Olfactory habituation in Drosophila-odor encoding and its plasticity in the antennal lobe.

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Olfactory Habituation in Drosophila—Odor Encoding and its Plasticity in the Antennal Lobe

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Isabell Twick*,1,2, John Anthony Lee*,1,2, Mani Ramaswami*,{ *

School of Genetics and Microbiology and School of Natural Sciences, Smurfit Institute of Genetics, Trinity College Institute of Neuroscience, Trinity College Dublin, Ireland { National Centre for Biological Science, Bangalore, India 1 Corresponding authors: Tel.: 00353 (0) 1 896 8530; Fax: 00353 (0) 1 896 3183, e-mail address: [email protected]; [email protected]

Abstract A ubiquitous feature of an animal’s response to an odorant is that it declines when the odorant is frequently or continuously encountered. This decline in olfactory response, termed olfactory habituation, can have temporally or mechanistically different forms. The neural circuitry of the fruit fly Drosophila melanogaster’s olfactory system is well defined in terms of component cells, which are readily accessible to functional studies and genetic manipulation. This makes it a particularly useful preparation for the investigation of olfactory habituation. In addition, the insect olfactory system shares many architectural and functional similarities with mammalian olfactory systems, suggesting that olfactory mechanisms in insects may be broadly relevant. In this chapter, we discuss the likely mechanisms of olfactory habituation in context of the participating cell types, their connectivity, and their roles in sensory processing. We overview the structure and function of key cell types, the mechanisms that stimulate them, and how they transduce and process odor signals. We then consider how each stage of olfactory processing could potentially contribute to behavioral habituation. After this, we overview a variety of recent mechanistic studies that point to an important role for potentiation of inhibitory synapses in the primary olfactory processing center, the antennal lobe, in driving the reduced response to familiar odorants. Following the discussion of mechanisms for short- and longterm olfactory habituation, we end by considering how these mechanisms may be regulated by neuromodulators, which likely play key roles in the induction, gating, or suppression of habituated behavior, and speculate on the relevance of these processes for other forms of learning and memory.

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These authors contributed equally to this work.

Progress in Brain Research, Volume 208, ISSN 0079-6123, http://dx.doi.org/10.1016/B978-0-444-63350-7.00001-2 © 2014 Elsevier B.V. All rights reserved.

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CHAPTER 1 Olfactory Habituation in Drosophila

Keywords olfactory habituation, habituation, antennal lobe, synaptic plasticity, learning, memory, recurrent inhibition, Drosophila, translational control, neuromodulator

1 INTRODUCTION Olfactory habituation describes the reduced behavioral response to an odorant after repeated or continuous exposure. Because olfactory habituation occurs in response to odor presentation alone (in the absence of associated reward or punishment), it is defined as a form of nonassociative, implicit memory (Thompson and Spencer, 1966; Wilson and Linster, 2008). Reduced perceptual or behavioral responses to familiar inconsequential odorants probably enable organisms to selectively focus on scents potentially relevant for an organism’s survival: for example, that may predict an approaching predator or a rich food source. Thus, olfactory habituation enables attention to be more selectively focused on novel stimuli or those associated with positive or negative consequences. Habituation to olfactory stimuli has been widely studied in mammals where two forms with different timescales (2 or 30 min) have been differentiated (Wilson and Linster, 2008). The shorter timescale form results from synaptic depression in cortical areas (Wilson, 2009). However, this mechanism cannot explain the 30-min behavioral decrement, which arises from plasticity in the olfactory bulb, the primary, mammalian olfactory processing center, and induced by longer-timescale habituation protocols (Chaudhury et al., 2010; McNamara et al., 2008). Deciphering the underlying circuit plasticity and the molecular underpinnings of these changes in mammals has been complicated by the increased complexity of mammalian brains. The olfactory system of Drosophila melanogaster is composed of genetically defined cell types, accessible not only to a variety of convenient cell-type targeted perturbations but also to many kinds of cell biological studies. Genetic and transgenic techniques for this organism have been developed over recent decades making it a powerful model for use in neural circuit analysis (Venken et al., 2011). These techniques facilitate repeated targeting of the same populations of neurons as well as the precise manipulation of their function. The Drosophila olfactory system, readily accessible for electrophysiology and imaging, is also well characterized at the level of anatomy (Laissue and Vosshall, 2008; Laissue et al., 1999; Tanaka et al., 2012) and function (Liang and Luo, 2010; Masse et al., 2009; Ng et al., 2002; Wilson, 2013) of component cell types. This has enabled detailed studies of basal olfactory processing mechanisms as well as its experience-dependent changes (Davis, 2011; Keene and Waddell, 2007; McGuire et al., 2005). The similarities between Drosophila and mammalian olfactory systems suggest a convergence of anatomical arrangement and coding mechanisms used for solving the same problems (Kaupp, 2010; Su et al., 2009; Wilson, 2013), that is, for transforming a complex odor space into a neural representation that reliably describes the

2 Architecture of the Drosophila Olfactory System

important aspects of an odor stimulus. This olfactory network allows the organism to both generalize and discriminate between different signals as needed, offering sufficient plasticity of function to adapt to immediate and long-term demands. Thus, Drosophila is a particularly useful model organism in which the mechanisms of olfactory perception and its plasticity are elucidated. In this chapter, we begin our discussion with a summary of current knowledge of the structure and function of the Drosophila olfactory system including what is known about how aspects of olfactory stimuli such as identity, concentration, and valence are coded. This lays a platform for considering how changes in the neural circuitry may potentially result in behavioral habituation. We elaborate on the behavioral characteristics of short-term habituation (STH) and long-term olfactory habituation (LTH) in terms of the molecular and cellular underpinnings of both forms. More speculatively, we discuss interactions of habituation mechanisms in the antennal lobe (AL) with neuromodulatory inputs that may drive, gate, or reverse habituation. We suggest that understanding circuit mechanisms of olfactory habituation may also illuminate molecular and circuit underpinnings of habituation in other brain structures and in other species.

2 ARCHITECTURE OF THE DROSOPHILA OLFACTORY SYSTEM In Drosophila, odors are detected by odorant receptors (ORs) expressed in 1300 olfactory sensory neurons (OSNs), which are housed in sensilla, hair-like structures located on two sensory organs, the antenna and maxillary palp (Fig. 1) (Larsson et al., 2004; Shanbhag et al., 1999). There are three classes of receptor expressed: ORs, gustatory receptors (GRs), and ionotropic receptors (IRs). The predominant and most studied class of receptors is a family of insect-specific ORs (Clyne et al., 1999; Vosshall et al., 1999), which differ in one crucial manner from mammalian metabotropic ORs (Joerg Fleischer, 2009; Lledo et al., 2005) in that they do not act exclusively through G-protein-coupled signaling. Insect ORs also have seven membrane-spanning domains, but are believed to function primarily as ligand-gated ion channels (Buck and Axel, 1991; Sato et al., 2008; Smart et al., 2008), although they may additionally activate downstream G-proteins (Wicher et al., 2008). Insect ORs operate as part of a heteromeric complex composed of a variable odorantbinding subunit, 1 of 62 ORs, and a universal coreceptor, the Or83b or Orco protein (Benton et al., 2006; Neuhaus et al., 2004). Some sensory neurons express other classes of receptors. GRs are distantly related to ORs and most frequently expressed in taste neurons; however, two of these receptors are co-expressed in one OSN type involved in the detection of CO2 (de Bruyne et al., 2001). IRs, believed to function in parallel with ORs, are structurally similar to ionotropic glutamate receptors except for a highly divergent ligand-binding domain, but their characteristics have only recently been described (Benton et al., 2009).

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FIGURE 1 The Drosophila olfactory system. Odor stimuli are detected at the periphery by OSNs, which are housed in hair-like structures called sensilla. These are located on the maxillary palps and antennae and project from here via the labial nerve (omitted for clarity) and the antennal nerve to the antennal lobe (AL), the fly’s primary olfactory processing center. A major subset of projection neurons (PNs) that project to the calyx of the mushroom body (MBs) and the lateral horn (LH) are the principal output from the AL. The MBs, among other things, are important for olfactory associative learning and memory. The LH is thought to be predominantly involved in innate olfactory behaviors.

