A Genetic Screen for Olfactory Habituation Mutations in Drosophila: Analysis of Novel Foraging Alleles and an Underlying Neural Circuit

Mark Eddison; Amsale T. Belay; Marla B. Sokolowski; Ulrike Heberlein

doi:10.1371/journal.pone.0051684

Abstract

Habituation is a form of non-associative learning that enables animals to reduce their reaction to repeated harmless stimuli. When exposed to ethanol vapor, Drosophila show an olfactory-mediated startle response characterized by a transient increase in locomotor activity. Upon repeated exposures, this olfactory startle attenuates with the characteristics of habituation. Here we describe the results of a genetic screen to identify olfactory startle habituation (OSH) mutants. One mutation is a transcript specific allele of foraging (for) encoding a cGMP-dependent kinase. We show this allele of for reduces expression of a for-T1 isoform expressed in the head and functions normally to inhibit OSH. We localize for-T1 function to a limited set of neurons that include olfactory receptor neurons (ORNs) and the mushroom body (MB). Overexpression of for-T1 in ORNs inhibits OSH, an effect also seen upon synaptic silencing of the ORNs; for-T1 may therefore function in ORNs to decrease synaptic release upon repeated exposure to ethanol vapor. Overall, this work contributes to our understanding of the genes and neurons underlying olfactory habituation in Drosophila.

Citation: Eddison M, Belay AT, Sokolowski MB, Heberlein U (2012) A Genetic Screen for Olfactory Habituation Mutations in Drosophila: Analysis of Novel Foraging Alleles and an Underlying Neural Circuit. PLoS ONE 7(12): e51684. https://doi.org/10.1371/journal.pone.0051684

Editor: Efthimios M. C. Skoulakis, Alexander Flemming Biomedical Sciences Research Center, Greece

Received: August 2, 2012; Accepted: November 5, 2012; Published: December 17, 2012

Copyright: © 2012 Eddison et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: Research was supported by the National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism(UH) and the Wheeler Center (to ME). This work was also supported by grants from the Canadian Institute for Health Research of Canada (CIHR) (to MBS), the Natural Sciences and Engineering Council of Canada (to MBS) and a CIHR training grant (to ATB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Habituation is a fundamental behavior that is often overlooked by researchers, yet its prevalence in the animal kingdom suggests it is essential for survival [1]. Habituation is an active process of progressive decline of reaction to a harmless stimulus [2], [3]. Habituation allows animals to ignore inconsequential stimuli and may serve as a building block for more complex forms of attention [4]. An inability to habituate has been linked to schizophrenia [5], [6], autism [7], [8] and fetal alcohol syndrome [9], [10]. Despite the biological and clinical importance of habituation, its behavioral simplicity and its first description over 100 years ago [11], few genes that govern habituation have been described to date.

Our understanding of the neural basis of habituation is most extensive in the sea snail Aplysia californica [12], [13], whose defensive gill-withdrawal reflex habituates to repeated mechanical stimulation [14]–[16]. Early work showed that habituation in this sensory-neuron to motor-neuron circuit is due to a presynaptic decrease in excitatory neurotransmission, likely due to the active silencing of presynaptic release [12]. Although this decrease in presynaptic release, termed homosynaptic depression, is a common mechanism of habituation, potentiation of inhibitory connections can also achieve the same behavioral output [17], [18].

A variety of paradigms have been used to study habituation in Drosophila including the gustatory-based proboscis extension reflex (PER) and several olfactory-mediated behaviors, such as the jump reflex or startle response [19]. Using reverse genetics, several well studied genes and pathways have been identified as important regulators of habituation in Drosophila. These include K⁺ channels, NMDA and GABA_A receptors, as well as the cAMP and cGMP second messenger systems [18], [19]. An unbiased forward genetic approach can be useful in identifying novel genes and pathways that regulate habituation. However, due to the labor-intensive nature of many habituation assays, this approach has only sparsely been used [20], [21].

Our laboratory has previously described a simple and efficient paradigm to study olfactory startle habituation (OSH) in freely moving adult Drosophila [22]. In this assay, the flies’ gradual decline of a locomotor startle response to short exposures of vaporized ethanol is measured using an automated video tracking system [22]. The organization of the Drosophila olfactory system shows remarkable similarities to that of vertebrates [23], [24], suggesting the principal genes, circuits and mechanisms of olfactory habituation maybe conserved. In flies, odors are detected by the olfactory receptor neurons (ORNs), most of which reside in the antennae and project to the antennal lobe (AL) where they synapse with glomerulus-specific projection neurons (PNs) and local interneurons (LNs) [24]. Both excitatory and inhibitory LNs are present in the AL and make intra- and inter-glomerular connections with the PNs, shaping the neural representation of odors from the 1^st to 2^nd order neurons [25]–[27]. The PNs project to the mushroom body (MB) and lateral horn. Importantly, the MB is critical for habituation [22], [28], associative olfactory learning [29], and modulating locomotor responses [30]. Recently, much progress has been made in deciphering the neural circuits and identifying several genes mediating olfactory habituation in Drosophila [17], [18], [20], [31], [32].

