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The role of three gustatory neuron populations in conditioned stimulus representation in a Drosophila multi-modal associative learning paradigm
Jessie Whitfield, Arpiar Saunders, and John Tuthill
Learning & Memory
December 13, 2005
Abstract
Courtship conditioning is an associative learning paradigm that consists of a male fly learning to depress courtship toward a virgin female after an unproductive courting experience with an already-mated female. Learning depends on a training session where the conditioned stimulus (CS) is associated with an unconditioned stimulus (US), and a testing session where the detection of the same CS evokes modified behavior. Evidence suggests that the CS and US have nonvolatile cuticular hydrocarbon components, and are tasted with gustatory receptors on the male fly. To address the role of gustatory receptors in courtship learning, three populations of taste neuron expressing receptors were silenced during training using a spatially restricted and temperature sensitive protein. By training and testing with two types of flies expressing different but overlapping pheromone profiles, we found that non-volatile pheromones picked up through the gustatory system are important for memory involved in courtship suppression. In order to understand the roles of specific pheromone inputs in CS association, future studies must thoroughly examine different training and testing experiences for each genotype.
Introduction
Associative learning involves pairing genetically hard-wired circuits with salient environmental predictors to modify behavior based on previous experience. In the Drosophilamelanogaster system, a simple associative paradigm in which an odor is paired with a shock provided the main framework for discovering the genetic underpinnings of learning and memory (Ejima et al., 2005; Tully, 1984; Tully and Quinn, 1985). While the odor-shock paradigm relies on cues that are singular and discreet, recent work has focused on how sensory systems extrapolate the cues which define more complex experiences (Wasserman and Miller, 1997). One such paradigm is Drosophila courtship conditioning.
In courtship conditioning a male fly learns to depress courtship toward a virgin female after an unproductive sexual experience with an already-mated female. The expression of this learning depends on both a training session, where the components of the conditioned stimulus (CS) are detected and linked to an unconditioned stimulus (US), and a testing session, where CS detection evokes modified behavior. The inability to pick up on the CS during either training or testing should interfere with the expression of a learned response.
The courtship of Drosophila is based on visual, auditory and chemosensory information which drives the male through a loose progression of stereotyped behaviors: first, the male follows the female (1). After approaching, he taps her abdomen with his foretarsi (2) and vibrates his wing to generate a “courtship song.” (3). Next, he licks her genitalia (4). Lastly, the male attempts to mount the female by curling his abdomen (5) (Greenspan and Ferveur, 2000). If successful, mating lasts for approximately 15 minutes.
Mated females display a robust reluctance to re-mate, using their wings and legs to ward off suitors. This behavior is part of a host of physiological changes induced by the activity of two peptides in the male sperm (Chen et al., 1988; Saudan et al., 2002), changes which are thought to comprise the US.
Since 1983, when Tompkins et al. (1983)showed that a male fly could learn from a training experience with a male or virgin female presented with chemical extracts from fertilized females, evidence has steered toward a pheromonal basis for the CS. In Drosophila, pheromones are detected by specialized cells in both the olfactory and gustatory systems through ancestrally related G-Protein Coupled Receptors (GPCRs). The olfactory system is located in two head appendages, which transduce pheromone and odorant ligands through olfactory receptor neurons (ORNs). The ORNs send bifurcating axons to the glomeruli of the antennal lobes where olfactory information is organized, associated with other inputs and fed to higher-order brain regions (Amrein, 2004; Yu et al., 2004).
The gustatory system is less centralized than the olfactory system; while the primary taste organ (the labellum) is located at the end of the proboscis, the fore legs also have distinct 2-4 cell taste “bristles.” Gustatory receptor neurons (GRNs) from both areas project to either the subesophageal or thoracic ganglions. Unlike the ORNs, the GRNs express heterogenous sets of receptors, thus distinguishing taste “population coding” at the level of the sensory cell from the olfactory “population coding” at the level of the glomeruli. While taste circuitry remains mostly unresolved, initial evidence suggests functionally distinct sets of neurons exhibit different projection pathways for sugar and bitter compounds(Thorne et al., 2004); a neuroanatomical division of labor which may be mirrored in the decision to court or not to court.
A major breakthrough in defining gustatory pheromone reception was achieved by pursuing sexual dimorphism of the GRNs in the fore tarsi. One such receptor, Gr68a, was shown to pick up aphrodisiac cues (Bray and Amrein, 2003). Since most courtship pheromones are thought to be detected through contact behaviors like tapping and licking (Greenspan and Ferveur, 2000), this discovery implicated other structurally similar GRNs in the legs and labellum as possible pheromone receptors and potentially contributors to the representation of the CS.
Ejima et al.(2005)provides evidence for a volatile pheromone basis of the CS through studies where male flies are successfully trained without contacting mated females. Furthermore, they show that CS representation is somewhat plastic: male flies express “trainer specific” learning, exhibiting significant differences in courtship suppression dependent on the similarity of age between female trainers and testers. With their olfactory systems removed, male learning was generalized, suggesting an additional role for visual or gustatory cues. Generalized learning has also been demonstrated in courtship conditioning paradigms using trainer and tester flies with largely different (though somewhat overlapping) non-volatile pheromone profiles. This result suggests an opposite conclusion from Eijma et al. (2005), that the CS is represented by non-volatile cues and is subject to little plasticity (Siwicki et al., 2005).