Axons of OSNs bundle together in the antennal nerve and project to the AL, a bilateral brain structure analogous to the olfactory bulb in mammals (Lledo et al., 2005). The AL is composed of 56 neuropil regions, called glomeruli (Laissue et al., 1999; Tanaka et al., 2012). Each of these receives excitatory cholinergic input from a particular population of OSNs that expresses a single OR type (Couto et al., 2005; Fishilevich and Vosshall, 2005). Within a given glomerulus, OSNs form synapses with projection neurons (PNs), the AL output neurons (Fig. 2), similar to mitral/tufted cells in mammals (Lledo et al., 2005). They also contact processes of local interneurons (LNs) whose arborizations are restricted to the AL: LNs are

2 Architecture of the Drosophila Olfactory System

FIGURE 2 Wiring of the olfactory system. In general, each odorant receptor (OR) defines its own OSN type consisting of approximately 50 neurons (represented here as specific colors). Two to four different OSN types are housed in a sensillum in a stereotyped manner and each OSN type projects consistently to the same glomerulus in the antennal lobe (AL) where they form synapses with projection neurons (PNs) and local interneurons (LNs). The four PN tracts exiting the AL are the medial (mALT), mediolateral (mlALT), lateral (lALT), and transverse (tALT) antennal lobe tracts (the latter two omitted for clarity). The majority of PNs run through the mALT, including the well-studied uniglomerular cholinergic AL–mPN1 class (depicted here in red and yellow) that terminates in the calyx of the mushroom bodies (MBs) and the lateral horn (LH). The mlALT connects the AL to the LH, bypassing the MB calyx. A large part of this tract contains the AL–mlPN2 class, which consists of multiglomerular GABAergic PNs (depicted here in pink). A substantial amount of processing of the olfactory code is performed by interneurons (LNs) whose arborizations are restricted to the AL. These LNs can be excitatory, inhibitory, or neuromodulatory in nature. A pan-glomerular LN is depicted here in orange. For a more detailed description of LNs, PNs, and other extrinsic AL neurons, see Tanaka et al. (2012).

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comparable to mammalian LNs in the olfactory bulb, the granule, and periglomerular cells (Lledo et al., 2005; Stocker et al., 1990; Tanaka et al., 2012). PNs also form feedback connections to LNs (Liu and Wilson, 2013; Sudhakaran et al., 2012; Tanaka et al., 2009). Approximately, 150 PNs in total leave the AL (Stocker et al., 1990; Tanaka et al., 2012). On average, three PNs leave a given glomerulus and bundle together in one of four AL tracts (Jefferis et al., 2001; Tanaka et al., 2012). A major class of PNs, AL-mPN1, are uniglomerular and cholinergic. This class receives synaptic input from OSNs and LNs and projects via the medial antennal lobe tract (mALT, Fig. 2) to the mushroom bodies (MBs), the lateral horn of the protocerebrum (LH), and other higher brain regions (Stocker et al., 1990; Tanaka et al., 2012). The MBs are well known to be required for olfactory learning and memory (Heisenberg, 2003; Heisenberg et al., 1985), whereas the LH is involved in directing olfactory-mediated innate behaviors (Heimbeck et al., 2001; Parnas et al., 2013). A second major class of PNs, AL-mlPN2, projects via the mediolateral antennal lobe tract (mlALT), which innervates the LH and bypasses the MB altogether (Jefferis et al., 2001; Lai et al., 2008; Stocker et al., 1990; Tanaka et al., 2012). Neurons of this class are multiglomerular and GABAergic (Liang et al., 2013; Okada et al., 2009; Parnas et al., 2013; Tanaka et al., 2012). The other two AL tracts called lateral (lALT) and transverse (tALT) are more diverse in terms of their neuronal projections, but less well studied (Lai et al., 2008; Tanaka et al., 2012). LNs are highly diverse in terms of morphology and neurotransmitter types. There are approximately 100 LNs that arborize unilaterally within one AL and another 100 that arborize bilaterally, with innervation within and between glomeruli (Chou et al., 2010; Das et al., 2008; Lai et al., 2008; Okada et al., 2009; Seki et al., 2010; Shang et al., 2007; Tanaka et al., 2012). The arborization pattern of LNs ranges from innervating just a few glomeruli to labeling all of them, though only 11% of LNs are estimated to innervate less than half of the glomeruli (Chou et al., 2010). Two main neurotransmitters released by LNs are glutamate and GABA, both of which are primarily inhibitory in the AL. Cholinergic and neuromodulatory LNs also exist (Chou et al., 2010; Huang et al., 2010; Na¨ssel and Homberg, 2006; Seki et al., 2010; Shang et al., 2007). Several different neuropeptides have been reported to be expressed in LN subsets and, in several cases, are believed to be co-released with one of the traditional neurotransmitters (Carlsson et al., 2010; Ignell et al., 2009; Na¨ssel and Homberg, 2006). Besides OSNs, LNs, and PNs, several extrinsic neurons innervate the AL and thereby link the neuropil to various other brain areas (Stocker et al., 1990; Tanaka et al., 2012) and effect top-down modulation of odor information processing. A diversity of neuromodulatory inputs that may modulate olfactory information processing in the AL have also been found, such as serotonergic (Carlsson et al., 2010; Dacks et al., 2009; Roy et al., 2007; Tanaka et al., 2012), octopaminergic (Busch et al., 2009), and neuropeptidergic inputs (Carlsson et al., 2010; Na¨ssel, 2002).

3 Layers of Odor Information Processing

3 LAYERS OF ODOR INFORMATION PROCESSING How are odor identity, concentration, and dynamics encoded in the Drosophila olfactory system? Recent technical progress such as the implementation of extracellular recordings of sensory neurons (Clyne et al., 1997) and whole-cell patch clamp of central olfactory neurons in Drosophila (Wilson, 2004) has enormously broadened our knowledge about how odor information are encoded in various cell types (Wilson, 2013). Below, we give a brief overview on how odor features are encoded in OSNs and PNs through interactions with inhibitory and excitatory LNs.

3.1 Olfactory Sensory Neurons Odor information coding in OSNs exhibits characteristic features (Kaupp, 2010; Su et al., 2009). Here, we focus on insights gained from studies in Drosophila, but similar observations have also been made in mammals (Lledo et al., 2005; Malnic et al., 1999; Reisert and Restrepo, 2009; Saito et al., 2009). In the fruit fly, odor identity is encoded to an extent by the combined activity of several OSN types, each expressing a specific unique OR. Most OSN types respond to a number of different odorants, and conversely, most odorants activate a number of different OSN types. Individual OSN types vary in levels of spontaneous activity and can display a broad range of odor response patterns, from being narrowly to broadly tuned in terms of their odorant specificity. Odorants usually, but not always, have an excitatory effect on OSN spiking (de Bruyne et al., 1999, 2001; Hallem and Carlson, 2006). Higher odor concentrations typically cause both an increase in individual OSN firing rates and recruitment of additional OSN types in Drosophila (Hallem and Carlson, 2006). In OSNs, the spike rate in response to an odor stimulus encodes both odor concentration and its rate of change (Nagel and Wilson, 2011). OSNs fire most strongly at odor onset, and recent advances suggest that within a certain concentration range for an odorant, the dynamics of OSN firing can be predicted by a linear– nonlinear model unique to each OR (Martelli et al., 2013; Nagel and Wilson, 2011). Outside these concentration ranges, OSNs exhibit tonic low-rate or prolonged high firing rate responses (Martelli et al., 2013). Thus, odors are encoded based on the subset of activated OSNs as well as temporal features of their spike responses. OSNs show time-dependent, reversible decreases in sensitivity in response to sustained odor stimulation. This sensory adaptation is observed as a rightward shift of the concentration–response curve and is an important property of OSN function (Zufall and Leinders-Zufall, 2000). As OSNs represent concentration and dynamics of odor stimuli, such sensory adaptation in OSNs can contribute to behavioral plasticity. In Drosophila, exposure to an odorant for 1 min can cause sensory adaptation (measured in activity in population recordings in the antennae) lasting almost 10 min as well as concurrently reduced odor avoidance (Sto¨rtkuhl et al., 1999). Studies of cross-adaptation between two cognate odorants for a single OSN indicate that the OSN adaptation extends to all ligands of that cell (de Bruyne et al., 1999). This

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cross-adaptation occurs through a mechanism downstream of the odorant receptor itself (Nagel and Wilson, 2011).