In this report we describe the results of a genetic screen using our OSH paradigm [22] and identify 26 mutations affecting OSH. We also further characterize two hypomorphic mutations in the gene foraging (for), which encodes a cGMP-dependent kinase (PKG). These new for mutations decrease the expression of a specific isoform of for, for-T1. We show that for-T1 normally functions to inhibit OSH in a subset of neurons that include ORNs and the MB. We also show that overexpression of for-T1 in ORNs, but not the MB, reduces OSH, suggesting that for-T1 principally functions in ORNs to regulate OSH. Finally, we show that synaptic transmission of ORNs is required to promote startle habituation. Taken together, our results raise the possibility that for-T1 may inhibit OSH by decreasing synaptic release in ORNs after their exposure to ethanol vapor.

Materials and Methods

Fly Strains

All flies were maintained on standard cornmeal molasses agar at 25°C and 70% humidity under constant dim light. The P element collection screened was a collective effort generated internally in the Heberlein Lab. NP2614 was obtained from GETDB (Drosophila Genetic Resource Centre in Kyoto Institute of Technology). Our two control strains, 4.59 and 16.57 have P elements inserted just 5′ of CG5630 and in Socs36E respectively, genes with no known association with habitation or PKG. UAS-TeTx and UAS-TeTxⁱⁿ strains were obtained from Sean Sweeney, pdf-GAL4 flies were obtained from Paul Tagert, and Orco-GAL4 from Leslie Vosshall. UAS-for-T1 and for^R, for^s and for^s2 were obtained from Marla Sokolowski. OK-107-GAL4, UAS-GFP-CD8 and the septate junction P elements were from the Bloomington Stock Centre. All strains, except the for polymorphisms, were backcrossed for at least five generations to a w¹¹¹⁸ Berlin stock.

Habituation Assay

The habituation assay is essentially the same as described in [22], except that we used the “booz-o-mat” [66] which allows simultaneous video recording of eight individual genotypes. Films were recorded using Adobe Premiere (Adobe Systems, San Jose, CA). To measure the locomotor tracking response to ethanol, films were analyzed with a modified version of DIAS 3.2 (Solltech, Oakdale, IA) that was controlled by the OneClick 2.0 scripting language (Westcode Software, San Diego, CA). Briefly, for each genotype, 20 2-4 day-old male flies were collected under CO₂ anesthesia and kept in fresh food vials for 2 days. Flies were placed into a 16×125 mm cylindrical tube with perforations clustered at the rounded base. Flies were left to acclimate for 7 min before the start of video recording. After a further two minutes the flies were administered the first 30-second pulse of vaporized ethanol (P1); subsequent 30-second pulses of ethanol vapor were administered every 5 minutes. One minute after the forth pulse (P4) the flies were dishabituated with a sudden mechanical shock (banging the apparatus). A final pulse of ethanol vapor was administered 4 minutes after the dishabituation. Ethanol vapor was produced with an evaporator [22], [66] and the concentration controlled by a flow meter (Cole Parmer). Mixtures of ethanol and air vapor are noted as ratios. For screening purposes, a ratio of ethanol/air of 65/77 was used. All subsequent testing was carried out at an ethanol/air ratio of 80/60, where 80 units of flux is equivalent to 2.7 liters/min. Habituation assays were repeated on 2 to 3 different days with new flies to incorporate the day-to-day variations in behavior. In all Figures, n corresponds to the number of experiments performed on an independent group of 20 flies.

Calculations and Statistics

The total movement travelled during odor exposures was calculated as the area under the pulse curve, i.e. summing the velocities measured during the 30-second exposure, at 5-second intervals and multiplying the sum by 5 seconds. The habituation index (HI) was calculated as 1-P4/P1, where P4 and P1 are the areas under the locomotor activity curve for the 4^th and 1^st pulse respectively, such that a HI of 1 indicates complete habituation and a HI of 0 indicates no habituation. In order to more easily compare the extent of habituation in all graphs the total movement was normalized to the magnitude of the first startle. Significance was established by one-way-ANOVA with post-hoc Newmans-Keuls comparisons. Error bars in all experiments represent standard error of the mean (SEM). Statistical significance was achieved where p<0.05.

