Research Articles, Systems/Circuits

A Drosophila Circuit for Habituation Override

Swati Trisal, Marcia Aranha, Ankita Chodankar, K. VijayRaghavan and Mani Ramaswami
Journal of Neuroscience 6 April 2022, 42 (14) 2930-2941; https://doi.org/10.1523/JNEUROSCI.1842-21.2022

Abstract

Habituated animals retain a latent capacity for robust engagement with familiar stimuli. In most instances, the ability to override habituation is best explained by postulating that habituation arises from the potentiation of inhibitory inputs onto stimulus-encoding assemblies and that habituation override occurs through disinhibition. Previous work has shown that inhibitory plasticity contributes to specific forms of olfactory and gustatory habituation in Drosophila. Here, we analyze how exposure to a novel stimulus causes override of gustatory (proboscis extension reflex; PER) habituation. While brief sucrose contact with tarsal hairs causes naive Drosophila to extend their proboscis, persistent exposure reduces PER to subsequent sucrose stimuli. We show that in so habituated animals, either brief exposure of the proboscis to yeast or direct thermogenetic activation of sensory neurons restores PER response to tarsal sucrose stimulation. Similar override of PER habituation can also be induced by brief thermogenetic activation of a population of tyrosine hydroxylase (TH)-positive neurons, a subset of which send projections to the subesophageal zone (SEZ). Significantly, sensory-neuron induced habituation override requires transmitter release from these TH-positive cells. Treatments that cause override specifically influence the habituated state, with no effect on the naive sucrose response across a range of concentrations. Taken together with other findings, these observations in female flies are consistent with a model in which novel taste stimuli trigger activity in dopaminergic neurons which, directly or indirectly, inhibit GABAergic cells that drive PER habituation. The implications of these findings for general mechanisms of attentional and sensory override of habituation are discussed.

SIGNIFICANCE STATEMENT Habituation can be overcome when a new context requires an enhanced response to a familiar stimulus. However, the underlying mechanisms remain incompletely understood. Previous studies have provided evidence that habituation of the sucrose-induced proboscis extension reflex (PER) in Drosophila occurs through potentiation of inhibition onto the PER pathway. This work defines controlled protocols for override of PER habituation and uses them to outline the underlying circuit mechanisms. The results presented support a model in which novel taste stimuli cause dishabituation by activating a subset of tyrosine hydroxylase (TH)-expressing neurons that inhibit GABAergic neurons whose potentiation underlies PER habituation. At a general level, these findings further highlight a central role for inhibition and disinhibition in the control of behavioral flexibility.

Introduction

Habituation is a form of nonassociative learning in which the response to a stimulus reduces after repeated or extended passive exposure. However, a latent ability to respond to the innocuous stimulus remains. “Dishabituation” is a classical, defining feature of the habituated state, distinguishing it from sensory or synaptic fatigue (Thompson and Spencer, 1966; Rankin et al., 2009; Ramaswami, 2014). In this article we often use the term “override” in place of dishabituation, which while largely synonymous, better acknowledges that behavioral responses can be reinstated by not only classic dishabituating (novel) stimuli, but also top-down attentional mechanisms (Cooke and Ramaswami, 2020). For instance, in volitional inspection of a familiar object on a shelf, habituation is overcome by attention to the object, and not by an extraneous novel stimulus.

Habituation and the associated phenomenon of dishabituation/override are well known across different phyla of animal kingdom (Glanzman et al., 1989; Zaccardi et al., 2004; Smith et al., 2009; Ramaswami, 2014). Two broad observations are relevant here. First, mechanisms underlying very short and longer lasting forms of habituation may differ; some forms of the latter are known to involve inhibitory potentiation (Ramaswami, 2014; Shen et al., 2020). Second, largely because of the difficulty of necessary behavioral experiments, in most (but not all, Semelidou et al., 2018) instances, habituation override is not distinguished from a potential confounding process of response sensitization (Castellucci et al., 1970; Hawkins et al., 1998; Asztalos et al., 2007a,b). Thus, although arguments and evidence support a disinhibitory mechanism (Fischer et al., 1997; Das et al., 2011; Kato et al., 2015; Ogg et al., 2018), the neural pathways and mechanisms of habituation override remain incompletely characterized. Here, we address this issue in the gustatory system of Drosophila.

Drosophila sample and taste potential foods via chemosensory hairs on their tarsi and their proboscis (Stocker, 1994; Montell, 2009). Sugars detected by sensory hairs trigger a proboscis extension reflex (PER), which enables feeding (Minnich, 1921; Dethier, 1976). The neural circuit for PER is only partially understood. Sensory information is processed in subesophageal zone (SEZ) and then communicated to command neurons whose activation triggers the motor program required for PER (Flood et al., 2013). Repeated sucrose stimulation of the tarsus under conditions where proboscis extension is futile, leads to reduced PER response through a process that shows several classic features of habituation (Duerr and Quinn, 1982; Le Bourg, 1983; Fois et al., 1991; Engel and Wu, 2009; Paranjpe et al., 2012). Importantly, in habituated animals, PER to sucrose is quickly restored if the fly is presented with a strong, novel sensory stimulus (Le Bourg, 1983; Fois et al., 1991; Paranjpe et al., 2012). Here, we investigate circuit mechanisms that drive override of PER habituation.

We first reproduced prior experiments providing key support for increased inhibition in the PER pathway being the core mechanism for PER habituation (Paranjpe et al., 2012). Thereafter, we addressed mechanisms of habituation override, which we found could be achieved by yeast stimulation of the proboscis, thermogenetic activation of yeast-responsive or bitter responsive sensory neurons, or activation of a dopaminergic neuron subpopulation. We show that sensory stimulation procedures induce habituation override through dopaminergic neuron activation. Crucially, each dishabituation protocol specifically affects the response of habituated animals; none sensitize the naive response. While the data do not yet conclusively define all elements of the dishabituation circuit or the mechanism by which dopaminergic neurons trigger override, they support a model in which novel stimuli induce dopamine release, which acts to directly or indirectly inhibit inhibitory neurons that drive PER habituation. We suggest that this work: (1) circumscribes core elements of a sensory-central circuit for habituation override; (2) provides evidence for a new disinhibitory pathway in the Drosophila brain; and (3) supports an emerging framework in which latent perceptions, memories and behaviors may be generally activated through disinhibition (Sridharan and Knudsen, 2015; Barron et al., 2017; Wang and Yang, 2018).