3.2 Projection Neurons About 50 OSNs expressing a specific OR form synapses onto about three PNs in a single AL glomerulus (Stocker, 1994; Tanaka et al., 2004). Due to the high convergence of OSNs onto PNs (50:3), PNs more accurately represent odor stimuli than their respective OSNs (Bhandawat et al., 2007). Furthermore, the divergence of each individual OSN onto every PN within its glomerulus (1:3) causes the activity in these sister PNs to be highly correlated (Kazama and Wilson, 2009). The temporal dynamics of odor-induced PN activity are affected by the characteristics of the OSN–PN synapse such as its high vesicular release probability and strong short-term depression (Kazama and Wilson, 2008; Wilson, 2013). Thus, PN responses generally peak earlier and decay more quickly making the odor response more transient than that of their presynaptic OSNs, which may also further increase the speed of odor processing (Bhandawat et al., 2007; Wilson, 2004). PNs are sensitive to small changes in OSN input when their presynaptic OSNs fire at a low rate, whereas they show little sensitivity to small changes in presynaptic input when OSNs fire strongly (Bhandawat et al., 2007; Olsen et al., 2010; Wilson, 2013). Lateral excitation from LNs in the AL (Olsen et al., 2007; Root et al., 2007; Shang et al., 2007) causes PNs to be more broadly tuned to odors than their presynaptic OSNs (Bhandawat et al., 2007; Olsen and Wilson, 2008). Lateral activity can also be inhibitory and suppress PN odor responses when additional activity in other glomeruli is recruited (Olsen et al., 2010; Silbering and Galizia, 2007).

3.3 Local Interneurons LNs within the AL have a key role in odor processing because of their extensive interaction with both the input and output of the AL. They affect the amplitude and dynamics of PN responses allowing them to encode a broader range of odor concentrations and to process more complex odor mixtures. The LNs principally involved in mediating these effects are inhibitory (iLNs) but others play a role including a small group of excitatory LNs (eLNs). The two main classes of iLNs are GABAergic (Chou et al., 2010; Okada et al., 2009; Tanaka et al., 2012) and glutamatergic (Liu and Wilson, 2013), which act on OSN and PNs via GABAB, GABAA, or GluCla receptors (Liu and Wilson, 2013; Olsen and Wilson, 2008; Root et al., 2008; Wilson and Laurent, 2005). Like GABAergic inhibition (Olsen and Wilson, 2008), glutamate mediates both preand postsynaptic inhibition. However, GluCla-mediated inhibition requires the coactivation of a number of LNs and shows slower timecourse than GABAergic inhibition (Liu and Wilson, 2013; Wilson, 2013). GABAergic inhibition influences the duration of the PN response. Blocking either ionotropic GABAA receptors with picrotoxin (since shown to block GluCla

4 Olfactory Coding of Different Properties of an Odor Stimulus

currents as well) or metabotropic GABAB receptors greatly extends the duration of the PN response (Wilson and Laurent, 2005). A second important function of GABAergic LNs is in gain control (Olsen and Wilson, 2008; Olsen et al., 2010; Root et al., 2008), that is, in determining the concentration dependence of the PN response. This is crucially carried out by lateral inhibition, wherein LN excitation in one glomerulus results in inhibition in other glomeruli (Silbering and Galizia, 2007). Such lateral inhibition appears to be largely mediated via GABAB receptor on OSN presynaptic terminals. Lateral inhibition prevents PN saturation, greatly extending their dynamic range (Olsen and Wilson, 2008; Olsen et al., 2010; Root et al., 2008). Drosophila iLN populations are diverse. For instance, not only do the two populations, LN1 and LN2, differ in their morphology (with the former sending processes into the glomerular core where PN dendrites but not OSN terminals are found and the latter sending processes predominantly to the glomerular rind, enriched in OSN terminals), but also functional differences between the two have been shown by several studies (Sachse et al., 2007; Tanaka et al., 2009).

4 OLFACTORY CODING OF DIFFERENT PROPERTIES OF AN ODOR STIMULUS The olfactory system encodes identity, intensity, and valence of odorants. Each of these parameters influences downstream behavior. Odor identity can be observed in the spatial pattern of neural activity in the primary olfactory processing center in Drosophila. This pattern is largely due to the types of OSNs recruited by the odor, but local processing does reshape the signal significantly (see section 3). Odor concentrations may be represented by both the levels of activity in odorant-responsive PN ensembles and in the identity of PN types that compose this ensemble (Bhandawat et al., 2007; Silbering et al., 2008). Valence, the assignment of a positive or negative percept to an odorant, may occur through several alternative mechanisms. One model for valence coding suggests that it is encoded by specific populations of PNs (Knaden et al., 2012; Semmelhack and Wang, 2009) (for other models, see Haddad et al., 2010; Niewalda et al., 2011; Parnas et al., 2013). This model for valence is particularly strongly supported by an analysis of glomeruli that determine attractiveness or aversiveness of different concentrations of apple–cider vinegar (Semmelhack and Wang, 2009). Here, genetic silencing of each responsive OSN type revealed that two glomeruli were important for coding attractiveness at low vinegar concentrations. At higher concentrations, the odor becomes repellent. This coincides with another glomerulus (DM5) being recruited, the activity of which appeared both necessary and sufficient for this change in valence (Semmelhack and Wang, 2009). Another study, performed with a broad range of odorants, showed that certain populations of PNs were associated with attractive odorants, and a different subset were involved in the representation of aversive odorants (Knaden et al., 2012). These studies suggest that valence may be represented in

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the AL and it is encoded in a PN ensemble distinct from, or at least not wholly overlapping with, PNs encoding identity. An understanding of how these odor stimulus features are encoded can aid in our interpretation of the underpinnings of olfactory habituation. If the behavior is due to changes in the perception of either valence or concentration of the odor, it could be observed physiologically as either changes to specific subsets of PN types or as a more general reduction in the intensity and extent of neural activity, respectively (Fig. 3). Neural representation of an odor in the antennal lobe

Cellular adaptations

Reduction in perceived intensity

Reduction in perceived valence

Reduced activity in valence-specific glomerulus

Behavioral habituation FIGURE 3 Potential perceptual changes underlying olfactory habituation. Cellular adaptations underlying habituation could involve pre- or postsynaptic efficacy, cellular signaling pathways, expression of ion channels, etc. These changes could alter the representation of an odor stimulus in the AL as shown here. A shift in the perceived valence could be coded by a subset of PNs that code for the stimulus, while intensity perception could be changed by a more general reduction in the breadth or amplitude of activity observed to the stimulus. Both could lead to a reduced behavioral response. Differences in how these stimulus features are encoded may aid in interpreting the physiological correlates of olfactory habituation.

5 Olfactory Habituation in Drosophila

5 OLFACTORY HABITUATION IN DROSOPHILA The knowledge of the Drosophila olfactory system described earlier provides a valuable foundation for the analysis of mechanisms of olfactory habituation. There are several behavioral assays for assessing olfactory habituation in Drosophila, which differ in how the olfactory response is measured, how habituation is induced, and the specific odorants tested (Chandra and Singh, 2005; Cho et al., 2004; Das et al., 2011; Sharma et al., 2005). Three of the assays have been used, to a greater or lesser degree, for identifying molecular components required for neural plasticity that underlies olfactory habituation. Below we detail these assays and analyses while overviewing their contribution toward understanding olfactory habituation. We pay particular attention to studies using an olfactory avoidance assay, which have progressed furthest in terms of elucidating the neural circuit mechanisms underlying olfactory habituation.

5.1 Olfactory Startle A simple measure of the Drosophila olfactory response is the odor-evoked locomotor startle response, wherein flies respond to sudden odor exposure with increased movement. For example, when exposed to ethanol vapor for 30 s, Drosophila increase their walking velocity: this locomotor startle response phenomenon is most simply quantified as net movement during the ethanol stimulus period. After four repeated pulses, with interstimulus intervals ranging from 3 to 18 min, a reduced locomotor startle response is observed. This form of habituation occurs more rapidly at higher stimulus frequencies. The observation that it does not occur in animals without antennae argues that it is an olfactory response and not one induced via nonolfactory targets of ethanol. Consistent with the classical definitions of habituation (Rankin et al., 2009; Thompson and Spencer, 1966), the behavior spontaneously recovers and can be dishabituated by a mechanical stimulus. Habituation to ethanol vapor showed cross-habituation to ethyl acetate and isoamyl alcohol. A number of the genes and at least one brain structure required for this form of habituation have been discovered. An adenylate cyclase disrupted in the rutabaga mutant, as well as the Drosophila ortholog of GSK-3 are necessary (Wolf et al., 2007). In the case of GSK, not only do loss-of-function mutations lead to deficits in habituation, but overexpression of the protein also leads to stronger habituation. More recently, the assay was used to screen 874 mutant fly lines for altered habituation of the locomotor startle response (Eddison et al., 2012). Each line contained a mutation induced by a random P-element transposon insertion. Thirty-one strains were found to have abnormal habituation. Two of these showed deficits in habituation with all other strains showing increased habituation. Many of the proteins that disrupt this form of olfactory habituation localize at septate junctions, which are required for normal formation of the Drosophila perineural sheath.