Genetic Screen

A total of 874 P-element insertion strains were initially screened in the habituation assay, (n = 2–4). The habituation index (HI) for each strain was calculated and ranged from 0.92 to –0.42. A frequency distribution of the habituation indices showed a near normal distribution with a mean of 0.58, median of 0.64 and mode of 0.65. From 874 strains screened, 93 were identified, 63 had pronounced habituation (with an HI >0.8) and 30 failed to habituate (with an HI <0.2). These strains were backcrossed for 5 generations to our w¹¹¹⁸ Berlin genetic background to eliminate unlinked mutations. After retesting in the habituation assay (n = 6), 26 strains maintained their habituation phenotype: 25 exhibited enhanced habituation (HI>0.8) and 1 was a non-habituator (HI<0.2). All mutant strains were considered to be within the normal range of a locomotor startle response as none were signficantly different than at least one of the control strains (see below). Further, all of these mutant strains appeared healthy, fertile and viable. Two representative backcrossed strains, 4.59 and 16.57, that had a normal HI similar to the screen median and mode (0.49 and 0.5, respectively) and initial startle (36.7 mm/fly and 29.5 mm/fly, respectively) were chosen as controls and used throughout the behavioral experiments, although only one control strain is shown.

Molecular Characterization of for Alleles

The location of the insertions was determined by inverse PCR. (The 11.247 P element is located in the first intron of for, 994 bp downstream of exon 1, and the NP2614 insertion is located in the same intron, 666 bp downstream of exon 1. Imprecise excision strains of 11.247 were generated through remobilization of the P[GawB] element by introduction of a stable transposase source. Several phenotypic revertants were obtained. The excision strains were screened by PCR on genomic DNA using primers 5′-ACTACGCTACGCTGGCAGAAAC-3′ and 5′-AACACGAACACGA AAGATTGG -3′, and several were found to be precise excisions.

RNA Analysis

Total RNA was extracted from adult flies using Trizol Reagent (Invitrogen). Poly A⁺ RNA was purified from total RNA using the Oligotex system (Qiagen). Probes for the Northern blot were generated by PCR using genomic DNA as a template. For the for-T1 probe, the primers used were 5′-ATCTGGTGGGTGGCATTGTGA-3′ and 5′-CATCCTTGTCGTATTTGGGAAA-3′. For the for-T2, the primers were 5′-AGGAACACGAACTGGAAG-3′ and 5′-GATACAGAAACCCTCCCCGTTA-3′. As a control for RNA loading, a tubulin 84B gene probe was amplified using primers 5′-ACAGCCGTCTCTAGCTCCG-3′ and 5′-CATCACCTCCGCCCACGGTCTTG-3′. Northern blots were performed using mRNA isolated from 2–4 day old adult heads and bodies (or heads only) and probed with ³²P labeled probes.

PKG Enzymatic Activity Assay and Immunohistochemistry

PKG enzyme assays and immunohistochemistry were performed as previously described [44].

Results

A Behavioral Screen for Mutations Affecting Olfactory Startle Habituation

In Drosophila, exposure to a high concentration of ethanol vapor provokes a transient olfaction-dependent increase in locomotor activity termed the olfactory startle response [22]. Subsequent pulses of ethanol vapor result in a reduced startle that shows characteristics of habituation, including dishabituation following a novel stimulus (Fig. 1A; [22]). In order to identify genes that regulate OSH, we screened 874 fly strains, each harboring a randomly inserted P element in the genome, for strains with altered OSH. As a simple measure for habituation we calculated a habituation index (HI), defined as the ratio between the magnitudes of the fourth and first startle response (see Methods). We defined a normal HI to be similar to the median and mode of the entire screen and selected two strains as controls (termed Ctrl, see Methods). Potential mutants were selected by a numerical cut-off point at both ends of the distribution of HIs obtained from the screen (see Fig. 1B; see Methods).

Download:

Figure 1. Drosophila habituates to an ethanol-induced olfactory startle.

A) The olfactory startle attenuates with the characteristics of habituation. Ethanol naïve flies exposed to a 30-sec pulse of ethanol vapor showed an olfactory mediated startle response, characterized by a transient increase in locomotor activity. In subsequent pulses of ethanol vapor the olfactory startle increasingly attenuated. To demonstrate habituation (and not sensory adaptation or fatigue) flies were dishabituated (arrow) with a mechanical stimulus before the final pulse of ethanol. B) The frequency distribution of habituation indices (HI) of all strains tested in the genetic screen. The habituation index was calculated as a ratio of the total movement in the fourth pulse (P4) and first pulse (P1) (HI = 1-P4/P1; a HI of 1 indicates complete habituation while a HI of 0 or lower indicate no habituation or sensitization, respectively). A frequency distribution of the habituation indices showed a near normal distribution with a mean of 0.58, median of 0.64 and mode of 0.65. A HI of 0.8 or higher was used as the cut-off for enhanced habituation, while a HI of 0.2 or lower was the cut-off for failure to habituate normally. Strains with low and high HIs were selected for further analysis.

https://doi.org/10.1371/journal.pone.0051684.g001

After eliminating unlinked mutations by backcrossing each potential mutant to the parental strain (wBerlin; see Methods), we identified 26 strains that had a normal initial startle response and retained an abnormal OSH (Table 1). Curiously, only one of these strains decreased OSH (strain 12.132, which essentially failed to habituate), while all other strains had enhanced OSH. Using inverse PCR and DNA sequencing followed by genomic database searches (www.flybase.org) we mapped the location of the transposon insertions and identified the candidate genes disrupted (Table 1). Classified by their molecular function, the largest categories were those including genes with predicted or unknown molecular function (lama, ckn, hebe, CG1806, CG8321, CG3967, CG42697, CG11357) and genes with functions related to nucleic acids (HmgD, tara, Camta, heph, snp). Smaller categories included genes involved in cell signaling (for, gish, wun, PNUTS), the regulation of the cytoskeleton (kl-2, RtnL1) or cell junctions (pyd, cora).