Materials and Methods

Experimental design

Drosophila stocks

Fly stocks were maintained on standard corn meal media. Canton S (CS) flies were used as wild-type controls unless otherwise stated. The stocks were obtained either from stock centres or as generous gifts from following sources: Ir25a-Gal4 was provided by Carlos Ribeiro (Champalimaud Center for the Unknown, Lisbon, Portugal), TH-C'-Gal4, TH-D'-Gal4, and TH-C-Gal80 were generously provided by Mark Wu (Johns Hopkins University, Baltimore, MD), Gad1-Gal4 was from Gero Miesenböck (Oxford University, Oxford, United Kingdom), TH-Gal4 was provided by Gaiti Hassan (National Center for Biological Sciences, Bangalore, India), Gr66a-LexA, lexAop-CD4::spGFP11; UAS-CD4::spGFP1-10 and LexAop-TRPA1 were provided by Kristin Scott (University of California, Berkley, CA). rut2080 and UAS-rut+ were provided by Martin Heisenberg. UAS-Shits was obtained from Toshi Kitamoto (University of Iowa, Iowa City, IA), UAS-TRPA1 was provided by Paul Garrity (Brandeis University, Waltham, MA). Wg/CyO; Gr66a-Gal4 (BL 57670), UAS-mCD8::GFP (BL 5130), UAS-CD8::RFP, LexAop-CD8::GFP (BL 32 229) were obtained from Bloomington Drosophila Stock Center. Dopamine receptors were downregulated using lines UAS-DopR1-miR, UAS-DopR2-miR, and UAS-D2R-miR obtained from Mark Wu (Johns Hopkins University) in Gad1-Gal4-expressing neurons (data not shown). Tyrosine hydroxylase (TH) was downregulated in TH Gal4 neurons using TH-miR-G/TM6, Sb and TH-miR-9/Cyo; TH-miR-2/TM6, Tb provided by Mark Wu (Johns Hopkins University; data not shown). However, the results were inconclusive.

Proboscis extension behavior

Proboscis extension behavior was conducted as described in (Paranjpe et al., 2012). Briefly, 3- to 4-d-old female flies were mounted individually, ventral side up, on a cover slip. The flies were kept in a humidified chamber for 2 h for recovery. Tarsal hairs were stimulated with 2% sucrose using 1-ml syringe needle. The naive response score was determined after stimulating tarsal hairs five times. Failed PER response was scored as zero and complete proboscis extension was scored as one. Flies that showed naive PER response less than three out of five times were discarded and not used for habituation experiments.

Habituation of PER

Tarsal hairs were stimulated with 10% sucrose for 10 min after which the legs were washed using distilled water. The habituated response or postexposure response was recorded by stimulating tarsus with 2% sucrose five times, similar to naive response.

Habituation override

Ten percent yeast was presented to the proboscis over 1 min, in a “spaced manner” to prevent consumption of yeast and satiation. In gist, the yeast paste was applied to labellar bristles for about a second but withdrawn rapidly before consumption could occur and this was repeated (approximately six times) over a 1-min period. After exposure to yeast, flies were allowed to groom for 1 min so that yeast sticking to the proboscis could be cleaned. PER responses were subsequently recorded as usual.

For habituation override using mechanical stimulus, flies mounted on the cover slip were placed in a Petri dish (35 mm) and vortexed for 1 min on a Scientific Industries Vortex-Genie 2 instrument at the highest setting. Flies were allowed to recover from the shock for 1 min before testing the response.

To distinguish override from sensitization, naive response of flies was recorded at concentrations of sucrose lower than 2%. This was done as the PER response is maximum at 2%, any difference as a result of manipulation would have been difficult to observe. Four concentrations of sucrose were tested: 0.1%, 0.5%, 1%, and 2%. Flies were then exposed to either stimulus used for override or heat, for 1 min. Postresponse was tested after 1-min rest at room temperature (RT).

Heat-mediated manipulation

For TRPA1 and Shits experiments, flies were transferred to 32°C and 34°C, respectively, on a dry bath for 1 min. In case of Shits experiments, proboscis was stimulated with 10% yeast while at 34°C. Flies were given 1-min rest at RT before testing PER response.

Immunohistochemistry

Adult brains were dissected in 1× PBS and fixed in 4% paraformaldehyde diluted in 1× PBS with 0.3% Triton X-100 (PTX) for 30 min at RT. Samples were washed with 0.3% PTX for 20 min three times at RT and incubated with primary antibody for 48 h at 4°C on a shaker. Samples were again washed three times with 0.3% PTX for 20 min each. Secondary antibodies conjugated with Alexa Fluor 488, Alexa Fluor 568, and Alexa Fluor 647 (1:500, Invitrogen), diluted in 0.3% PTX, were added and samples were incubated for 24 h. Brain samples were again washed repeatedly three times for 20 min each and mounted in Vectashield (H-1000, Vector Laboratories) on a glass slide with spacers. Images were acquired using Zeiss LSM 510 Meta microscope and Olympus Fluoview (FV- 3000).

Following primary antibodies were used: rabbit anti-GFP (1:100, Invitrogen), mouse anti-Bruchpilot (n82; 1:20, DSHB), mouse anti-GFP (Sigma, 1:100), rabbit anti-TH (1:1000, Invitrogen), chick anti-GFP (Abcam, 1:5000), rabbit anti-DsRed (1:100, Clontech).

Statistical analysis

Nonparametric tests were used to analyze data. Friedman test was used to analyze habituation override experiments which had three matched groups, followed by Dunn's multiple comparisons post hoc test to determine difference among different treatments. Wilcoxon signed-rank test was used for comparing two paired groups, whereas Mann–Whitney U test was used to compare two unpaired groups in Figure 1C. All the data were analyzed using GraphPad Prism v8.4.2 software.

