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- Materials and methods
Members of many species tend to congregate, a behavioral strategy known as local enhancement. Selective advantages of local enhancement range from efficient use of resources to defense from predators. While previous studies have examined many types of social behavior in fruit flies, few have specifically investigated local enhancement. Resource-independent local enhancement (RILE) has recently been described in the fruit fly using a measure called social space index (SSI), although the neural mechanisms remain unknown. Here, we analyze RILE of Drosophila under conditions that allow us to elucidate its neural mechanisms. We have investigated the effects of general volatile anesthetics, compounds that compromise higher order functioning of the type typically required for responding to social cues. We exposed Canton-S flies to non-immobilizing concentrations of halothane and found that flies had a significantly decreased SSI compared with flies tested in air. Narrow abdomen (na) mutants, which display altered responses to anesthetics in numerous behavioral assays, also have a significantly reduced SSI, an effect that was fully reversed by restoring expression of na by driving a UAS-NA rescue construct with NA-GAL4. We found that na expression in cholinergic neurons fully rescued the behavioral defect, whereas expression of na in glutamatergic neurons did so only partially. Our results also suggest a role for na expression in the mushroom bodies (MBs), as suppressing na expression in the MBs of NA-GAL4 rescue flies diminishes SSI. Our data indicate that RILE, a simple behavioral strategy, requires complex neural processing.
All social behavior requires conspecifics to be close enough to interact with one another. In many species, the proximity of conspecifics appears to result from a tendency to assemble into groups. This behavior, which is called local enhancement, has been observed in a wide range of species and can aid in defense against predators (Rohlfs & Hoffmeister 2004), utilization of natural resources (Stamps 1991) and learning (Zentall 2006). More broadly, local enhancement may be a prerequisite for the initiation of competitive or cooperative interactions among conspecifics, depending on the particular configuration of social cues in a given context. Despite a possible central role in modulating complex social interactions, neither genetic nor neural determinants of local enhancement are known. However, recent studies of local enhancement in Drosophila suggest that this genetically tractable organism, which has been successfully used to study the neural circuits underlying behaviors including sleep (Harbison et al. 2009), learning and memory (Pitman et al. 2009; Waddell & Quinn 2001) and courtship and mating (Villella & Hall 2008), may be a useful model for elucidating the genetic and neural bases of local enhancement.
Previous studies have shown that Drosophila form groups in the presence of a resource or in response to pheromones. These studies have focused primarily on aggregation in response to cis-vaccenyl acetate (cVA) (Bartelt et al. 1985; Ha & Smith 2006) and in the presence of resources such as potential mates (Chen et al. 2002), egg-laying substrates (Battesti et al. 2012; Mery & Kawecki 2002; Prokopy & Duan 1998; Sarin & Dukas 2009) or food (Hoffmann 1990; Lefranc et al. 2001; Saltz & Foley 2011). While such gatherings of conspecifics are examples of local enhancement, it is not clear whether these animals are responding primarily to the presence of conspecifics or to other cues.
Although some studies provide evidence that grouping is not simply governed by an individual's attraction to the resource (Tinette et al. 2004), few have examined local enhancement in the absence of environmental resources. An early study by Navarro and del Solar (1975) provided evidence for gregarious behavior in Drosophila in such conditions, but only recently has rigorous analysis established local enhancement as a robust behavior in Drosophila under such conditions (Simon et al. 2012). Simon et al. (2012) eliminated possible environmental confounds associated with most other fly behavioral studies, and were thus able to characterize a simple, resource-independent form of local enhancement for the first time. Another recent study performed in the absence of a food source showed that flies within groups similar to those described by Simon et al. (2012) form social networks (Schneider et al. 2012). To distinguish this form of local enhancement from local enhancement observed in the presence of environmental resources (and often accompanied by more complex behaviors), we refer to it here as resource-independent local enhancement (RILE). This study elucidates for the first time neural and genetic mechanisms involved in Drosophila RILE and paves the way for a better understanding of the mechanisms involved in local enhancement.
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- Materials and methods
Local enhancement in D. melanogaster has been described to (1) occur in the absence of environmental stimuli; (2) be dependent on vision and (3) be independent of chamber geometry and orientation (Simon et al. 2012). To study RILE in Drosophila under conditions in which we could conveniently test animals exposed to general anesthetics, we built an apparatus similar to that described by Simon et al. (2012), modified to allow laminar airflow through horizontally oriented chambers.