Download:

Table 1. OSH mutants isolated from genetic screen.

https://doi.org/10.1371/journal.pone.0051684.t001

Several Olfactory Startle Habituation Mutations are Associated with Septate Junctions

One candidate gene we identified in our screen, coracle (cora), encodes an integral component of septate junctions [33]. In the insect nervous system, septate junctions are known to seal neighboring glial cells together to protect axons from the high K⁺ environment of the hemolymph [34]. Septate junctions are found in the fly’s blood-brain barrier, between perineurial and peripheral glia, and also between peripheral glia and axons [35]. An analogous structure in mammals is the paranodal junction found at the nodes of Ranvier, which enables rapid saltatory conduction of action potentials [36]–[38]. Interestingly, a parallel screen for OSH mutations identified gliotactin (gli) (B. Cho and U.H, unpublished data), another component of the septate junction [39], [40]. The identification of two septate junction genes in our screens suggested that the structure and/or function of the septate junction might be important for OSH. To test this hypothesis, we selected fifteen P element mutations in eight known genes whose products localize to the septate junctions, normalized their genetic background and tested their OSH response. Of these, seven strains, representing five of the eight genes tested (cora, dlg, fas3, gli, nrx-IV), showed an abnormal OSH (Table S1). Further, one strain (EP809, inserted in nrx-IV) failed to habituate, a phenotype rarely seen in the original screen. Therefore, septate junctions maybe an important regulator OSH, though future work will be necessary to reveal its specific function in modulating this form of behavioral plasticity.

Foraging Regulates Olfactory Startle Habituation

In Drosophila, foraging (for/dg2) encodes a cGMP-dependent kinase (PKG) that regulates habituation of the giant-fiber neurons (involved in an escape reflex) [41] and the PER [42]. We identified strain 11.247, carrying a P element insertion in the 5′ region of the for locus (from hereon called for^11.247), that exhibited enhanced habituation (Fig. 2A, 2B). This phenotype is robust as it was maintained in two different genetic backgrounds (Fig. S1A). Moreover, normal habituation was restored upon precise excision of the P element in for^11.247 (Fig. 2C), indicating that the insertion is responsible for the mutant phenotype. We also identified an additional strain that carries a P element insertion near for^11.247, NP2614 (called for²⁶¹⁴; see Fig. 3A), which also enhanced OSH (Fig. 2A, 2B). In both of these for alleles, the magnitude of the initial startle was normal (Fig. S1B). Therefore, we conclude that for regulates OSH.

Download:

Figure 2. for alleles enhance olfactory startle habituation.

A) for^11.247 and for²⁶¹⁴ show enhanced OSH. A reduction of distance traveled (compared to Ctrl) was seen in both alleles at pulse 2 (p<0.01), 3 and 4 (p<0.001;(n = 12). B) for^11.247 and for²⁶¹⁴ have an enhanced HI (indicating more habituation). Significant difference was seen between Ctrl and for^11.247 or for²⁶¹⁴ (p<0.001; n = 12). C) Compared to Ctrl, the precise excision, for^Δ11.247, had a normal HI (p>0.05; n = 6). Unless indicated, significance was established by a One-Way-ANOVA with post-hoc Newmans-Keuls tests in all figures (*p<0.05, **p<0.01, ***p<0.001).

https://doi.org/10.1371/journal.pone.0051684.g002

Download:

Figure 3. Molecular characterization of for alleles.