Results

Habituation of the PER to sucrose

Two percent sucrose applied to tarsal hairs of immobilized, naive flies induces robust and reproducible proboscis extension response. However, following extended 10-min exposure to 10% sucrose solution, PER decreases from 92.41% to 28.96% (***p < 0.0001, Wilcoxon signed-rank test; Fig. 1A,B). No change in naive PER response is seen if water is presented for 10 min instead of 10% sucrose (Fig. 1B). A previous study concluded that plasticity in central GABAergic neurons is required for PER habituation (Paranjpe et al., 2012). Given their significance for the inferred inhibitory mechanism for PER habituation, and because these observations were made exclusively in male flies, we independently repeated key experiments to re-examine: first, the need for the rutabaga-encoded adenyl cyclase in PER habituation; and second, its reported sufficiency in GAD1-Gal4 expressing, predominantly GABAergic neurons for this function in our hands and in female flies. Our results were consistent with and confirmed previously reported observations (Fig. 1C).

Figure 1.
Figure 1.

Gustatory habituation of PER to sweet taste. A, Behavioral protocol of PER habituation to tarsal stimulation following sucrose exposure. B, Tarsal exposure to 10% sucrose for 10 min leads to decrease in PER response (**p < 0.0001, Wilcoxon signed-rank test), whereas exposure to water does not affect the response (p = 0.6133, Wilcoxon signed-rank test). C, As previously described (Paranjpe et al., 2012), gustatory habituation is dependent on the Rutabaga adenylate cyclase since it is impaired in rut2080 mutants. Habituation can be restored by expressing wild-type rut in inhibitory Gad1-Gal4 neurons (**p < 0.006, Mann–Whitney U = 227). D, The habituated response can be overridden by a novel mechanical stimulus like vortexing (p < 0.001, F statistic = 47.08, Dunn's multiple comparison post hoc test, ***p < 0.001, *p = 0.01). E, Vortexing does not have an effect on naive response to sucrose at concentrations 0.1% (p = 0.0518, Wilcoxon signed-rank test), 0.5% (p = 0.6880, Wilcoxon signed-rank test) and 1% (p = 0.1626, Wilcoxon signed-rank test) as, determined by testing PER response prevortexing and postvortexing at different sucrose concentrations. Therefore, the enhanced PER response after vortexing habituated animals is not a result of general sensitization but instead represents a specific override of habituation to sucrose. Bars represent mean ± SEM, triangles represent individual data points, ns represents not statistically significant, p > 0.05. %PER represents: number of complete proboscis extension/Total number of tarsal stimulations (5).

Habituation is often distinguished from sensory or muscular fatigue by demonstrating the rapid reinstatement of the sensory response by strong, novel stimuli (Rankin et al., 2009; Ramaswami, 2014). Consistently, PER-habituated animals retain the ability to respond relatively robustly to sucrose, as evidenced following strong or novel sensory stimulation. Similar to olfactory habituation, strong mechanical stimulation (vortexing in a dish) for 1 min substantially reinstates the sucrose-induced PER in habituated flies (p < 0.001, F statistic = 47.08, Dunn's multiple comparisons post hoc test, *p = 0.01), without affecting baseline sucrose sensitivity in naive animals (Fig. 1D,E; Das et al., 2011; Paranjpe et al., 2012). Thus, the effect of mechanical stimulation on PER is selective on the habituated state and represents a form of habitation override and not sensitization. In order to address the underlying circuit mechanisms, we first looked to identify more precisely defined sensory stimuli that could cause override of PER habituation.

Novel stimuli override per habituation

Drosophila are attracted by the taste of yeast. Gustatory receptor neurons (GRNs) that respond to yeast components have been recently identified (Fischler et al., 2007; Wisotsky et al., 2011; Ganguly et al., 2017; Steck et al., 2018). To examine whether the novel taste of yeast could influence PER habituation to sucrose, we applied a 10% yeast solution to the fly labellum, which has previously been shown to possess yeast-responsive GRNs (Steck et al., 2018), and tested whether this resulted in override of sucrose habituation. Application of yeast to the labellum substantially reinstated the PER response to tarsal sucrose stimulation in PER-habituated animals (p < 0.0001, F statistic = 31.15, Dunn's multiple comparison post hoc test, *p = 0.014; Fig. 2A,B). In contrast, 10% sucrose solution, which should be familiar to the flies, when applied to the labellum of habituated flies had no effect on PER (Fig. 2B). Thus, a brief experience of a novel and in this case, attractive stimulus appears capable of inducing habituation override.

Figure 2.
Figure 2.

Habituation override mediated by novel stimulus. A, Behavior protocol of PER habituation and its override by presenting stimulus to the labellum. B, Presentation of novel 10% yeast stimulus to labellum restores PER response (p < 0.0001, F statistic = 31.15, Dunn's multiple comparisons post hoc test, ***p < 0.0001, *p = 0.014) whereas 10% sucrose, which is a familiar stimulus, does not have an effect on habituated response (p < 0.0001, F statistic = 43.49, Dunn's multiple comparisons post hoc test, ***p < 0.0001, p > 0.99). C1, Expression pattern of Ir25a-Gal4 in the adult brain. Ir25a-Gal4 is expressed in GRNs, predominantly bitter and sweet sensory neurons in the subesophageal zone (SEZ), as well as some olfactory receptor neurons in the antennal lobe (AL). C2, Directly activating yeast-responsive Ir25a-Gal4 cells expressing heat activated channel TRPA1 for 1 min after habituation at 32°C is sufficient to override PER habituation to sucrose (p < 0.0001, F statistic = 38.66, Dunn's multiple comparisons post hoc test, ***p < 0.0001, **p = 0.0012), whereas at permissive temperature, Ir25a>TRPA1 flies do not show an increase in habituated response (p < 0.0001, F statistic = 22.62, Dunn's multiple comparisons post hoc test, ***p = 0.0002, p > 0.99; Ir25a-Gal4/+ p < 0.0001, F statistic = 38.99, Dunn's multiple comparison post hoc test, ***p < 0.0001, p > 0.99, UAS-TRPA1/+, p < 0.0001, F statistic = 33.88, Dunn's multiple comparisons post hoc test, ***p < 0.0001, p > 0.99). D1, Expression pattern of Gr66a-Gal4 in the SEZ region of adult brain. The bitter sensory neurons marked by Gr66a form a ringed structure; they project to the anterior region of the SEZ. D2, Thermogenetic activation of bitter Gr66a-marked GRNs for 1 min after habituation at 32°C also overrides sweet-taste habituation (p < 0.0001, F statistic = 39.85, Dunn's multiple comparisons post hoc test, ***p < 0.0001, **p = 0.0042), whereas no difference is observed in controls (Gr66a>UAS-TRPA1 at RT p < 0.0001, F statistic = 17.56, Dunn's multiple comparisons post hoc test, ***p = 0.0009, p > 0.99, Gr66a-Gal4/+ p < 0.0001, F statistic = 44.60, Dunn's multiple comparisons post hoc test, ***p < 0.0001, p = 0.97, UAS-TRPA1/+, p < 0.0001, F statistic = 40.22, Dunn's multiple comparison post hoc test, p < 0.0001, p > 0.99). Together, the data show that stimulus-novelty drives override of habituation. Bars represent mean ± SEM, triangles represent individual data points, ns represents not statistically significant, p > 0.05. %PER represents: number of complete proboscis extension/Total number of tarsal stimulations (5).