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5.2 Olfactory Jump Reflex A brief puff of the aversive odorant benzaldehyde causes flies to exhibit a jump reflex. The odor-evoked jump response habituates upon repeated exposure (Asztalos et al., 2007a; Boynton and Tully, 1992; Sharma et al., 2005). With 4-s long stimuli given at intervals of 0.25–20 min, it takes approximately 2–15 pulses for the flies to habituate. Consistent with classical properties of behavioral habituation, the reduction in response exhibits dishabituation upon mechanical stimulation, becomes more difficult to invoke with increasing odorant concentration, and shows spontaneous recovery in the absence of the odorant. Dunce, encoding cAMP phosphodiesterase, and rutabaga mutants show decreased habituation despite exhibiting some motor fatigue. Five potassium channel subunits were shown to be involved in olfactory jump habituation (Joiner et al., 2007). Disruption of a fly homolog of bruton tyrosine kinase causes more rapid habituation while showing normal dishabituation (Asztalos et al., 2007b), which suggests that the cause of this phenotype is due to impaired sensitization. A high-throughput method, capable of screening 250 genotypes/month, for assaying this behavior has also been published with 36 genotypes showing deficits in the behavior (Sharma et al., 2009).

5.3 Olfactory Avoidance The use of an olfactory avoidance paradigm, termed the “Y-maze” assay (Rodrigues and Siddiqi, 1978), has been most successful in identifying potential neural mechanisms underlying olfactory habituation in Drosophila so far (Das et al., 2011). The Y-maze apparatus, a glass maze in form of a “Y,” is positioned upright (Fig. 4). One of its arms contains an odorant and the other one contains air. Flies placed in an entry tube on the bottom of the maze climb upward due to their innate negative geotaxis. At the Y-junction of the two arms, the flies have a choice to enter either the odor arm or the air arm (Das et al., 2011). Innate avoidance of the flies is characterized by a response index, which represents the proportion of flies choosing the air arm rather than the odor arm (Rodrigues, 1980; Rodrigues and Siddiqi, 1978). If the flies are exposed for 30 min to ethyl butyrate (EB) and subsequently tested for odor preference, they show a diminished aversion for EB (Das et al., 2011). Documented for EB, CO2, and 3-octanol (3-OCT), this STH recovers spontaneously with a half-life of 30 min and can be dishabituated by either a mechanical stimulus or a puff of yeast odor. Flies habituated to EB, CO2, or 3-OCT do not show cross-habituation to any of the other odorants, indicating that this form of olfactory habituation arises from plasticity in neural circuit elements selectively used for one or other odorant channel (Das et al., 2011; Sudhakaran et al., 2012). In addition to STH, LTH is elicited by 4-day exposure to either odorant, lasts up to 6 days and once again shows odorant selectivity (Das et al., 2011; Devaud et al., 2001). An important observation is that 4-day exposure to EB or CO2 not only induces LTH but also reduces odor-evoked physiological responses in respective odorantresponsive PNs, which can be measured using GCaMP-based imaging of odorevoked calcium fluxes in odor-responsive PNs (Das et al., 2011; Sachse et al., 2007). That odor-evoked calcium transients are also reduced after STH is strongly

5 Olfactory Habituation in Drosophila

Air

Odor

Air

Odor

Odor exposure

Naive response

Habituated response

FIGURE 4 Y-maze assay for measuring olfactory avoidance. An odorized and a pure air stream flow through the arms of the Y-maze apparatus. Flies placed at the bottom walk up due to negative geotaxis and encounter the two streams at the junction. If the odor is aversive, the majority of flies choose to climb up the arm containing air. After exposure to the odor (30 min for STH and 4 days for LTH), the aversive response is no longer elicited.

predicted by several behavioral genetic observations (Das et al., 2011) but is yet to be experimentally tested. Several observations strongly indicate that STH and LTH of olfactory avoidance response arise from central mechanisms rather than peripheral neuronal changes (Das et al., 2011). Activation of genetically targeted subsets of OSNs using transgenically expressed TRPA1 (Pulver et al., 2009), a heat-activated cation channel, for either 30 min or 4 days, to substitute for odorant exposure, still elicited odorselective STH or LTH, respectively (Das et al., 2011). As such OSN stimulation bypasses normal activation of ORs and subsequent signaling, the reduced olfactory avoidance response must occur through a process downstream of olfactory signal transduction (Das et al., 2011). More striking, TRPA1-mediated direct 30-min stimulation of PN subsets responding to particular odors elicited an odor-selective behavioral decrement that showed typical characteristics of STH (Sudhakaran et al., 2012). Thus, neither odorant stimulation nor cellular adaptations in OSNs are necessary for the formation of STH. Two other studies looking at 4-day odor exposure showed that olfactory transduction, as measured by electroantennograms, remains normal (Devaud et al., 2001) and found neither altered OSN morphology nor function when measured using calcium imaging (Sachse et al., 2007). Taken together, these results imply that circuit plasticity that underlies olfactory habituation occurs independently of cellular changes in OSNs.

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6 POTENTIAL MECHANISMS OF OLFACTORY HABITUATION IN DROSOPHILA 6.1 A Recurrent Inhibitory Circuit Motif Underlying Olfactory Habituation Several observations indicate that reduced olfactory avoidance behavior after prolonged odor exposure results from increased input from inhibitory LNs onto odorant-selective PNs (Das et al., 2011; Larkin et al., 2010; Sadanandappa et al., 2013). This potentiation of LN transmission is driven by a recurrent inhibitory circuit motif (Fig. 5), wherein feedback from PNs onto LNs increases the release of the inhibitory neurotransmitter GABA back onto PNs (Sudhakaran et al., 2012). Observations in support of these conclusions, as well as the differences in the molecular underpinnings specific of STH and LTH, are addressed below.

FIGURE 5 Odorant-selective habituation is driven by a recurrent inhibitory motif. The current model suggests that habituation of olfactory avoidance arises from selective potentiation of inhibitory transmission between a population of LNs and PNs. Odor presentation activates a number of different receptors. Here, we show EB activating one of its cognate receptors, Or85a, which projects to the DM5 glomerulus. Odor-evoked activity in PNs drives activity in LN1 cells, a subpopulation of GABAergic iLNs that co-release glutamate and GABA across the AL. Potentiation of this recurrent inhibition occurs during prolonged odor exposure due to activation of NMDA receptors expressed on PNs. These receptors require coincident PN depolarization and bound glutamate for their activation. Thus, in nonresponsive glomeruli, here the V glomerulus, no potentiation of inhibition occurs. These changes are also dependent on rutabaga activation, which could occur due to neuromodulatory input extrinsic or intrinsic to the AL.

6 Potential Mechanisms of Olfactory Habituation in Drosophila

Drosophila rutabaga mutants, which have defects in a range of associative and nonassociative forms of learning (Aceves-Pinã et al., 1983; Asztalos et al., 2007a; Cho et al., 2004; Duerr and Quinn, 1982), show strong disruption of normal STH and LTH of olfactory avoidance (Das et al., 2011). In particular, the rutabaga mutant rut2080, a hypofunction allele, neither shows behavioral habituation nor diminished odor-evoked PN calcium transients after 4-day odor exposure, as observed in wildtype flies (Das et al., 2011). As the rutabaga gene encodes for a calcium–calmodulindependent adenylate cyclase (Levin et al., 1992; Livingstone et al., 1984) which are believed to act as coincidence detectors of G-protein signaling and neuronal depolarization (Anholt, 1994; Gervasi et al., 2010; Impey et al., 1994; Tomchik and Davis, 2009), this indicates a role of cAMP signaling in STH and LTH. Spatially and temporally controlled rutabaga transgene expression, achieved with the Gal4/UAS system combined with a Gal80ts construct (Brand and Perrimon, 1993; McGuire, 2003), indicates that rutabaga is required in a specific LN subset for olfactory habituation. Tissue-restricted and adult-specific expression of the rutabaga gene in OSNs, PNs, or MB neurons of rut2080 did not alter defects in STH or LTH. In contrast, expressing a wild-type rutabaga transgene specifically in either GABA-releasing neurons expressing glutamate acid decarboxylase (GAD1) or the LN1 subtype of mainly GABAergic, pan-glomerular LNs rescued both STH and LTH defects of rutabaga mutants (Das et al., 2011). This result showed that expression of rutabaga solely in the LN1 population is sufficient for habituation of olfactory avoidance. The necessity of rutabaga expression in LN1s is demonstrated by the observation that adult-specific knockdown of rutabaga in GAD1 or LN1 cells (using stage- and cell-type-specific expression of a rutabaga RNAi transgene) blocks STH as well as LTH in otherwise wild-type flies (Das et al., 2011). Not only do these experiments determine chemical pathways involving rutabaga to be important in this behavior, but they also pinpoint the inhibitory neural population in which cAMPdependent plasticity must occur for behavioral habituation. As cAMP is frequently a positive regulator of transmitter release, this suggested that increased inhibitory transmission drives habituation, a suggestion supported by other experiments. Inhibitory transmitter release from the LN1 population is necessary and the potentiation of LN1 activity is theoretically sufficient for the reduced olfactory avoidance response (Das et al., 2011). Rapidly blocking synaptic output from LN1 cells, achieved using targeted expression of a shibirets transgene expressing temperature-sensitive dynamin (Kitamoto, 2001), restored naive behavior to flies habituated by either 30-min or 4-day odorant exposure (Das et al., 2011). Thus, LN1 output is essential for the display of habituated behavior. Conversely, brief activation of LN1 cells, accomplished using genetically expressed TRPA1, immediately reduced olfactory avoidance behavior of naive flies, indicating that LN1 neurons form sufficiently strong and influential connections onto PNs to drive habituation (Das et al., 2011). LN1restricted and adult-specific expression of an RNAi transgene targeted to degrade GAD1 mRNA prevents the diminished avoidance response observed in STH and LTH, showing that GABA release from LNs is required. Consistently, GABAA receptors in odorant-responsive PNs are necessary for habituated behavior (Das et al., 2011).