A) Schematic of the for transcription unit, with insertion sites of for^11.247 and for²⁶¹⁴. Blue bars represent translation start/stop sites, grey bars represent region probed for for-T1/T3 and for-T2 transcripts. The 3 major for isoforms, collectively called for-T1/T2/T3 have a total of nine splice forms, all encoding a common kinase domain at the 3′ end. FOR-T1 is a 1088 amino acid (aa) protein encoded by for-RA/RH/RI, FOR-T2 is a 894 aa protein encoded by for-RC/RD/RF/RG/RK, and FOR-T3 is a 742 aa protein encoded by for-RB. B) Northern blot of adult fly mRNA using probes specific to for-T1/T3 or for-T2 transcripts. B, top panel) In the control strain (wBerlin) we detected two bands with the for-T1/T3 probe. Based on its size, the upper, more intense, band corresponds to for-T1 transcripts, while the lower, less intense band, to the for-T3 transcript. Compared to wBerlin and for^Δ11.247(a precise excision of for^11.247) a reduced intensity of for-T1, but not for-T3 transcripts, was seen in for^11.247 and for²⁶¹⁴. B, middle panel) Using a for-T2 probe we detected no differences in levels of for-T2 transcripts in either for^11.247 or for²⁶¹⁴. B, bottom panel) A tubulin probe was used to compare total mRNA levels. C) Quantification of Northern Blot showing reduced for-T1, but not for-T2 or for-T3, in for^11.247 and for²⁶¹⁴. Levels were calculated as a ratio between for and tubulin band intensity. D) Quantification and representative Western blot of extracts from adult heads analyzed with an antibody that recognizes FOR-T1. Compared to controls, we saw a reduction of FOR-T1 in both for^11.247 and for²⁶¹⁴ (p<0.001; n = 3).

https://doi.org/10.1371/journal.pone.0051684.g003

Molecular Characterization of for Alleles that Disrupt Olfactory Startle Habituation

The for locus produces 11 transcripts that encode four protein isoforms [43]. Of these transcripts, nine encode the three major FOR protein isoforms FOR-T1, T2 and T3 (Fig. 3A; see [43] for alternative nomenclature). To determine the molecular nature of our for alleles, we performed Northern blots using mRNA derived from adult flies. In control flies we observed two bands using probes specific for the for-T1 and for-T3 transcripts (Fig. 3B, top panel). The more intense, larger molecular weight band corresponds to the three for-T1 transcripts, for-RA/RI/RH, while the lower molecular weight less intense band corresponds to for-T3 (or for-RB). With a probe specific to for-T2 transcripts we detected a doublet corresponding to for-RD/RF and for-RC/RG/RK (Fig. 3B; middle panel).

In both for alleles we observed a reduced intensity of the for-T1 band and did not detect a change in either the for-T3 or for-T2 transcripts (Fig. 3B, 3C). Therefore, both for^11.247 and for²⁶¹⁴ have specifically reduced expression of for-T1 transcripts. The precise excision of the P element in for^11.247 that showed normal habituation (Fig. 2C) also restored for-T1 transcripts to control levels (Fig. 3B, 3C, top panel). Therefore, our data suggest that for-T1 functions to inhibit OSH.

Using a FOR antibody [44], we next determined levels of FOR-T1 and FOR-T3 in the adult heads of flies carrying these for alleles; we were unable to determine FOR-T2 levels with this antibody. We observed a significant reduction in FOR-T1 protein expression in for^11.247 and for²⁶¹⁴ (Fig. 3C), while levels of FOR-T3 appeared normal (Fig. S1C). Therefore, consistent with the Northern blot, we conclude that, both for^11.247 and for²⁶¹⁴ have reduced expression of FOR-T1 in the adult head.

We next attempted to measure the level of PKG activity in the heads of for^11.247 and for²⁶¹⁴ flies. However, the levels of PKG activity in our P element control strain and genetic background control were significantly different (Fig. S2A), precluding any definitive conclusions about relative levels of PKG activity in for^11.247 and for²⁶¹⁴ (which were also significantly different from each other). We also failed to see a difference in OSH in the natural variants of for (for^R, for^s and for^s2) that do subtly but significantly differ in PKG activity [44], [45] (Fig. S2B). Therefore, for reasons we do not currently understand, we were unable to find a correlation between PKG activity, levels of for-T1 and OSH. In summary, we conclude that for^11.247 and for²⁶¹⁴ have reduced levels of for-T1 in the adult head suggesting that for-T1 functions to inhibit OSH.

for^11.247-GAL4 Expression Partially Recapitulates the FOR Expression Pattern

The neuronal expression pattern of all FOR isoforms has been reported previously and includes specific neuroanatomical loci, namely the ellipsoid body (EB), mushroom body (MB), dorsal posterior cells (DPC) and clusters of neurons situated laterally [44], [46]. Since the P elements in the for alleles drive GAL4 expression [47], its insertion in/near the 5′ end of for-T1 may capture the endogenous for-T1 expression pattern. Expression of GFP with for^11.24⁷-GAL4 or for²⁶¹⁴-GAL4 (in flies of genotype for-GAL4/+;UAS-GFP/+) revealed expression in the aristae (AR), a subset of ORNs of the 3^rd antennal segment, discrete glomeruli of the antennal lobe (AL), the pars intercerebralis (PIs), and very weakly in the MB and lateral cell (LC) (Fig. 4A, 4B, data not shown). In for^11.247 homozygotes (flies of genotype for-GAL4;UAS-GFP/+) we observed stronger GFP expression in the MB, as well as additional expression in the EB and the pigment-dispersing factor (PDF)-expressing ventral lateral neurons (LN_vs) (Figs. 4C, S3A–C).

Download:

Figure 4. Expression pattern of for^11.247-GAL4.