To validate that flies in this chamber exhibited local enhancement, we first tested Canton-S male flies in our rectangular testing chamber. We observed that after an initial period of vigorous walking lasting approximately 5 min, Canton-S flies began to settle into groups that were stationary for up to 45 min. In our rectangular chamber, most (80.8% ± 3.3%, n = 9 trials) young male Canton-S flies are within 1 mm of at least two other flies (data not shown). A greater percentage of flies (90.3% ± 1.7%, n = 9) settle within 5 mm of their nearest neighbor (Fig. 1a,b). Chamber geometry is known to affect exploratory and spontaneous activity of flies (Liu et al. 2007). To test whether chamber geometry affects local enhancement under our assay conditions, we tested behavior in a circular chamber and found that 94.9% ± 1.8% flies (n = 8 trials) are within 5 mm of their nearest neighbor (Fig. 1c,d). To provide a simpler measure of local enhancement, we calculated the SSI that is obtained from the histograms of fly distributions by subtracting the second bin from the first (Simon et al. 2012). The SSI of male Canton-S flies in our rectangular and circular chambers (84.7 ± 3.2, n = 9 and 93.3 ± 2.5, n = 8, respectively) did not statistically differ from one another (Student's t-test, Fig. 1e). Young female Canton-S flies show behavior similar to the males in our rectangular arena (SSI = 84.4 ± 3.8, n = 4, data not shown), consistent with observations by Simon et al. (2012). As expected, on the basis of previous studies that have shown flies in an arena will spend a large part of the time near the arena walls (Soibam et al. 2012; Valente et al. 2007), flies in each of our arenas showed a preference for settling in corners or along edges after their initial exploratory behavior.
Figure 1. Resource-independent local enhancement of flies in different chambers or under conditions of visual impairment. (a and b) Wild-type Canton-S (CS) males in humidified air in rectangular arena. A representative image is shown (a). Scale bar represents 30 mm. The histogram (b) represents the mean percentage of flies (±SEM) at the indicated distance from their nearest neighbors (n = 9). (c and d) The CS males in humidified air in circular arena. A representative image is shown (c). Scale bar represents 30 mm. The histogram (d) represents the mean percentage of flies (±SEM) at the indicated distance from their nearest neighbors (n = 8). (e) Social space indices (SSIs) for CS flies in the two different chambers shown in a and c (error bars represent ±SEM; no significant difference, Student's t-test). (f and g) A representative image of CS males tested in humidified air in dim red light (f). Scale bar represents 30 mm. The histogram (g) shows the percentage of flies at given distance intervals from their nearest neighbor (mean ± SEM, n = 7). (h) Summarized mean SSI (±SEM) data for CS flies tested in dim red light (white bar) and for the three visual mutants tested in ambient light: trp301 (n = 6), trpl302 (n = 5) and trpl302;trp301 double mutants (n = 6). The dashed horizontal line represents the mean SSI of CS flies tested in ambient light and is shown for comparison. *P < 0.05 compared with CS control.
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We also tested whether SSI under our testing conditions was vision-dependent, as reported by Simon et al. (2012). The SSI for flies tested under dim red light is 45.5 ± 6.4 (n = 7; P < 0.05 compared with flies tested under ambient light) (Fig. 1f–h). In dim red light, Canton-S males introduced into our chamber walk for a considerably longer time (∼20 min) than flies tested under ambient light (∼5 min). However, flies finally settle between 22 and 25 min. Interestingly, the strong preference flies exhibit for corners and edges is notably diminished, with flies often settling down in the middle area of the arena.
To further examine the dependence of RILE on vision, we examined the behavior of visual mutant flies with partial or complete impairment of the phototransduction machinery. trp mutants (trp301), which are nearly completely blind, carry perturbations in the transient receptor potential (trp) gene product, a subunit of the cation channel that carries a major component of the light-induced current in fly eyes (Montell 2005). A separate minor component of light-induced current is mediated by Trpl protein (Niemeyer et al. 1996; Reuss et al. 1997). In our arena, trp flies tested under ambient light and in humidified air show significantly reduced RILE compared with wild-type flies (SSI = 16.8 ± 12.5, n = 6, P < 0.05; Fig. 1h). In contrast, the SSI of trpl flies (trpl302) (SSI = 91.3 ± 1.7, n = 5), which have relatively minor visual impairments, did not differ from that of wild-type flies (Fig. 1h). Given the difference in visual impairment between trp and trpl flies, the difference in their behavior is not surprising. Flies carrying both the trp and trpl mutations (trpl302;trp301) did not statistically differ in SSI from trp flies, indicating that the trp mutation itself accounts for the behavioral deficit in the double mutants.