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LNs can be stimulated by OSNs or by PNs. To ask whether habituation was driven by OSN activity or PN activity, Sudhakaran et al. (2012) used TRPA1 to activate either EB- or CO2-responsive PNs for 30 min and found that this was sufficient to induce STH in the complete absence of OSN stimulation. This PN-induced habituation also showed odorant selectivity, spontaneous recovery within an hour, and dishabituation in response to mechanical stimulation or a strong yeast puff (Sudhakaran et al., 2012). This PN-induced habituation appears to occur by the same pathway as normal odor-induced habituation, which can be argued by the following two lines of data. First, PN output is necessary for STH, as indicated by the observation that STH does not occur when transmitter release from PNs is blocked during odorant exposure. Second, PN-induced habituation also requires rutabaga function in LN1 neurons, indicating that habituation occurs through potentiation of a recurrent (feedback) inhibitory pathway in the AL (Sudhakaran et al., 2012). PN excitation of iLNs could occur due to either direct excitatory dendrodendritic connections from PNs to LN1 neurons, or indirectly by excitation from eLNs or neurons in the MB sending centrifugal projections to the lobes that were recently shown to occur (Hu et al., 2010). The latter mechanism was ruled out because blocking MB output using shibirets did not lead to any changes in habituation (Sudhakaran et al., 2012). However, shakB mutants with disrupted gap junctions were shown to have deficits in habituation consistent with a model where the feedback signaling is mediated by eLNs (Sudhakaran et al., 2012). These findings show that olfactory habituation is driven by recurrent inhibition, where feedback from PNs onto LNs drive an enhanced inhibition of PNs, which results in a decreased odor-evoked activity in PNs, leading to an attenuated olfactory avoidance response. However, a recurrent inhibitory circuit motif on its own cannot account for the observed odor specificity in STH and LTH. Most of the neurons within the LN1 subset are pan-glomerular, activation of LN1 neurons would therefore inhibit most PNs and thereby cause habituation to most odors as shown by stimulating LN1 cells using TRPA1. Indeed, forced activation of LN1 neurons resulted in a nonselective reduction in the behavioral response decrement to EB and CO2 (Das et al., 2011). Odorant-selective habituation requires glomerulus-selective inhibitory potentiation. How is this achieved in the olfactory circuit? Several genetic experiments suggest that NMDA receptors, which would be only active on odorant-responsive PNs, restrict LN1 synapse plasticity to active odorresponsive glomeruli. Targeted expression of an RNAi transgene against the NMDA receptor subunit NR1 in EB-responsive GH146 PNs prevents STH and LTH to EB but not to CO2 (Das et al., 2011). In contrast, RNAi-mediated knockdown of NMDA receptor in CO2-responsive V PNs prevents habituation to CO2 but not to EB (Das et al., 2011). This is consistent with a model for odorant-selective habituation in which NMDA receptors, activated only in PNs that are depolarized in response to an odorant, mediate glomerulus-selective potentiation of LN1 GABA release. Glutamate required for NMDA receptor activation could come from multiple sources but one essential source appears to be LN1 neurons themselves. RNAi-mediated knockdown of DVGLUT, a widely expressed, vesicular glutamate transporter in LN1 cells


or GABAergic GAD1-expressing cells blocks STH and LTH (Das et al., 2011). This indicates that postsynaptic NMDA receptors on PNs are activated by glutamate coreleased (with GABA) from the LN1 population, while the odor-selective PNs are depolarized at the same time. NMDA signaling, which would be restricted to odor-responsive PNs, then potentially mediates, possibly through retrograde signaling, the enhancement of GABAergic transmission onto these PNs. In summary, the current model suggests that habituation of olfactory avoidance arises from the selective potentiation of inhibitory transmission between LNs and PNs in the Drosophila AL. Before prolonged odor exposure, the LN–PN synapse is comparatively weak. Initial odorant stimulation causes a strong innate response because it strongly activates odor-responsive PNs. Continuous odor exposure causes PNs to drive activity in LNs. The coincidence of LN and PN activation sensed by NMDA receptors only occurs in odorant-responsive glomeruli. This results in glomerulus-selective, rutabaga-dependent inhibitory potentiation in active glomeruli, causing reduced odor-evoked PN activity. This would cause a reduction in the perceived intensity of the signal. Additional detailed analyses of how exposure alters transmission in a large sample of odorant responsive and control PNs will be necessary to formally test this model and to more clearly discriminate between a reduced perceived concentration or altered valence mechanism for habituation.

6.2 Differences of Olfactory STH and LTH While both STH and LTH appear to arise through potentiation of iLN–PN synapses, there are some clear differences in the underlying mechanisms. STH but not LTH requires the function and phosphorylation of synapsin in LN1 neurons (Fig. 6A). As synapsin phosphorylation is thought to increase the pool of synaptic vesicles available for neurotransmitter release, this suggests that a transient change in the active pool of synaptic vesicles underlies short- but not long-term habituation (Sadanandappa et al., 2013). In contrast, LTH is specifically associated with the growth of odor-responsive glomeruli and LTH but not STH is blocked in response to perturbation of various transcription and translation factors (Fig. 6B) (Das et al., 2011; McCann et al., 2011; Sachse et al., 2007; Sudhakaran et al., 2012). We overview experimental observations that have begun to elucidate the specific mechanisms of short (30–60 min) and long-term (days) habituation of the olfactory avoidance response. Synapsins are a conserved family of synaptic vesicle associated proteins predominantly associated with reserve pools of synaptic vesicles. When phosphorylated by kinases such as calcium-dependent protein kinases (CaMKs), protein kinase A (PKA), or MAPK/Erk, they allow synaptic vesicles to detach from the reserve pool and enter an active pool. By making synaptic vesicles available for transmitter release, synapsins induce presynaptic facilitation (Bykhovskaia, 2011). The Drosophila genome encodes only one synapsin gene (Klagges et al., 1996) which is required only in LN1 cells for STH. Syn97 mutant defects in STH are rescued by synapsin wild-type cDNA synþ expression in the LN1 subset or GABA-releasing neurons

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FIGURE 6 Differences between STH and LTH. While both STH and LTH appear to arise through potentiation of iLN–PN synapses, the two forms differ in a substantial manner. (A) STH requires the function and phosphorylation of the synaptic vesicle associated protein synapsin. Synapsin phosphorylation by CaMKII and potentially other kinases is thought to increase the pool of synaptic vesicles available resulting in an increased GABA release from LN1s onto PNs. (B) LTH is associated with glomerular growth of odor-responsive glomeruli and dependent on CREB-induced transcription and miRNA-mediated translational regulation. This translational control, occurring both at the pre- and postsynaptic, involves the RNA binding proteins Atx2 and FMRP. A mutual target of translational control of these RNA binding proteins is the kinase CaMKII.

(Sadanandappa et al., 2013). In addition, RNAi-mediated knockdown of synapsin mRNA in LN1 cells or GABA-releasing neurons blocks STH. The known function of synapsins in presynaptic facilitation and the requirement of Drosophila synapsin in LN1 cells suggests that phosphorylation of synapsin mediates facilitation of GABA release at the LN1–PN synapse during olfactory STH (Fig. 6A).