A, B) Expression of for^11.247-GAL4/+;UAS-GFP/+ flies. A) In the antenna, GFP (green) was expressed in the arista (AR) and a sub-population of olfactory receptor neurons (ORNs) in the third antennal segment. B) In the CNS, GFP was expressed in specific glomeruli in the antennal lobe (AL), par intercrebalis (PI) neurons, and weakly in the mushroom body (MB) and lateral cells (LC). C) In for^11.247-GAL4;UAS-GFP/+ flies strong GFP expression was seen in MB, PIs, LC and sub-oesophageal ganglion (SOG), as well as the ventral lateral neurons (LN_vs), the giant dorsal interneuron (DGI), parts of the antennal lobe (AL) and ellipsoid body (EB). D) Higher magnification of for^11.247-GAL4;UAS-GFP/+ flies showing partial co-localization with a FOR antibody (red) in the MB and LC, but not in DGI.

https://doi.org/10.1371/journal.pone.0051684.g004

This GFP expression pattern partially overlaps, in the MB and LN, with that observed with the FOR antibody [44]. We did not, however, detect GFP expression in the DPCs, which stain with a FOR antibody (Fig. S3D); thus DPCs may not express the FOR-T1 isoform of FOR. Expression of GFP was also observed in regions not labeled by the FOR antibody, including the ORNs, LN_vs and PI neurons. However, our behavioral data suggests that FOR is likely expressed in ORNs (see below). Furthermore, mammalian PKG is expressed in ORNs and the suprachiasmatic nucleus [48]. Therefore, FOR may not be expressed at levels detectable by this FOR antibody in the ORNs, LN_vs and some neurons of the PI.

for-T1 Functions in for^11.247-GAL4 Neurons to Inhibit Olfactory Startle Habituation

In order to test if for-T1 functions in the neurons defined by for^11.247-GAL4, we attempted to rescue the enhanced habituation of for^11.247 by expressing for-T1 with for^11.247-GAL4. Indeed, expressing for-T1 in homozygous for^11.247-GAL4 flies restored normal OSH (Fig. 5A, 5B). We conclude that the enhanced OSH of for^11.247 flies is due to reduced for-T1 expression and that for-T1 function in for^11.247 -GAL4 neurons is sufficient for flies to show normal habituation.

Download:

Figure 5. for^11.247 -GAL4 flies expressing for-T1 have normal olfactory startle habituation.

A) Habituation profile of functional rescue of for^11.247-GAL4 by expressing UAS-for-T1. No significant difference in distances travelled were seen between for^11.247-GAL4;UAS-for-T1/+ and either Ctrl or UAS-for-T1/+. At pulse 2, 3 and 4, a significant difference was only seen between for^11.247–GAL4;UAS-for-T1/+ and for^11.247 (p<0.01; n = 12). B) HI of for^11.247 rescue. No significant differences were seen between for^11.247-GAL4;UAS-for-T1/+ and either Ctrl or UAS-for-T1/+, but were observed between for^11.247-GAL4;UAS-for-T1/+ and for^11.247 (p<0.01; n = 12).

https://doi.org/10.1371/journal.pone.0051684.g005

Synaptic Silencing of ORNs Inhibits Olfactory Startle Habituation

We next investigated whether the activity of neurons expressing for^11.247-GAL4 directly regulates OSH. To test this, we first blocked synaptic release in for^11.247-GAL4/+ expressing neurons with tetanus toxin light chain (TeTx) [49]. Blocking synaptic release by expressing TeTx in for^11.247-GAL4/+ expressing neurons reduced OSH (Fig. 6A), without affecting the initial startle (Fig. S4A). Further, expressing an inactive form of TetTx (TeTx-ⁱⁿ) in for^11.247-GAL4/+ expressing neurons did not alter OSH (Fig. 6A). These data suggest that synaptic activity of the ORNs, a few MB neurons and/or PI neurons promotes habituation. To further define the neurons regulating OSH, we next silenced specific subsets of for^11.247-GAL4/+ expressing neurons. As blocking synaptic release in the MB is already known to regulate OSH [22], we focused on other neurons of the for^11.247-GAL4-expression pattern, specifically the ORNs and LN_vs.

Download:

Figure 6. Analysis of neuronal circuitry implicated in olfactory startle habituation.