We further looked to identify a single GRN class responsive to yeast components that may be sufficient to drive dishabituation. To do this, we expressed heat-activated cation-permeable TRPA1 channels in yeast-responsive GRNs and tested whether heat-induced activity in these cells, which bypassed the need for normal ligand-receptor interactions, would result in dishabituation (Hamada et al., 2008). Such experiments showed that “thermogenetic” activation of the Ir25a class of sensory neurons is sufficient to cause PER habituation override.

TRPA1 channels open at temperatures above 25°C; thus, activation of TRPA1-expressing neurons can be temporally controlled by exposing experimental animals to temperatures where the channel is either closed (RT) or open (above 25°C). We expressed TRPA1 in yeast responsive Ir25a-Gal4 positive sensory neurons (Steck et al., 2018). These flies were habituated to sucrose at RT (21°C), transferred to 32°C posthabituation for 1 min and then tested for PER. Thermogenetic activation of Ir25a-expressing GRNs was sufficient to cause rapid override of sucrose habituation (Fig. 2C). Thus, after Ir25a activation, PER-habituated animals showed significantly increased PER to tarsal sucrose stimulation (p < 0.0001, F statistic = 38.66, Dunn's multiple comparison post hoc test, **p = 0.0012). Similarly, 32°C exposure of genetic control animals not expressing TRPA1 did not affect PER habituation (Fig. 2C).

To distinguish between the novelty and attractiveness of yeast taste as being primarily instrumental in override, we also examined, whether thermogenetic activation of bitter-compound responsive Gr66a-expressing GRNs could similarly affect override of PER habituation. Thus, we expressed TRPA1 in Gr66a-Gal4-positive neurons and examined how sucrose-responsiveness of flies habituated at RT was altered after a brief 1 min shift to 32°C. Sucrose induced PER increased from 6.67% to 56.29% (p < 0.0001, F statistic = 39.85, Dunn's multiple comparison post hoc test, **p = 0.0042), indicating that brief activation of bitter-taste sensing Gr66a-positive neurons is sufficient to induce dishabituation. The observation that perception of novel bitter taste as well as novel yeast taste can promote override of PER habituation strongly indicates that it is the novelty of the dishabituating stimulus, rather than its attractiveness, that drives habituation override (Fig. 2D). We also tested the effect of thermogenetic activation of olfactory and visual sensory neurons by expressing TRPA1 in all Or83b-Gal4-positive olfactory sensory neurons (OSNs), in subsets (Or85a-Gal4 or Gr21-Gal4 positive) of OSNs that mediate strong repulsion, or Rh1-Gal4 expressing photoreceptors, but we did not observe an effect on PER habituation (data not shown). Therefore, we did not explore these stimuli further.

Does novelty-induced override of PER habituation represent a specific effect on the habituated state or a general sensitization of the sensory response? To differentiate between these possibilities, we tested whether and how yeast exposure, Ir25a-neuron activation or Gr66a-neuron activation altered PER in naive flies to a range of sucrose concentrations. If the increase in PER response in habituated flies were because of sensitization, then we would expect an increase in PER after the novel stimulus is applied to naive animals. However, as shown in Figure 3A–C, there is no significant difference in PER response before and after presentation of yeast or Ir25a or Gr66a-GRN activation, respectively. Thus, it can be concluded that the reinstatement of PER response that we observe in our experiments is because of a process that specifically acts on neural correlates of PER habituation, not broadly on taste perception.

Figure 3.
Figure 3.

Novelty induced habituation override does not induce sensitization of sensory response. A, Labellar exposure to 10% yeast does not increase the naive PER response to 0.1%, 0.5%, 1%, or 2% sucrose stimulation (probabilities of PER being unchanged using the Wilcoxon signed-rank test: p = 0.6404, p = 0.6731, p = 0.1957, and p = 0.1208 at the 4 respective sucrose concentrations). B, Thermogenetic activation of Ir25a-Gal4 does not sensitize response to sucrose tested at concentration 0.1% (p > 0.99, Wilcoxon signed-rank test), 0.5% (p = 0.5938, Wilcoxon signed-rank test), 1% (p = 0.7588, Wilcoxon signed-rank test), 2% (p = 0.4814, Wilcoxon signed-rank test). C, Thermogenetic activation of Gr66a-Gal4 does not have an effect on response to 0.1% (p = 0.5, Wilcoxon signed-rank test), 0.5% (p > 0.99, Wilcoxon signed-rank test), 1% (p = 0.375, Wilcoxon signed-rank test), 2% (p = 0.3828, Wilcoxon signed-rank test) concentrations of sucrose. Points represent mean ± SEM, ns represents not statistically significant, p > 0.05. %PER represents: number of complete proboscis extension/Total number of tarsal stimulations (5).

Artificial activation of TH-expressing neurons overrides per habituation

Across species, novel stimuli trigger central release of neuromodulators that confer or enhance their salience (Ranganath and Rainer, 2003; Kafkas and Montaldi, 2018). In particular, the activity of dopaminergic neurons has been implicated in the novelty response both in insects and in mammalian systems (Hattori et al., 2017; Morrens et al., 2020). We therefore investigated whether thermogenetic activation of Drosophila TH-expressing central dopaminergic neurons would be (1) sufficient and (2) necessary for novel-taste induced override of PER habituation.