Overall, our data, which show that RILE in flies is independent of chamber geometry, occurs in the absence of environmental cues and is vision dependent, are consistent with those of Simon et al. (2012).
Apart from its dependence on visual cues, little is known about the neural mechanisms that mediate RILE in Drosophila. Because social interactions require responses to diverse cues, we were interested in compromising the fly's ability to coordinate such neural processing. To determine whether the mechanisms that govern RILE are sensitive to the effects of general anesthetics, which, at non-immobilizing concentrations, target neural circuits involved in central processing and decouple higher order brain processes from simpler neural functions (van Swinderen 2006; van Swinderen et al. 2004), we examined RILE of wild-type flies exposed to a low concentration of the GVA halothane. It has been shown previously that both flies are sensitive to clinical doses of GVA and that simple reflexes in Drosophila are preserved at subclinical doses (Allada & Nash 1993; Campbell & Nash 1994, 1998; Gamo et al. 1981). We exposed flies to halothane for 30 min before testing to allow the anesthetic to reach equilibrium in their nervous systems. Flies were equilibrated and tested at 0.15% halothane, a concentration that was low enough to allow flies to walk unhindered on horizontal surfaces (data not shown).
When introduced into the testing arena with halothane, flies exhibit an initial exploratory period lasting 15–20 min, which was longer than the period of exploration in Canton-S flies tested in humidified air alone. By 25 min, most flies have stopped walking. In these conditions, flies settle into a dispersed array with substantially reduced clustering (Fig. 2). Compared with flies tested in humidified air, significantly fewer flies were found within 5 mm of their nearest neighbor (62.6% ± 2.6% in halothane, n = 6; P < 0.001) and about 25% of flies are 5–10 mm from their nearest neighbor (Fig. 2b), resulting in a significantly lower SSI compared with flies tested in humidified air alone (SSI = 37.3 ± 4.1, n = 6; P < 0.001, Fig. 2b). Thus, exposure to the GVA halothane at low doses disrupts normal RILE in Canton-S flies.
Figure 2. Resource-independent local enhancement of flies tested in the presence of halothane. (a) A representative image of flies in the testing chamber in the presence of 0.15% (volume) halothane is shown. Scale bar represents 30 mm. (b) The histogram shows the mean percentage of Canton-S flies (±SEM) at the indicated distance from their nearest neighbors (n = 6) during testing in halothane. The SSI (±SEM) is also indicated.
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Given the above results, we hypothesized that the neural circuitry that governs RILE employs signaling molecules that are sensitive to halothane. Halothane sensitivity is thought to be mediated, at least in part, by the ion channel encoded by the na gene, and the har38 allele (nahar38) causes pronounced changes in sensitivity to this anesthetic (Guan et al. 2000; Krishnan & Nash 1990; Nash et al. 1991). We therefore tested the behavioral phenotype of nahar38 flies.
Although nahar38 flies are deficient in their climbing ability in ambient air and will alternate between hesitant walking and freezing (Humphrey et al. 2007), we did not observe this locomotor phenotype during the exploratory activity on our horizontal testing surface. However, nahar38 flies are significantly deficient in RILE with an SSI of only 22.2 ± 4.1 (n = 9, Fig. 3a), an effect that is fully rescued by expression of a UAS-NA construct under the control of NA-GAL4 (Lear et al. 2005), which drives transgene expression in cells that normally express na (nahar38; +; NA-GAL4/UAS-NA, SSI = 74.4 ± 4.5, n = 7, Fig. 3a). Neither the NA-GAL4 driver line by itself nor the UAS-NA rescue construct by itself, each in the nahar38 mutant background, provides behavioral rescue (Fig. 3a).
Figure 3. Social space indices of nahar38 mutant and rescue flies. (a) The SSIs are shown for nahar38 (n = 9); nahar38;+;NA-GAL4/UAS-NA rescue (n = 7) and control lines nahar38; +; UAS-NA/+ (n = 7) and nahar38; +; NA-GAL4/+ (n = 7) flies. The CS SSI (mean) represented by dashed horizontal line for reference. **P < 0.001 compared with har38. (b) The SSIs are shown for nahar38; Cha-GAL4/+; UAS-NA/+ (black, n = 7); Cha-GAL4 driver line by itself (dark gray, n = 9); nahar38; dVGlut-GAL4/+; UAS-NA/+ (light gray, n = 9) and dVGlut-GAL4 driver line by itself (white, n = 7). Mean SSI of nahar38 flies represented by dashed horizontal line for reference. **P < 0.001 compared with nahar38 flies.