A) Blocking synaptic activity in for^11.247-GAL4 neurons reduces OSH. Heterozygous for^11.247-GAL4 flies expressing tetanus toxin (TeTx) had reduced OSH. Significant differences were seen between for^11.247-GAL4/+ or UAS-TeTx/+ and for^11.247-GAL4/+;UAS-TeTx/+ (p<0.001; n = 9). No significant difference was seen in flies expressing inactive TeTx (TeTxⁱⁿ) with for^11.247-GAL4/+ (p>0.05; n = 9). B) Synaptic silencing of ORNs inhibits OSH. Expressing UAS-TeTx with Orco-GAL4 significantly reduced OSH. Differences were observed between Orco-GAL4/+ or UAS-TeTx/+ and Orco-GAL4/+;UAS-TeTx/+ (p<0.01; n = 6). No effect was seen upon expressing UAS-TeTxⁱⁿ with Orco-GAL4/+ (p>0.05; n = 9). C) for-T1 overexpression in ORNs inhibits OSH. Expressing UAS-for-T1 with Orco-GAL4, but not with MB driver OK107-GAL4, reduced OSH. Significant differences were observed between Orco-GAL4/+ or UAS-for-T1/+ and Orco-GAL4/+;UAS-for-T1/+ (p<0.001; n = 14), but not between controls and OK107-GAL4/+;UAS-for-T1/+ (p>0.05; n = 8).

https://doi.org/10.1371/journal.pone.0051684.g006

To test if the activity of the ORNs promotes habituation, we silenced them by expressing TeTx with the Odorant receptor co-receptor-GAL4 (Orco-GAL4) driver, which is expressed in ∼80% of ORNs [50]. Like for^11.247-GAL4/+ expressing neurons, synaptic silencing of the ORNs also significantly suppressed OSH (Fig. 6B), without affecting the initial startle (Fig. S4B). Therefore, neurotransmission in the ORNs defined by Orco-GAL4 is required to promote OSH.

Since the PDF-expressing LN_vs are also labeled by for^11.247–GAL4 (Fig. S3A-C), we next tested if synaptic activity of the LN_vs neurons regulates OSH. However, silencing neurotransmission in the LN_vs, by expressing TeTx with Pdf-GAL4 [51], did not affect OSH (Fig. S4C). Therefore, our data suggest that LN_v neurons do not regulate OSH. In summary, our data indicate that synaptic activity of ORNs promotes OSH.

FOR-T1 Overexpression in the ORNs Inhibits Olfactory Startle Habituation

We have shown that for-T1 inhibits OSH (Fig. 5) and that blocking synaptic release in ORNs, which likely express for-T1 (Fig. 4A), also reduces OSH (Fig. 6B). Therefore, it is possible that for-T1 inhibits OSH by decreasing synaptic release in ORNs. If this was the case, increasing levels of for-T1 in ORNs should reduce OSH. Indeed, similar to the effect of silencing ORNs with TeTx, overexpression of for-T1 with Orco-GAL4 significantly reduced OSH (Fig. 6C). Therefore, for-T1 may inhibit OSH by reducing synaptic release in ORNs. We also tested whether for-T1 overexpression in MB affects OSH. However, flies expressing for-T1 with the pan-MB driver OK107-GAL4 had a normal OSH (Fig. 6C), suggesting that in the MB for-T1 may not regulate OSH. To conclude, our data suggest that for-T1 may act primarily in ORNs to inhibit OSH and a possible FOR-T1 function here is reduction of synaptic release after an initial exposure to ethanol vapor.

Discussion

We describe the isolation of Drosophila mutants that disrupt olfactory startle habituation (OSH); of these 26 mutants, the majority showed enhanced OSH. Additional targeted analysis also identified several strains carrying mutations in genes that play a role in septate junctions thus implicating this structure in regulating OSH. We characterized two mutations in for that enhanced OSH due to reduced expression of a specific for product, FOR-T1. We show that for-T1 limits OSH by functioning in a subset of neurons that include ORNs and the MB. Our data further map for-T1 function primarily to ORNs, implying that OSH can occur in the sensory neurons of the olfactory circuit.

for encodes several isoforms of protein kinase G (PKG), a cGMP-dependent serine/threonine kinase that regulates neuronal excitability [52] and leaning and memory [46]. With respect to habituation, the natural variant (for^s) with reduced PKG activity [45] also has reduced habituation of the giant-fiber system, which mediates escape responses to visual stimuli [41] and the gustatory-based PER [42], implying that for limits these behaviors. We now show that for also limits OSH; thus for appears to be a central suppressor of habituation, regardless of sensory modality. A question remains as to whether for isoforms and their function is similar in these separate neuronal populations. Interestingly, the mammalian PKG with highest homology to for, PRKG1, [53] has been associated with Attention Deficit/Hyperactivity Disorder [54], a condition characterized by a persistent lack of attention possibly due to a failure to habituate to large amounts of information received from the environment [55].

Ethanol activates several olfactory receptors (ORs): OR7a, OR22a, OR35a, OR85b (http://neuro.uni-konstanz.de/DoOR). Although, curiously, activity of the ORNs expressing these ORs does not appear to be needed for flies to initially sense the smell of ethanol, as the magnitude of the initial startle response was unaffected by synaptic silencing using Orco-GAL4. Interestingly, one glomerulus that appeared labeled in for^11.247-GAL4 heterozygotes is VC31, which expresses OR35a, the OR most strongly activated by acute ethanol. Therefore, VC31 maybe a glomerulus mediating ethanol-induced OSH. It is also worth noting that, in addition to activating particular ORs, ethanol is also a known GABA_A receptor agonist [56] and may also act on GABA_A receptors expressed in LNs and PNs that promote OSH [18], [32], [57].