In PER-habituated TH-Gal4>UAS-TRPA1 animals, 1-min exposure to 32°C to drive thermogenetic activation of TH-Gal4-expressing neurons resulted in rapid habituation override (Fig. 4A). In PER-habituated animals, sucrose-induced PER was close to 12%; in these same animals, activation of TH-expressing neurons increased PER to 60% (p < 0.0001, F statistic = 50.54, Dunn's multiple comparisons post hoc test, ***p = 0.0004). Significantly, activation of TH Gal4 neurons had no effect on the innate response to sucrose in naive flies (Fig. 4B). Thus, activation of these modulatory neurons causes habituation override, not general taste sensitization.

Figure 4.
Figure 4.

Activation of TH-expressing neurons overrides habituation. A, Thermogenetic activation of TH-expressing neurons for 1 min at 32°C after habituation results in habituation override (p < 0.0001, F statistic = 50.54, Dunn's multiple comparisons post hoc test, ***p < 0.0001, ***p = 0.0004; TH-Gal4/+, p < 0.0001, F statistic = 49.56, Dunn's multiple comparisons post hoc test, ***p < 0.0001, p > 0.99, UAS-TRPA1/+, p < 0.0001, F statistic = 37.31, Dunn's multiple comparisons post hoc test, ***p < 0.0001, p > 0.99, TH-Gal4/UAS-TRPA1 temperature control, p < 0.0001, F statistic = 26, Dunn's multiple comparisons post hoc test, ***p < 0.0001, p = 0.8665). B, Activation of TH-expressing neurons does not lead to sensitization of gustatory response tested at 0.1% (p = 0.09, Wilcoxon signed-rank test), 0.5% (p = 0.1, Wilcoxon signed-rank test), 1% (p = 0.14, Wilcoxon signed-rank test), 2% (p = 0.1367, Wilcoxon signed-rank test). C, Activation of a subset of TH-expressing cells marked by TH-C'-Gal4 for 1 min after habitation at 32°C is sufficient to override habituated response to sucrose (p < 0.0001, F statistic = 69.50, Dunn's multiple comparisons post hoc test, ***p < 0.0001, **p = 0.0042; TH Gal4/+, p < 0.0001, F statistic = 59.70, Dunn's multiple comparisons post hoc test, ***p < 0.0001, p = 0.4914, UAS-TRPA1/+, p < 0.0001, F statistic = 63.52, Dunn's multiple comparisons post hoc test, ***p < 0.0001, p > 0.99; TH-C'-Gal4 temperature control, p < 0.0001, F statistic = 21.41, Dunn's multiple comparisons post hoc test, **p = 0.0017, p > 0.99). D, Activation of TH-D' subset of neurons, which marks subset of neurons that does not overlap with TH-C' for 1 min after habituation at 32°C does not override habituation (p < 0.0001, F statistic = 42.70, Dunn's multiple comparisons post hoc test, ***p < 0.0001, p> 0.99; UAS-TRPA1/+, p < 0.0001, F statistic = 76.79, Dunn's multiple comparisons post hoc test, ***p < 0.0001, p = 0.3710). E, Combining TH-C-Gal80 along with TH Gal4 blocks the expression in TH-C' subset of neurons only, as observed by GFP expression (SEZ, subesophageal zone). F, The flies carrying TH-C-Gal80 along with TH-Gal4 fail to show habituation override when these are activated at 32°C after habituation further confirming the role of TH-C' subset of neurons (p < 0.0001, F statistic = 35.74, Dunn's multiple comparisons post hoc test, ***p < 0.0001, p > 0.99; TH-Gal4/UAS-TRPA1, p < 0.0001, F statistic = 45.65, Dunn's multiple comparisons post hoc test, ***p < 0.0001, **p = 0.0025; F statistic = 35.38, TH-C-Gal80/+, p < 0.0001, F statistic = 35.38, Dunn's multiple comparisons post hoc test, ***p < 0.0001, p = 0.9061; UAS-TRPA1/+, p < 0.0001, F statistic = 30.55, Dunn's multiple comparisons post hoc test, ***p < 0.0001, p > 0.99). G, Activation of TH-C' Gal4 neurons does not affect the naive response to sucrose tested at 0.1% (p = 0.5635, Wilcoxon signed-rank test), 0.5% (p = 0.3748, Wilcoxon signed-rank test), 1% (p = 0.7338, Wilcoxon signed-rank test), 2% (p = 0.3125, Wilcoxon signed-rank test). Bars represent mean ± SEM, triangles represent individual data points, points in B, G represent mean ± SEM, ns represents not statistically significant, p > 0.05. %PER represents: number of complete proboscis extension/Total number of tarsal stimulations (5).

TH-Gal4 labels around 200 neurons in the Drosophila brain (Friggi-Grelin et al., 2003). In order to more tightly define TH-expressing cells involved in PER-habituation override, we tested two nonoverlapping subsets of TH-Gal4-positive neurons, marked by TH-D'-Gal4 and TH-C'-Gal4 drivers (which labels ∼54 ± 5 and ∼45 ± 3 neurons, respectively) for their potential roles. While 1 min thermogenetic activation of TH-D' neurons had no effect on PER habituation, similar activation of neurons labeled by TH-C'-Gal4 significantly increased PER response from 28.72% in control habituated flies, to 57.81% after TH-C' activation (p < 0.0001, F statistic = 69.50, Dunn's multiple comparisons post hoc test, **p = 0.0042; Fig. 4C). Moreover, TH-Gal4-driven habituation override required activity in TH-C' cell population; thus, 32°C exposure did not trigger PER habituation override in TH-C-Gal80; TH-Gal4> UAS TRPA1 flies, in which Gal80 expression prevented TRPA1 expression in the TH-C' subset of TH-Gal4 neurons (Fig. 4D). This indicates that activity in TH-C' subset of cells is necessary and sufficient to cause habituation override. To further support this conclusion, we checked and confirmed that activation of TH-C' neurons had no significant effect on the PER of naive flies across a range of sucrose concentrations (Fig. 4E) confirming that TH-C' neurons specifically influence mechanisms of habituation, rather than general taste perception.