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Our data suggest that the NA ion channel is involved in mediating RILE. To begin to elucidate its neuronal site of action, we selectively restored na gene expression in various neuronal subtypes in nahar38 mutants using specific GAL4 drivers. We tested these flies for RILE under ambient light in humidified air. We found that expression of na in cholinergic neurons (nahar38; Cha-GAL4/+; UAS-NA/+) completely rescues the behavioral defect (SSI = 89.7 ± 2.8, n = 7, **P < 0.01 compared with nahar38, Fig. 3b), whereas restoration of na expression in glutamatergic neurons (nahar38; dVGlut-GAL4/+; UAS-NA/+) only partially rescues the RILE phenotype (SSI = 43.9 ± 5.9, n = 6, **P < 0.01 compared with nahar38, Fig. 3b). While both cholinergic and glutamatergic rescue flies have SSI that significantly differs from that of the mutant nahar38 flies (Fig. 3b), the glutamatergic rescue flies' SSI also differs from that of the control CS flies, thus suggesting a behavioral rescue midway between wild-type and nahar38 flies. Neither of the driver lines expressed by itself in the nahar38 background affected the phenotype (Fig. 3b).
In addition to determining in what type of neurons na expression is needed for normal RILE behavior, we were also interested in determining in what central brain structure(s) na expression is needed. NA-GAL4 has a strong expression pattern in the mushroom body (MB). Additionally, RILE may depend in part on learning, as socially isolated flies have a significantly lower SSI than socially enriched flies (Simon et al. 2012). Various forms of learning in Drosophila, including context generalization in visual learning (Liu et al. 1999; Zars 2000), have been shown to require the MB. Thus, we hypothesized that inhibition of na expression in the MB in the NA-GAL4 rescue flies would prevent behavioral rescue.
We used an MB-GAL80 line (Krashes et al. 2007) to selectively decrease na expression in the MB of nahar38; +; NA-GAL4/UAS-NA rescue flies. We first verified the effectiveness of the MB-GAL80 line by comparing the expression of a nuclear marker, RedStinger, in the MB in the presence (w1118; MB-GAL80/UAS-RedStinger; NA-GAL4/UAS-NA) or absence (w1118; UAS-RedStinger/+; NA-GAL4/UAS-NA) of the MB-GAL80 transgene by examining whole-mount brains with confocal imaging (Fig. 4). The nerve fibers of the Kenyon cells of the MB form the peduncle and calyces, which appear as bilaterally symmetric holes toward the dorsal side of the brain (Fig. 4a). We labeled the MB with anti-DC0 antibody (Skoulakis et al. 1993), thus providing a readily identifiable landmark by which to identify distally located MB cell bodies. In the control fly brains (from flies without MB-GAL80), we saw robust expression of RedStinger (under control of the NA-GAL4 driver) in the MB calyces (Fig. 4b; dashed lines denote circumference of anti-DC0 labeling). However, in the presence of MB-GAL80, the signal was reduced in the MB (Fig. 4c; dashed lines denote circumference of anti-DC0 labeling). The functional effect of blocking na expression in the MB of the NA-GAL4 rescue flies was to reduce the SSI (SSI = 45.0 ± 4.3, n = 8) compared with that of the nahar38; +; NA-GAL4/UAS-NA control flies (Fig. 4d). Thus, na expression in the MB plays a role in normal RILE behavior.
Figure 4. Social space indices of na-rescue flies in which na expression is blocked selectively in the mushroom bodies. (a) Schematic representation of the fly brain showing the relevant orientation and position of the MB calyces, peduncles and Kenyon cell bodies. The dashed oval around the Kenyon cell bodies and the calyx in the right hemisphere correspond to the area enclosed in dashed circles in (b) and (c). (b and c) Expression of both UAS-RedStinger and anti-DC0 labeling was visualized in whole-mount adult brains. Representative images of whole brain from w1118; UAS-RedStinger/+;NA-GAL4/UAS-NA (b, n = 4) and from w1118;MB-GAL80/UAS-RedStinger;NA-GAL4/UAS-NA (c, n = 4) show the expression of RedStinger. The MB calyces, as marked by anti-DC0 labeling [green, as shown in inset in (c)], are outlined in white dashed lines. (d) The histogram shows the mean percentage of nahar38; MB-GAL80/UAS-RedStinger; NA-GAL4/UAS-NA flies (±SEM) at the indicated distance from their nearest neighbors (n = 8). The SSI (±SEM) is also indicated.
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