Consistent with this model, CaMKII/PKA consensus phosphorylation sites on synapsin are necessary in LN1 neurons for the protein’s activity, and CaMKII not only phosphorylates these residues of Drosophila synapsin in vivo but also is required for habituation (Sadanandappa et al., 2013). The surprising observation that synapsin mutants are capable of LTH but not STH indicates that signaling pathways for STH and LTH diverge upstream of synapsin, and that synapsin is a STH-specific target protein. A simple model to account for synapsin’s specific function in STH would propose the following. cAMP and calcium-activated kinases mediate both STH and LTH. However, their phosphorylation of synapsin is only necessary for STH. Other targets of synaptic signaling pathways will be proteins specifically required for LTH. The model is consistent with published and unpublished data. LTH is associated with increased volume of odorant-responsive glomeruli. Fourday exposure to EB causes a volume increase in the EB-responsive glomeruli DM2 and DM5 but not in the CO2-responsive V glomerulus (Das et al., 2011). Four-day CO2 exposure, in contrast, results in a selective volume increase in the V glomerulus (Das et al., 2011; Sachse et al., 2007). Like behavioral LTH, the glomerulus-selective volume growth is odor-selective, recovers spontaneously over several days, and requires the same cellular mechanisms as the behavior (Das et al., 2011; Sachse et al., 2007). The morphological changes require functional rutabaga in LN1s, DVGLUT in GABA-releasing neurons and NMDA receptors in odorant-selective PNs for odorspecific glomerular growth (Das et al., 2011). Long-term memory is classically distinguished from short-term memory on the basis of its dependence on: (a) the growth of new stable synapses; and (b) translationally and transcriptionally regulated gene expression (Kandel, 2001). Increased glomerular volume observed after LTH is consistent with synaptic growth. Additional observations confirm and outline transcriptional and translational control mechanisms specifically required for LTH but not STH. A key regulator of transcription during long-term plasticity is the cAMPresponsive element binding protein (CREB), which binds the cAMP response element in gene regulatory sequences and thereby increases or decreases gene transcription (Sakamoto et al., 2010). LTH requires the transcription factor CREB, shown using an inhibitory isoform of CREB, termed CREB2. One-hour CREB inhibition, through a heat-inducible hsCREB2b transgene, blocked LTH without affecting STH (Das et al., 2011). CREBs requirement specifically in the LN1 subset was tested by transiently induction of CREB2b in LN1 cells with the use of Gal80ts temporal and Gal4/UAS spatial control. Transient inhibition of CREB in LN1 cells blocked LTH, showing that CREB function in the LN1 subset is required for LTH (Das et al., 2011). In line with this observation, transient CREB inhibition in LN neurons also blocked LTH-associated morphological changes (Das et al., 2011). Activity regulated local translation of synaptic mRNAs that are normally stored in a repressed state also contributes to the synapse-specificity of long-term plasticity (Bramham and Wells, 2007; Costa-Mattioli et al., 2009; Martin and Zukin, 2006). This process involves mRNA binding proteins (RBPs) and microRNAs (miRNAs) that control the translation of synaptic RNAs and thereby the levels of proteins

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required for synaptic plasticity. Some RBPs bind specific sequences in the 30 untranslated region of the mRNA to positively or negatively regulate translation. Others interact with components of the miRNA pathway such as members of the RNA-induced silencing complex (RISC) like Ago1 and GW182. The RISC complex facilitates miRNAs binding to sequences on the mRNA 30 -UTR to either suppress translation or facilitate degradation of their target mRNA. Additional observations are consistent with the regulation of local translation as being necessary for LTH. Several known and recently identified translation factors such as the fragile X mental retardation protein (FMRP), encoded by the dFMR1 gene, and the Ataxin-2 (Atx2) protein implicated in spinocerebellar ataxia-2 (McCann et al., 2011; Nonhoff et al., 2007; Sidorov et al., 2013) are required in LN1 neurons and in PNs for LTH but not STH. Thus, transgenic RNAi-mediated, adult-specific knockdown of Atx2 or dFMR1 in LN1 cells prevented LTH to both EB and CO2, whereas it had no effect on STH (Sudhakaran et al., 2013). Adultspecific knockdown of these same proteins in odor-responsive PNs again selectively blocked LTH, but only to the odorant senses by the targeted PN (McCann et al., 2011; Sudhakaran et al., 2013). Together, these results show that dFMR1 and Atx2 are necessary both in LNs and in odor-responsive PNs for the decrement in olfactory avoidance behavior underlying LTH. In line with this, glomerular-selective physiological changes associated with LTH are also repressed in flies that lack Atx2 or dFMR1 in odor-selective PNs (McCann et al., 2011; Sudhakaran et al., 2013). While not tested for dFMR1, Atx2 knockdown in odor-specific PNs also blocked LTH-associated volume increase in odor-selective glomeruli (McCann et al., 2011). Additional genetic, biochemical, and cell biological studies further establish the importance of translational control for long-term habituation by providing independent support for dFMR1 and Atx2 function with known and newly identified miRNA pathway components. The key genetic evidence is the observation that all tested double heterozygotes for mutations in the atx2, dfmr1, me31B, and ago1 show completely normal STH but no significant LTH, a striking observation given that the single heterozygotes show completely wild-type habituation (McCann et al., 2011; Sudhakaran et al., 2013). A spectrum of coimmunoprecipitation experiments indicate the strong likelihood that the proteins associate in vivo (Sudhakaran et al., 2013). However, the most direct evidence is that not only do Atx2 and dFMR1 physically associate with the same CaMKII target mRNA, but also LN1 or PN knockdown of any of five proteins, dFMR1, Atx2, Me31B, Dicer 1, or the Ago-1associated GW182, results in increased synaptic expression of a GFP-based translational reporter under the control of the CaMKII 30 -UTR (Sudhakaran et al., 2013). Together, these findings show that LTH accompanied by morphological changes in odor-responsive glomeruli is dependent on transcriptional and translational control mechanisms (Fig. 6B). NMDA receptor activation in PN dendrites may trigger local translational control at the postsynapse, through a pathway that involves Atx2 and dFMR1 interactions with the miRNA pathway. Cellular changes occurring at the postsynapse could give rise to a retrograde signal from PN to LNs that in turn


stimulates transcriptional regulation mediated by CREB and translational control once more through Atx2, dFMR1, and the miRNA pathway. One target of translational control at the pre- and postsynapse is the kinase CaMKII. Such a working model would be consistent with information on translational control mechanisms in mammalian and Aplysia synapses; however, the underlying cellular pathways still need to be identified in more detail.

6.3 Open Questions The findings presented above point to an inhibitory recurrent circuit motif driving odor-selective olfactory habituation where short- and long-timescale habituation underlie distinct synaptic adaptations. However, the cellular pathways and neuronal circuits depicted are predominantly inferred from genetic and behavioral experiments. While genetic techniques such as cell-directed RNAi-mediated knockdowns certainly give a good indication of the requirement of the respective proteins for olfactory habituation, these experiments cannot directly show physiological changes such as increased GABA release or decrements in PN activation. Electrophysiological recordings and functional imaging experiments are needed to confirm the model inferred by behavioral observations. So far, the only evidence for a reduction in odorevoked PN activity comes from calcium imaging after 4-day EB or CO2 exposure. These experiments were restricted to LTH, studied only two habituating and one control odorant, and also measured calcium changes in very few glomeruli (Das et al., 2011; Sachse et al., 2007). To further improve our understanding of the physiological changes underlying olfactory habituation, calcium changes in several odor-responsive PNs and to a variety of different odors will need to be studied for both STH and LTH. Only if a decreased odor-evoked PN activity can be observed in a number of odor-responsive glomeruli and to a selection of odors, we can be certain that olfactory habituation represents a reduction in the perceived odor intensity rather than a change in encoded odor valence. Testing numerous odors will furthermore give us an idea of how odorspecific olfactory habituation really is and may even show that some odors generalize. Functional imaging could likewise be used to elucidate the cellular signaling pathways that mediate increased GABA release from LN1 cells. Olfactory STH and LTH both require the adenylate cyclase rutabaga in LN1 neurons. Adenylate cyclases are known to initiate cellular signaling pathways by converting ATP into cyclic AMP (cAMP). Functional imaging using a genetically encoded cAMP reporter could confirm that rutabaga also increases cAMP levels during prolonged odor exposure (Gervasi et al., 2010; Tomchik and Davis, 2009). Increased transmitter release from the LN1 terminals after odorant exposure could be shown with the help of the genetically encoded synapto-pHluorin indicator of vesicle release (Ng et al., 2002). Since GABA and glutamate are believed to be co-released from the LN1 population, this indicator would give an estimate of the combined GABAergic and glutamatergic transmitter release from LN1 neurons. The increased inhibition of

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odor-evoked PN activity associated with olfactory habituation could furthermore be measured using a genetically encoded chloride sensor expressed in PNs (Berglund et al., 2011; Markova et al., 2008). Further research is also required to elucidate the way in which glutamate is released from the LN1 population. While VGLUT expression in LN1 cells has a critical role in behavioral habituation and co-release of glutamate with other neurotransmitters is well known (Mestikawy et al., 2011), there could also be a small subpopulation of neurons within the LN1 population that releases glutamate but not GABA. Prospective research will also need to address the details of STH and LTH. Thus, is presynaptic plasticity initiated by a retrograde signal from the odor-selective postsynaptic PN? If so, how is the retrograde signal transmitted and are there different retrograde signals for STH and LTH? Specifically for STH: does 30-min exposure also cause postsynaptic plasticity? For LTH: How does CREB activation cause longterm potentiation of the presynapse? What are the signaling pathways involved? What are the pathways for miRNA-mediated translational control at the synapse? What roles do Atx2 and FMRP play? Which mRNAs are being translationally regulated at the synapse? And what are the exact changes at the synapse in terms of synaptic plasticity? The discussion about pre- and postsynaptic synapses is confounded by the fact that LNs and PNs may form dendrodendritic synapses, as shown for granule and mitral cells in the olfactory bulb (Isaacson and Strowbridge, 1998), where both release and receive neurotransmitters within the same synapse. A clear assignment of the terms “presynaptic” and “postsynaptic” would then not be possible.