Significantly, a small subset of TH-C' neurons (∼13 ± 6) are present locally in the SEZ, an area which not only receives inputs from taste sensory neurons, but also houses interneurons and motor neurons involved in proboscis extension (Gordon and Scott, 2009; Kain and Dahanukar, 2015). Thus, some TH-C' neurons are well positioned to mediate sensory-driven novelty signals that influence mechanisms of PER habituation.

A subset of TH-expressing neurons mediates novelty-induced habituation override

In order to test whether the activity of TH-Gal4 and TH-C'-Gal4 neurons is necessary for novel-taste induced override of PER habituation, we examined whether novel-taste stimulation could cause habituation override under conditions where synaptic output from TH-Gal4 or TH-C'-Gal4 neurons was blocked. To achieve this, we expressed the temperature-sensitive, dominant-negative Shits1 mutant form of dynamin in these neurons and tested whether a 1-min exposure to 10% yeast at 34°C could override habituation in TH-Gal4, UAS-Shits1 and TH-C'-Gal4 UAS-Shits1 at temperatures restrictive for Shits1 dynamin function.

At permissive (room) temperatures, TH-Gal4, UAS-Shits1 and TH-C'-Gal4, UAS-Shits1 flies behaved similarly to wild-type flies, showing both robust PER habituation after 10 min of tarsal sucrose exposure (Fig. 5A) and significant PER dishabituation following brief (1 min) exposure of their labella to 10% yeast solution (Fig. 5A). The respective efficiency of PER in habituated and dishabituated animals (before and after yeast exposure) were 34.05% and 77.29% (p < 0.0001, F statistic = 42.47, Dunn's multiple comparisons post hoc test, ***p = 0.0008) and 13.46% and 48.46% (p < 0.0001, F statistic = 41.01, Dunn's multiple comparisons post hoc test, *p = 0.045), respectively (Fig. 5A).

Figure 5.
Figure 5.

Activity of TH-C' subset of neurons is necessary for novelty induced override. A, At permissive temperature (RT = 21°C), presentation of 10% yeast to the labellum overrides habituation in both TH-C' Gal4/UAS-Shits1 (p < 0.0001, F statistic = 41.01, Dunn's multiple comparisons post hoc test, ***p < 0.0001, *p = 0.0457) and TH-Gal4/+;UAS-Shits1/+ flies and (p < 0.0001, F statistic = 42.47, Dunn's multiple comparisons post hoc test, ***p < 0.0001, ***p = 0.0008), respectively. However, at restrictive temperature (34°C), blocking the synaptic transmission of TH-Gal4 and TH-C'-Gal4 neurons by expressing Shits1 during presentation of novel yeast stimulus after habituation to sucrose impairs habituation override (TH-Gal4/+;UAS-Shits1/+, p < 0.0001, F statistic = 102.8, Dunn's multiple comparisons post hoc test, ***p < 0.0001, p = 0.3897; TH-C'-Gal4/UAS-Shits1, p < 0.0001, F statistic = 37.98, Dunn's multiple comparisons post hoc test, ***p < 0.0001, p > 0.99), respectively. The genotypic controls show override of habituation to 10% yeast presented to the labellum (TH-Gal4/+, p < 0.0001, F statistic = 35.83, Dunn's multiple comparisons post hoc test, ***p < 0.0001, **p = 0.0015; UAS-Shits1/+, p < 0.0001, F statistic = 56.96, Dunn's multiple comparisons post hoc test, p < 0.0001, ***p = 0.0009; TH-C'-Gal4/+, p < 0.0001, F statistic = 46.90, Dunn's multiple comparisons post hoc test, ***p < 0.0001, ***p = 0.0001). B, Blocking TH-Gal4 neurons and TH-C'-Gal4 neurons does not have an effect on PER itself (p = 0.1909 and p = 0.9141, respectively, Wilcoxon signed-rank test). Bars represent mean ± SEM, triangles represent individual data points, ns represents not statistically significantly, p > 0.05. %PER represents: number of complete proboscis extension/Total number of tarsal stimulations (5).

In contrast, if after PER habituation the same flies were shifted to and exposed to 10% yeast at 34°C, where the essential function of dynamin in transmitter release would be compromised in Shits1-expressing cells, then habituation override was significantly impaired as compared with controls (Fig. 5A). Thus, yeast-induced dishabituation of control flies not expressing Shits1 at 34°C was substantially more efficient (p < 0.0001, F statistic = 35.83, Dunn's multiple comparisons post hoc test, **p = 0.0015) than of TH-Gal4, UAS-Shits1 flies at the same temperature (Fig. 5A). Similarly, dishabituation of control flies not expressing Shits1 was also substantially more efficient at 34°C (p < 0.0001, F statistic = 46.90, Dunn's multiple comparisons post hoc test, ***p = 0.0001), than of TH-C'-Gal4, UAS-Shits1 flies, expressing temperature-sensitive mutant dynamin in THC'-Gal4 neurons. These data argue that presynaptic activity in TH-C'-Gal4 and TH-Gal4-positive cells is required for yeast-induced override of PER habituation (Fig. 5A).

An important caveat to the above conclusion is that dopaminergic neuron activation may be associated with motivation or reward prediction and therefore be fundamentally required for high levels of PER. In such a scenario, inactivation of TH-expressing cells would be expected to generally reduce levels of PER. To address this issue and test whether the apparently reduced override in Figure 5A is an artifact of blocking dopaminergic neurons during yeast exposure, we measured the innate response to sucrose in naive flies with and without blockage of synaptic transmission in dopaminergic neurons (Fig. 5B). There was no significant difference in the innate response to 2% sucrose, confirming a more restricted role for these TH-positive cells in the override of habituation.

Sensory neurons projections in SEZ overlap spatially with projections of TH neurons

In the mushroom body, dopaminergic neurons potentially compute novelty by combining excitatory inputs from olfactory sensory channels with inhibitory inputs driven by familiarity encoding interneurons (Zhao et al., 2021; see Discussion). If TH-positive cells were to play a similar role in the SEZ, then they would be predicted to receive direct or indirect inputs from taste sensory neurons as well as input from familiarity-representing inhibitory neurons. Previous work has shown that processes from some TH-positive neurons are present in the SEZ which also contains presynaptic endings of taste sensory neurons, processes of local interneurons and dendrites of motor neurons involved in proboscis extension (Fig. 6A; Liu et al., 2012). Therefore, we considered the possibility that taste sensory neurons make contacts with TH-positive processes.