This study attempts to elucidate the role in courtship learning of GRNs defined by three populations of potential pheromone receptors (Gr68a, Gr39a.A and Gr39a.D). A first attempt at identifying this role was made by selectively preventing neurotransmission in all cells expressing a given receptor during the testing phase of the paradigm (unpublished). These preliminary results were confounded in two ways. Silencing a population of pheromone-transducing cells during testing dramatically altered courtship levels of untrained males by disrupting non-learning dependent aphrodisiac and anti-aphrodisiac pathways. Secondly, having multiple gustatory receptors within a cell means that silencing cells based on the expression of a single receptor type may lead to overlapping silenced populations, obscuring any direct relationship between ligand, receptor and cellular pathway.
This study addresses the first problem by silencing taste neurons during the training phase of the paradigm. If learning does not occur during testing, and it is shown that training with a silenced population of taste neurons is vigorous enough to cause memory, then it can be inferred that the specific pheromone input lacking during training was essential for representation of the CS such that when a similar but non-identical CS is perceived during testing with all taste neurons available, no association between the training and testing CS is made.
This study also examines the plasticity of CS representation by using two different types of trainers and testers which express different pheromone profiles. In the case of heterogeneous training and testing flies, if CS representation does not generalize when a certain population of taste neurons is silenced, it is possible to contrast the pheromone profiles of each fly type and pursue what shared cue was lacking.
Materials and Methods
Drosophila strains and maintenance
CSB, Tai2 and UAS-shitsDrosophila stocks were originally obtained from the Bloomington Fly Center while the transgenic Gal4- driver lines (p[Gr68a]-Gal4 and p[Gr39a.A]-Gal4) were provided by the lab of Hubert Amrein (Duke University). Drosophila stocks were raised on standard cornmeal-agar-molasses medium and incubated at 25C on a 12hr light: 12hr dark cycle (except for p[Gr--]-Gal4/ UAS-shits crossed lines and UAS-shits/+ crossed lines, which were incubated at 18C). All flies for behavioral assays and crosses were collected within eight hours of eclosion using CO2 anaesthesia. Male Gal4/UAS-shits subjects were placed in individual tubes to preserve social naivete. Virgins were collected in large vials in groups of 10 to 20.
Crosses
Control strains (UAS-shits/+) were generated by crossing UAS-shits males with virgin CSB females and by crossing virgin UAS-shits females with CSB males. To create subject strains (p[Gr68a]-Gal4/UAS-shits and p[Gr39a.A]-Gal4/UAS-shits), males from each Gal4-driver line were crossed with UAS-shits virgin females, and vice versa.
Behavioral training and testing
All male subjects used for training and testing were collected 5-6 days before training and testing session. Trainer flies and testing targets consisted of 3-5 day old, wing-clipped Tai2 males and 3-5 day old mated CSB females, which were mated the night before by pairing virgin CSB females with socially naive CSB males. All training and testing sessions took place between 8 am and 1 pm. Room temperature during training was between 25° C and 28° C.
Training Profiles: To ensure that adequate courtship activity occurred during training sessions at both the restrictive (30C) and permissive (18C) temperatures, p[Gr68a]-Gal4/UAS-shits and p[GR39a.A]-Gal4/UAS-shits male subjects were individually exposed to a one-hour training session at either temperature. Training sessions took place in individual training chambers (which were created by placing a watch glass on a glass plate) on temperature-regulated platforms. Healthy male subjects were transferred by gentle aspiration to a training chamber and paired with either a single Tai2 male or a single mated CSB female for 60 minutes. Courtship activity was scored by measuring the time spent courting during six 5-minute windows within a 60-minute period. Courting behavior included winging, tapping, licking and attempts to copulate. Sniffing behavior was also observed but did not contribute to the courtship index. To generate a courtship index (CI), time spent courting was divided by total time for each interval.
Training and Testings: Prior to testing, p[Gr68a]-Gal4/UAS-shits and p[Gr39a.A]-Gal4/UAS-shits male subject flies were trained with either a single 3-5 day old, wing-clipped Tai2 male or a single 3-5 day old mated CSB female in a 60-minute training session. The subject male was transferred by aspiration to the watch glass training chamber before the target was added. To train the flies at the permissive temperature, training chambers with the subject/trainer pairs were placed in the 18° C incubator for the full 60 minutes. Training sessions at the restrictive temperature took place in a 30° C oven. Before the trainer fly was added at the start of the restrictive-temperature sessions, male subjects in their training chamber were allowed two minutes of isolation inside the oven to allow the dominant shibire mutation to take effect (Kitamoto 2002). Training plates at both permissive and restrictive temperatures were periodically observed to ensure adequate training occurred.