7 COMPARISON OF OLFACTORY HABITUATION IN MAMMALS AND DROSOPHILA Similar to olfactory habituation in Drosophila (Das et al., 2011), mammalian olfactory systems show different forms of olfactory habituation that act over different timescales (McNamara et al., 2008) and can be induced using different paradigms (Wilson and Linster, 2008). One of them resembles olfactory STH in Drosophila in terms of its neuronal localization within the olfactory bulb, the mammalian brain structure analogous to the AL, and its persistence for at least 30 min (McNamara et al., 2008). This form of olfactory habituation is induced by repeated 50-s odor presentations with 5-min intertrial intervals and has been mainly studied using a behavioral paradigm that assesses the investigation of scented objects (McNamara et al., 2008; Wilson and Linster, 2008). Here, an object scented with a novel odor is presented, the duration that the animal spends to investigate the object monitored and compared with its investigation time on subsequent presentations of the object scented with the same odor. With prolonged continuous exposure or repeated brief presentation, the length of the time spent investigating the odor decreases (Cleland et al., 2002; Yadon and Wilson, 2005).

7 Comparison of Olfactory Habituation in Mammals and Drosophila

Thirty-minute habituation shows strong cross-adaptation to similar odors (Cleland et al., 2002; McNamara et al., 2008) and can be blocked by infusion of the NMDA receptor antagonist MK-801 into the olfactory bulb, suggesting that the olfactory bulb is a critical locus involved (McNamara et al., 2008). This observation is further supported by a study that found a neuronal activity correlate of 30-min olfactory habituation in the olfactory bulb (Chaudhury et al., 2010). Thus, spiking responses of mitral cells in the rat adapt to and recover from repeated odorant stimulation similar to behavioral 30-min habituation (Chaudhury et al., 2010). Moreover, this neuronal correlate also required functional NMDA receptors in the olfactory bulb. To explain 30-min olfactory habituation, changes in inhibitory transmission within in the external plexiform layer of the olfactory bulb have been proposed as a potential mechanism (Chaudhury et al., 2010; McNamara et al., 2008). It has been suggested that inhibitory input from granule cells onto mitral cells is increased after repeated odor stimuli presentation, leading to a suppression of mitral cell responses to odorants (Chaudhury et al., 2010). Enhanced transmission from granule cells could thereby result either from glutamatergic feedback connections from mitral cells or from centrifugal glutamatergic inputs (Balu et al., 2007; Chen et al., 2000). Thus, NMDA receptors in either mitral cells (Salin et al., 2001) or granule cells could be required to strengthen the respective synapse. Activation of mitral cells has been shown to result in transmitter release of the excitatory neurotransmitter glutamate onto granule cells which, in turn, release the inhibitory neurotransmitter GABA back onto mitral cells (Balu et al., 2007; Chen et al., 2000; Halabisky et al., 2000). This proposed model shows considerable similarity to the neuronal circuit underlying STH in Drosophila. In both, increased input from inhibitory LNs onto PNs/ mitral cells is suggested to result in a diminished behavioral response to an odor stimulus after habituation occurred. Thus, the findings obtained in Drosophila can be potentially helpful for deciphering the underlying mechanisms of 30-min olfactory habituation in mammals. Another much shorter form of olfactory habituation, that has not yet been studied in D. melanogaster, could be identified to result from synaptic depression within the mammalian piriform cortex (McNamara et al., 2008). This transient form can be induced by repeated 20-s stimulation with 10-s intertrial intervals and persists for at least 2 min but has recovered after 10 min (McNamara et al., 2008). Two-minute habituation is mainly studied using the odor-evoked heart-rate orienting reflex (HROR) (Fletcher and Wilson, 2002). HROR is based on the observation that novel sensory stimuli such as unfamiliar odor presentations evoke a bradycardia reflex, which habituates with repeated stimulation. The HROR is driven by a polysynaptic connection from the olfactory bulb and the olfactory cortex, a major subdivision of the piriform cortex innervated by mitral cells, to the cardiac pacemaker cells (Wilson, 2009). Habituation of HROR recovers spontaneously with time, shows stimulus specificity with very little cross-habituation (Fletcher and Wilson, 2002), and can also be dishabituated (Smith et al., 2009).

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Recent advances have identified piriform cortex adaptation as the neuronal correlate of HROR habituation. In the piriform cortex, pyramidal cells, targets of second-order mitral cells, adapt to repeated or prolonged odor exposure (Wilson, 1998a). Similar to behavioral odor-evoked HROR, cortical adaptation is odorspecific with only minimal cross-adaptation (Wilson, 2000), can be dishabituated, and recovers spontaneously within a similar time span as HROR (Best and Wilson, 2004; Smith et al., 2009). In vivo intracellular recordings indicated that piriform cortex adaptation results from depression of the glutamatergic mitral– pyramidal cell synapse (Wilson, 1998b). An analogous depression of this synapse introduced by electric stimulation of mitral cell axons in vitro, in a pattern mimicking odor-evoked activity, showed that this depression is homosynaptic and can be blocked by an antagonist of group III metabotropic glutamate receptor mGluIII (Best and Wilson, 2004). mGluIII receptors have been found presynaptically on mitral cell axon terminals (Wada et al., 1998). The mGluIII receptor antagonist blocked not only synaptic depression in vitro but also adaptation of cortical odor responses in vivo (Best and Wilson, 2004). Intrapiriform cortex infusion, furthermore, prevented HROR habituation (Best et al., 2005). These results demonstrate that odor-evoked HROR habituation results from depression of the mitral–pyramidal cell synapse in the piriform cortex and that this synaptic depression requires mGluRIII receptors in presynaptic mitral cell terminals.

8 INTERACTIONS WITH NEUROMODULATORY SYSTEMS Several neuromodulators are expressed in the Drosophila AL (Busch et al., 2009; Carlsson et al., 2010; Chou et al., 2010; Dacks et al., 2009; Na¨ssel, 2002; Roy et al., 2007), suggesting that neuromodulatory processes heavily modulate olfactory processing within the AL. We speculate that three different classes of neuromodulators must influence olfactory habituation in Drosophila (Fig. 7). First, since rutabaga is responsive to G-protein signaling, there must be a “default” neuromodulatory signal on LN1 neurons that occurs with odorant exposure and plays a role in rutabaga activation. Neuromodulators transmit their signals through binding of metabotropic G-protein-coupled receptors (GPCRs), which causes intracellular activation of G-proteins (Pierce et al., 2002). Thus, we speculate that a neuromodulator, here termed NM1, is released during prolonged odorant exposure and thereby causes the activation of GPCRs expressed in the LN1 neuronal subsets (Fig. 7). Initiation of intracellular G-protein signaling and simultaneous LN1 depolarization would activate rutabaga, which in turn would increase cAMP levels resulting in the synaptic changes observed for olfactory habituation. NM1 would therefore be required for the initiation of olfactory habituation. Second, olfactory habituation can be dishabituated by unexpected stimuli. Exposure to yeast odorant or mechanical stimulation can dishabituate Drosophila STH to EB and CO2. This is probably accomplished through a different class of neuromodulatory input that perhaps inhibits inhibitory neurons that drive habituation (Fig. 7).

