08.005 . OpenUrl CrossRef 2. ↵ Lee , C.R. , Chen , A. , and Tye , K.M . ( 2021 ). 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Share Socialization causes long-lasting behavioral changes Beatriz Gil-Martí , Julia Isidro-Mézcua , Adriana Poza-Rodriguez , Gerson S. Asti Tello , Gaia Treves , Enrique Turiégano , Esteban J. Beckwith , Francisco A Martin bioRxiv 2024.04.25.591071; doi: https://doi.org/10.1101/2024.04.25.591071 Share This Article: Copy Citation Tools Socialization causes long-lasting behavioral changes Beatriz Gil-Martí , Julia Isidro-Mézcua , Adriana Poza-Rodriguez , Gerson S. Asti Tello , Gaia Treves , Enrique Turiégano , Esteban J. 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