Figure 6.
Figure 6.

TH-Gal4 neurons are functionally connected to GRNs. A, Expression pattern of Gr66a with respect to TH-expressing neurons. B, GRASP between Gr66a and TH-expressing neurons show GFP signal in animals expressing both the components of split GFP. No signal is observed in controls lacking either of split GFP component. C, Controls do not show any significant increase in response at 34°C (Gr66a LexA/+; TH-Gal4/+, p < 0.0001, F statistic = 55.06, Dunn's multiple comparisons post hoc test, ***p < 0.0001, p = 0.9079, LexAop-TRPA1/+; UAS-Shits1/+, p < 0.0001, F statistic = 53.41, Dunn's multiple comparisons post hoc test, ***p < 0.0001, p = 0.0580). Activation of Gr66a while inhibiting TH-expressing neurons simultaneously fails to reinstate habituated response (Gr66a LexA/LexAop-TRPA1; TH-Gal4/UAS-Shits1, p < 0.0001, F statistic = 52.57, Dunn's multiple comparisons post hoc test, ***p < 0.0001, p = 0.9494), whereas activating Gr66a solely overrides habituation (Gr66a LexA/LexAop-TRPA1; TH-Gal4/+, p < 0.0001, F statistic = 44.02, Dunn's multiple comparisons post hoc test, ***p < 0.0001, *p = 0.0205). Control at permissive temperature does not show any difference in habituated response (Gr66a LexA/LexAop-TRPA1; TH-Gal4/+, ***p = 0.0004, F statistic = 15.50, *p = 0.0322, p > 0.99, Dunn's multiple comparisons post hoc test). Bars represent mean ± SEM, triangles represent individual data points, ns represents not statistically significantly, p > 0.05, SEZ, subesophageal zone. %PER represents: number of complete proboscis extension/Total number of tarsal stimulations (5).

To test whether taste-sensory neurons carrying dishabituating signals form connections, direct or indirect, with TH-neuron processes in the SEZ, we used the GFP-reconstitution across synaptic partners (GRASP) technique, which requires the use of dual binary transcription systems (based on LexA and Gal4 transcription factors) to separately express two complementing fragments of GFP, one in sensory neurons and the other in TH neurons. Limited by the immediate availability of transgenes, we were technically restricted to analyzing TH connections with neurons in which gene expression could be controlled by LexA. This was possible for bitter-taste responsive Gr66a neurons but not for the yeast-responsive Ir25a class.

We first examined whether Gr66a axonal projections and TH-Gal4 marked processes were present in close proximity within the SEZ, by examining the relative localization of GFP driven in Gr66a-expressing neurons with RFP in TH-positive cells. Processes of sensory neurons expressing Gr66a-LexA-driven GFP and dopaminergic neurons expressing TH-Gal4 driven RFP showed close proximity (Fig. 6A). Further, GRASP experiments showed that two halves of split GFP, one expressed in Gr66a neurons and the other in TH-positive cells could combine to reconstitute GFP fluorescence within the SEZ (Fig. 6B). Robust fluorescence reconstitution was seen in 8/13 experimental animals compared with 0/15 total animals expressing only one split-GFP component. Thus, GRASP experiments confirm that the processes of Gr66a axons and TH-expressing cells come in close proximity of each other. However, because of the limitation of the GRASP technique used it cannot be established certainly if there is a direct synapse between the two cell types.

These data predict that habituation override induced by thermogenetic activation of Gr66a cells should require activity in TH-Gal4-positive cells. We tested this prediction by creating and analyzing PER habituation and dishabituation in Gr66a-LexA/LexAop-TRPA1; TH-Gal4/UAS-Shits1 and control Gr66a-LexA/LexAop-TRPA1; TH-Gal4/+ lines at temperatures permissive and restrictive for Shits1 dynamin. As shown (Fig. 6C), both lines showed robust PER and PER habituation. One-minute exposure to 34°C in control flies permissive for synaptic transmission from TH-positive neurons resulted in significant (p < 0.0001, F statistic = 44.02, Dunn's multiple comparisons post hoc test, *p = 0.02) override of habituation. However, similar 34°C thermogenetic stimulation of Gr66a neurons in experimental flies where synaptic transmission from TH-positive neurons is blocked did not result in override of PER habituation. Together the anatomic and behavioral data indicate that Gr66a-expressing bitter taste sensory neurons are functionally connected to TH-expression modulatory neurons whose activity is required for PER habituation override.

Control experiments demonstrating that blocking synaptic output from TH-expressing cells has no effect on basal PER response in naive animals further confirm that transmitter release from TH-neurons is required for override of neuronal mechanisms of habituation, not for the innate response to sucrose (Fig. 5B).

Discussion

Animal behavior is profoundly flexible. Thus, at any time, multiple potential behavioral programs remain dormant, while a subset relevant to specific contexts are active. A growing body of evidence suggests that inhibitory inputs play a major role in preserving perceptions, behaviors and memories in dormant form until required (Barron et al., 2017). Thus, a given context, by recruiting disinhibitory circuits may override inhibition to release latent perceptions, motor programs and memories appropriate to that context. While the overarching principles are increasingly appreciated, there is still limited understanding of how these are implemented in cells and circuits. While there are many potential reasons for this, the difficulty in studying mechanisms of override has been substantially caused by the paucity of systems in which both mechanisms of habituation or cognitive silencing can be addressed as well as where robust override can be experimentally achieved.

The results we present outline essential elements within a Drosophila circuit that overrides habituation of the sucrose-evoked PER. In doing so, they connect sensory neurons mediating override to neuromodulatory neurons projecting to the SEZ, which may override habituation by silencing inhibitory neurons that drive habituation.