8 Interactions with Neuromodulatory Systems

FIGURE 7 Putative neuromodulatory control. A number of neuromodulators are expressed within the Drosophila AL that could modulate olfactory habituation. We suggest neuromodulatory inputs that could be required for the initiation (NM1), reversion (NM2), and suppression (NM3) of habituation behavior. (NM1) A neuromodulatory role in the initiation of habituation is indicated by the requirement of G-protein activated rutabaga in LN1 cells. Neuromodulatory binding to a G-protein-coupled receptor expressed in LN1 neurons could stimulate G-protein activation of rutabaga and thereby initiate olfactory habituation. (NM2) A second neuromodulator NM2 could mediate a dishabituating stimulus as shown in mammalian olfactory habituation. To be able to rapidly reverse olfactory habituation, this neuromodulator may inhibit LN1 activity and thereby rapidly suppress GABAergic release from LN1s. (NM3) Associating an odorant with reward or punishment as well as motivational changes may trigger a third neuromodulator that could suppress the formation of olfactory habituation. This could be achieved by either suppressing the activity of NM1 neurons or by direct reduction of cAMP levels within the LN1 population.

Dishabituation is a rapid process wherein the dishabituating stimulus almost immediately reverses habituation. Thus, we propose that another neuromodulator, here termed NM2, acts directly on GABA release from LN1 neurons. NM2 release could hereby rapidly restore the unhabituated avoidance response. Third, habituation occurs more easily in the absence of other consequential stimuli such as punishment, reward, or motivational inputs. This argues that associated neuromodulators may act to suppress (gate) the processes of habituation (Fig. 7). Thus, one may predict that dopaminergic, serotonergic, or octopaminergic inputs may prevent the establishment of olfactory habituation in Drosophila, a prediction that has not yet been formally tested. We propose that this “gating” could be achieved by a neuromodulator, here termed NM3, that either suppresses the activity of NM1 neurons or directly reduces cAMP levels within LN1 cells. Suppression of NM1 release would prevent rutabaga activation and its associated increase in cAMP levels, which mediate synaptic plasticity required for olfactory habituation. Interference with cAMP levels in LN1 cells would have the same effect.

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In line with our speculations, olfactory habituation in mammals is subject to neuromodulation. Two-minute olfactory habituation in rats can be reversed by a dishabituating auditory stimulus, which has been shown to drive noradrenergic transmitter release within the piriform cortex. Blockage of noradrenergic receptors within this area prevented auditory-induced dishabituation (Smith et al., 2009), demonstrating that noradrenergic release within the piriform cortex mediates dishabituation. Mice trained according to the 30-min olfactory habituation protocol failed to habituate when their cortical noradrenergic fibers were lesioned by treatment with a noradrenergic neurotoxin. Bilateral infusion of noradrenaline into the olfactory bulb restored normal habituation behavior (Gue´rin et al., 2008), showing that noradrenaline release can also suppress olfactory habituation. Further evidence comes from the sea slug Aplysia californica, possibly the best studied organism in terms of neuromodulatory effects on habituation. In Aplysia, serotonin release reverses or counteracts synaptic depression of the sensory neuron to motor neuron synapse shown to underlie habituation of the gill withdrawal reflex although in this case it is unclear if habituation is being suppressed or overridden by a superposed sensitization that operates via a different mechanism (Glanzman et al., 1989; Marinesco and Carew, 2002). Suppression or overriding of the gill withdrawal reflex can likewise be induced by the neuromodulator dopamine (Ruben and Lukowiak, 1983).

9 RELEVANCE TO OTHER FORMS OF LEARNING Habituation is considered to be a building block for higher forms of learning (Rankin et al., 2009). Olfactory habituation, by reducing responses to familiar, inconsequential odor stimuli, allows an animal to focus on novel and potentially relevant odor stimuli. This allows more accurate and efficient formation of associative memories. There are two potential levels of interaction between habituation and associative memory formation. First, in a complex environment, olfactory habituation allows familiar odors to be filtered out and salient odors to be selectively associated with appetitive or aversive consequences. This phenomenon may be relevant to, and underlie, the process of latent inhibition, which in honey bees has been shown to arise from a process that occurs in the AL and modeled to arise from inhibitory synapse potentiation (Bazhenov et al., 2013; Locatelli et al., 2012). The term describes the observation that preexposure to an odorant without positive or negative consequences prior to olfactory appetitive or aversive conditioning reduces the efficiency with which it can be paired with reward or punishment (Lubow, 1973). While not yet studied in Drosophila, the honey bee Apis mellifera shows reduced appetitive learning in the proboscis extension reflex paradigm after prior odor exposure to the conditioned odor (Chandra et al., 2010). Neuromodulatory involvement has been indicated for this form of latent inhibition (Fernańdez et al., 2012). It seems likely that latent inhibition arises due to olfactory habituation induced during preexposure to the odorant, which, through the inhibition of responses to a familiar odor, would

References

reduce its perceived salience. Consistent with a model in which inhibition gates olfactory memory formation, reduction of GABAA receptor levels in the MB increases the efficiency of olfactory memory formation (Liu et al., 2007, 2009). The prediction that this increased efficiency could come at the expense of accuracy, that is, increased generalization to odorants that should not be salient, has not yet been tested. A second level of interaction between habituation and associative memory pertains to the effect of neuromodulators that mediate reward and punishment on the process of habituation that has been alluded to in section 8. In Drosophila, associative memories are formed within the MB neuropil downstream of the AL (Heisenberg, 2003). Associative learning occurs due to odor-evoked activity within the MB Kenyon cells and coincident neuromodulatory signaling representing the rewarding or punishing stimulus. Both reward and punishment are mainly signaled by dopamine release, but an octopaminergic component has also been implicated for appetitive learning (Burke et al., 2012; Liu et al., 2012; Riemensperger et al., 2005; Schwaerzel et al., 2003; Waddell, 2013). The upstream location of the AL within the olfactory system raises the question whether olfactory habituation is suppressed by potential neuromodulatory input (as discussed in section 8) or whether it is overwritten within the MB neuropil. Theoretically, olfactory habituation could occur to any prolonged or repeated odor exposure, independently of whether the odor stimulus is relevant or not. Thus, associative memory may arise from neural plasticity in downstream brain regions, the MBs, that overrides habituation. Alternatively, and we suggest more likely, associative memory formation may involve a second process that suppresses AL plasticity that underlies habituation. The Drosophila olfactory system allows the above questions to be directly addressed by in vivo experimentation. Such future work may not only advance understanding of olfactory habituation mechanisms and their relevance but also make fundamental contributions toward understanding other forms of learning and memory.

Acknowledgments Research in the laboratories is funded by Science Foundation Ireland. The authors like to thank Jens Hillebrand for allowing the use of the Y-maze figure and Alex Crean for his invaluable help with the other figures.

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Sexual dimorphism and phenotypic plasticity in the antennal lobe of a stingless bee, Melipona scutellaris.

Semaphorin-1a prevents Drosophila olfactory projection neuron dendrites from mis-targeting into select antennal lobe regions.

Synaptic organization of the Drosophila antennal lobe and its regulation by the Teneurins.

Antennal lobe organization and pheromone usage in bombycid moths.

Network plasticity in adaptive filtering and behavioral habituation.

Olfactory abnormalities in temporal lobe epilepsy.

Habituation of stimulus-elicited investigation in gerbils after olfactory bulbectomy.

Firing and intrinsic properties of antennal lobe neurons in the Noctuid moth Agrotis ipsilon.

Antennal transcriptome and expression analyses of olfactory genes in the sweetpotato weevil Cylas formicarius.

Analysis of the Antennal Transcriptome and Insights into Olfactory Genes in Hyphantria cunea (Drury).

Pheromone modulates plant odor responses in the antennal lobe of a moth.

The anatomical basis for modulatory convergence in the antennal lobe of Manduca sexta.

Functional integration of a serotonergic neuron in the Drosophila antennal lobe.

Mechanisms Underlying Population Response Dynamics in Inhibitory Interneurons of the Drosophila Antennal Lobe.

Antennal olfactory sensilla responses to insect chemical repellents in the common bed bug, Cimex lectularius.

Modelling Odor Decoding in the Antennal Lobe by Combining Sequential Firing Rate Models with Bayesian Inference.

Digital in vivo 3D atlas of the antennal lobe of Drosophila melanogaster.

Identification and expression pattern of candidate olfactory genes in Chrysoperla sinica by antennal transcriptome analysis.

Cortical Plasticity and Olfactory Function in Early Blindness.

FMRP and Ataxin-2 function together in long-term olfactory habituation and neuronal translational control.

Commentary: Cortical Plasticity and Olfactory Function in Early Blindness.

Modification of Male Courtship Motivation by Olfactory Habituation via the GABAA Receptor in Drosophila melanogaster.

Decreases in frontal and parietal lobe regional cerebral blood flow related to habituation.

Identification of candidate olfactory genes in Chilo suppressalis by antennal transcriptome analysis.