Following the 60-minute training period, the male subject was transferred for testing to a clean testing chamber (watch glass and glass plate) on a 18° C temperature-regulated platform. The fly was allowed to rest in isolation for 5 minutes to consolidate any memory. After the 5-minute consolidation, a decapitated Tai2 or a decapitated CSB mated female was aspirated into the chamber. Targets were decapitated at the intersection between thorax and head using a razor; any targets that would not stand were discarded. The 10-minute testing session started upon the target’s introduction to the chamber. All the courtship and non-courtship behaviors that occurred over the 10-minute period were tallied. Courtship behaviors tallied included wingings, lickings, tappings and attempted copulations. Sniffing behavior was also marked and contributed to a interest index, calculated by dividing the number of courtship behaviors and sniffing behaviors by the total marks made. The courtship index (CI) was generated by dividing the number of courtship behaviors by the total tallies.
Data analysis
All indices of courtship were first analyzed by training or temperature (in the case of “training profiles”) to assess the normality of distribution for a given condition. If either condition being compared was non-normal, the Wilcoxon test was used to assess significance of difference. When data was normal, pooled t-tests were used. All statistics were don in JMP.
Results
Training profiles were created for Gr68a and Gr39a.A subjects during experience with mated CSB and Tai 2 trainers respectively (Figures 1, 2). In both scenarios, subjects trained at the restrictive temperature courted significantly less than those at the permissive temperature. However the training profiles were well within values known to induce courtship suppression (Figures 3,4).
Figure 1: Over the course of a 60-minute training session, Gr68a subject males decreased their courtship intensity of a mated CSB female trainer fly at both permissive (blue) and restrictive (red) temperatures. Subject males courted mated CSB trainers significantly less at the restrictive temperature than the permissive temperature. Data are expressed as mean ± SEM.
Figure 2: Over the course of a 60-minute training session, Gr39a.A subject males decreased their courtship intensity of a Tai2 trainer fly at both permissive (blue) and restrictive (red) temperatures. Subject males court Tai2 trainers significantly less at the restrictive temperature than the permissive temperature. Data are expressed as mean ± SEM.
Figure 3: Over the course of a 60-minute training session, wild-type male flies decreased their courtship intensity of three trainer genotypes: mated CSB females (tangerine), Tai 2 males (spinach), and Tp2602-tra males (eggplant). Data are expressed as mean ± SEM.
Figure 4: Following a 60-minute training period, wild-type courtship of decapitated targets relative to naïve controls for the three genotypes represented in Figure X (above). Solid bars indicate naïve flies; bars with diagonal hatching indicate flies trained and tested with: mated CSB females (tangerine), Tai 2 males (spinach), and Tp2602-tra males (eggplant). Trained flies exhibit courtship suppression in each case. Data are expressed as mean +/- SEM (*p<.05).
Gr68a subjects showed significant learning with decapitated Tai 2 targets after both permissive and restrictive training (Figure 5). With virgin CSB female targets, Gr68a subjects showed learning after permissive but not the restrictive training (Figure 6). For both target types, sniffing was significantly reduced after restrictive training (Figures 5, 6). Statistical comparisons for courtship and sniffing indices of each genotype are displayed in Tables 1 and 2 respectively.
Gr39a.A subjects showed significant learning with decapitated Tai 2 targets after both permissive and restrictive training with Tai 2 males (Figure 7, left panel). With virgin CSB female targets, Gr39a.A subjects showed a non-significant reduction in courtship in the trained condition after permissive training; after restrictive training, no learning was exhibited (Figure 8). Sniffing levels mirrored those of courtship: with virgin CSB targets, subjects experiencing restrictive training showed no difference in sniffing between Naïve and Trained conditions, while a near significant difference was seen after permissive training (Figure 7, right panel). With Tai 2 targets, significant reductions in sniffing in the trained condition were present for training sessions at both temperatures (Figure 8).
Gr39a.D subjects showed significant learning with virgin CSB targets after both restrictive and permissive training (Figure 9, right panel). Oppositely, no sniffing differences were exhibited between naïve and trained conditions at either training temperature (Figure 9, left panel).
UAS-shits/+ control subjects showed significant learning with Tai 2 male targets after both permissive and restrictive training with Tai 2 males (Figure 10, left panel). With virgin CSB female targets, learning was seen after the permissive but not the restrictive training temperature (Figure 10, left panel). Sniffing indices showed no differences between naïve and trained flies for either trainer/target combination (Figure 10, right panel).
Figure 5. Following 60 minutes of experience with CSB mated female, trained subject (Gr68a) courtship of decapitated CSB virgin females relative to naïve controls. Dotted bars indicate naïve flies; diagonally striped bars indicate trained flies. (Left panel) Courtship indices for male Gr68a subjects at the permissive (blue) and restrictive (red) temperature. Relative to naïve subjects, trained subjects significantly suppress courtship at the permissive but not the restrictive temperature. (Right panel) Sniffing indices for male Gr68a subjects at the permissive and restrictive temperature. Results were similar to courtship indices in A. Data are expressed as mean +/- SEM (***p<.005).