Previous work in Drosophila concluded that PER habituation arises from increased sucrose-evoked inhibition onto neurons that drive proboscis extension (Paranjpe et al., 2012). Two findings, which closely mirror observations on olfactory habituation, provided key support for this conclusion (Das et al., 2011; Paranjpe et al., 2012). First, the rutabaga-encoded adenyl cyclase is required specifically in inhibitory neurons for PER habituation, an observation that we have independently confirmed in this study (Fig. 1). Second, experimental silencing of inhibitory neurons causes override of habituation. Together these observations indicate first, that increased GABAergic activity is required for the expression of PER habituation and second, that disinhibition could serve as strategy for habituation override. How might such disinhibition be biologically achieved? Our current experiments show that novel sensory experience induces override of PER habituation through a pathway that requires activity in the TH-C' class of dopaminergic neurons. As thermogenetic activation of TH-C' cells is also sufficient to override PER habituation, the data suggest a framework in which sensory stimuli activate TH-C' neurons, which directly or indirectly, inhibit GABAergic neurons responsible for habituation (Fig. 7).

Figure 7.
Figure 7.

Model for habituation override in the Drosophila gustatory system. As shown by Paranjpe et al. (2012), potentiation of GABAergic (Gamma-Aminobutyric acid) neurons (“strong inhibition”) underlies PER (Proboscis extension reflex) habituation to a familiar stimulus (right side). A novel stimulus (left side) activates TH-C' subset of (likely) dopaminergic neurons which in turn inhibits GABAergic neurons that are potentiated during habituation. The resulting disinhibition restores strong PER to familiar stimulus (habituation override). Familiar stimuli cause only weak activation of TH-C' neurons (potentially through increased inhibition onto TH-C') and do not alter the habituated PER response.

An important question to address is why novel but not familiar taste stimuli are effective for override? In physiological terms, how could activation of a novel subset of (Ir25a or Gr66a) sensory-neurons result in strong excitation of TH-C' cells, while similar levels of activation of sucrose-response sensory-neurons causes weaker excitation of the same TH-C' cells? We suggest that this occurs because familiar stimuli also result in decreased activation (or likely increased inhibition) of TH-C' neurons. A schematic circuit level model is shown in Figure 7. More detailed information on the anatomic and physiological connectivity across key elements of the dishabituation circuit will be required to test and elaborate on this broad model. The subtypes and connectivities of relevant inhibitory neurons in the SEZ remain unknown and crucially important to establish. It is particularly important to know whether different classes of SNs activate different subgroups of iLNs in the SEZ. In addition, the exact subset of TH-C' cells involved as well as the mechanism by which they function and modulate GABAergic iLNs need to be identified. While the relevant TH-C' cells are marked by three independent dopaminergic reporter lines, TH-Gal4, TH-C' and TH-C-Gal80, and therefore probably dopaminergic, it remains unclear whether dopamine release is required for habituation override. Our attempts to address the last issue via knock-down of TH through RNAi, or by various genetic manipulations of dopamine receptor expression did not yield definitive results (Materials and Methods), often because these manipulations affected baseline levels of PER and PER habituation.

Despite the above lacunae, given that the SEZ, which contains gustatory neurons axons as well as dendrites of motor neurons that drive PER, is likely to be numerically simple, the most parsimonious model for override would posit that taste sensory neurons trigger direct excitation of dopaminergic processes, which in turn acts within the SEZ to directly inhibit GABAergic cells (Pimentel et al., 2016) whose potentiation drives PER habituation.

The model above is consistent with observations on habituation override in mouse and Aplysia brains (Bristol and Carew, 2005; Smith et al., 2009; Kato et al., 2015; Ogg et al., 2018). In mouse, long-term auditory habituation to a passively experienced tone is accompanied by increased activity in tone-responsive SOM+ neurons that inhibit similarly tuned pyramidal cells in the auditory cortex. However, if habituated mice are coaxed to attend to the same tone (by a reward for successful engagement), then the behaving mice show overriding inhibition of SOM+ neurons and increased activity of downstream L2/3 pyramidal neurons (Kato et al., 2015). It appears likely that this disinhibition is accomplished by modulatory inputs onto upstream VIP+ neurons. A more recent analysis showed that cholinergic inputs into the mouse olfactory bulb could cause override of a fast form of olfactory habituation. Thus, electrical or optogenetically induced acetylcholine release into the bulb caused mice to override habituation and investigate a previously ignored odor (Ogg et al., 2018). While sensitization is not formally excluded here, these studies are consistent with an emerging theme wherein neuromodulators released in response to novel or meaningful stimuli (Vankov et al., 1995; Giovannini et al., 2001; Ranganath and Rainer, 2003; Hattori et al., 2017; Kafkas and Montaldi, 2018; Morrens et al., 2020) result in disinhibition which can either enhance learning or override habituation.

The experimental results described here provides multiple lines of circumstantial evidence in support of a novelty-induced dopaminergic pathway for disinhibition of sensory perception. It outlines a habituation override circuit all the way from sensory neurons that detect stimulus, to motor neurons that mediate behavioral response. In context of the increasingly widely appreciated role for disinhibition in the control of perception, cognition, and behavior (Letzkus et al., 2015; Sridharan and Knudsen, 2015; Barron et al., 2017; Wang and Yang, 2018), we suggest that this work provides a valuable intellectual and biological foundation for future studies to comprehensively identify neurons and mechanisms involved in a central pathway for behaviorally important disinhibition.

Footnotes

  • This work was supported by a Wellcome Trust Investigator Award and a Science Foundation Ireland Investigator Program grant (M.R.), by core support from the National Center for Biological Sciences (Tata Institute of Fundamental Research; K.V.), and by a Council of Scientific and Industrial Research postgraduate fellowship and a Biocon-Trinity scholarship (S.T.). We thank Ali Asgar Bohra, Camilla Roselli, Tamara Boto, and James Cooke for comments on the manuscript; Frederic Marion-Poll and members of Ramaswami and VijayRaghavan labs for advice, support, and useful discussions; Carlos Ribeiro for valuable discussions and reagents; and Pushkar Paranjpe for help setting up the PER assay as well as the Bloomington stock center and our many colleagues (mentioned in the text) for various Drosophila stocks used.

  • The authors declare no competing financial interests.

  • Correspondence should be addressed to Mani Ramaswami at mani.ramaswami{at}tcd.ie

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A Drosophila Circuit for Habituation Override
Swati Trisal, Marcia Aranha, Ankita Chodankar, K. VijayRaghavan, Mani Ramaswami
Journal of Neuroscience 6 April 2022, 42 (14) 2930-2941; DOI: 10.1523/JNEUROSCI.1842-21.2022
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