How might for-T1 function in ORNs to limit OSH? Since for-T1 overexpression in ORNs, or their synaptic silencing, reduced OSH, for-T1 may limit OSH by decreasing synaptic release. Indeed, cultured neurons of for^s flies with reduced PKG activity [45] exhibit increased excitability, resulting in increased spontaneous and evoked activity [52]. for-T1 may achieve decreased synaptic release by modulating cAMP levels, as PKG does in mammalian ORNs [58], [59]. Alternatively, as in the mammalian neurons, it may phosphorylate a number of possible substrates including: TRPC channels, which regulate Ca²⁺ influx [60], SEPTIN3, a regulator of vesicle targeting or tethering [61], [62], or transporters of serotonin [63], [64], a neurotransmitter implicated in presynaptic inhibition in the AL [57].

Finally, our data suggest that olfactory habituation can occur in the 1^st order neurons of the olfactory circuit (the ORNs), while several recent papers demonstrate that the 2^nd order neurons of the olfactory circuit (the LNs and PNs) are key players in olfactory habituation [17], [18], [27], [32]. MB silencing and ablation experiments also suggest that these 3^rd order neurons are also involved [20], [22]. Indeed, studies in the rat show that olfactory cortex and not peripheral circuits, regulate olfactory habituation [65]. Therefore, the capacity to habituate to olfactory cues appears to be distributed throughout the olfactory circuit. Indeed, synaptic silencing of either the ORNs (this study) or the MB [22] did not completely block OSH, as one might expect if habituation occurred at a singular point in the circuit. This distributed mechanism of habituation may allow the fruit fly a greater flexibility in the interplay between its innate responses and learnt experience.

Supporting Information

Figure S1.

for^11.247 has enhanced OSH in two genetic backgrounds. A) for^11.247 in the wBerlin background has enhanced OSH (p>0.001; n = 7, Unpaired t-test). for^11.247 in the 2202U isogenic background has enhanced OSH (p>0.0134; n = 7, Unpaired t-test). B) for alleles have a normal initial startle response. Total movement during the first ethanol pulse was similar between Ctrl, for^11.247 and for²⁶¹⁴ (p>0.05; n = 8). C) FOR-T3 are unaffected in for^11.247 and for²⁶¹⁴. Representative Western blot of adult heads using an antibody that recognizes FOR-T3. Compared to controls, no differences in levels of FOR-T3 were observed.

https://doi.org/10.1371/journal.pone.0051684.s001

(TIF)

Figure S2.

PKG activity levels do not correlate with OSH or for-T1 levels. A) Levels of PKG activity levels were significantly different between control strains wBerlin and Ctrl (p<0.001; n = 5), precluding informative conclusions about PKG activity in for^11.247 and for²⁶¹⁴, which were also significantly different from each other (p<0.001; n = 5). B) for^R, for^s and for^s2 did not show significant differences in OSH (p>0.05; n = 6–8).

https://doi.org/10.1371/journal.pone.0051684.s002

(TIF)

Figure S3.

for^11.247-GAL4 is expressed in PDF expressing neurons, but not DPC neurons. A) Expression of GFP (green) in for^11.247-GAL4 flies revealed expression in the lateral ventral neurons (LN_vs), identified in (B) by a PDF antibody (red). C) Co-localization of GFP and PDF in for^11.247–GAL4;UAS-GFP/+ flies. D) Co-staining of for^11.247-GAL4;UAS-GFP/+ flies with FOR antibody (red), revealed no co-localization in the dorsal posterior cells (DPCs).

https://doi.org/10.1371/journal.pone.0051684.s003

(TIF)

Figure S4.

for^11.247-GAL4 and Orco-GAL4 neurons expressing TeTx have a normal initial startle. A) No difference in total movement in the initial startle was seen between for^11.247-GAL4/+;UAS-TeTx/+, for^11.247-GAL4/+ and UAS-TeTx/+ (p>0.05; n = 9). B) No difference in total movement of the initial startle was seen between Orco-GAL4/+;UAS-TeTx/+, Orco-GAL4/+ and UAS-TeTx/+ (p>0.05; n = 6). C) Expressing Tetanus Toxin in PDF neurons did not alter OSH. No significant difference in HI was seen between Pdf-GAL4/+;UAS-TeTx/+ and Pdf-GAL4/+ or UAS-TeTx/+ (p>0.05; n = 8–12).

https://doi.org/10.1371/journal.pone.0051684.s004

(TIF)

Table S1.

Habituation Index of P elements inserted in or 5′ to septate junction genes.

https://doi.org/10.1371/journal.pone.0051684.s005

(DOCX)

Acknowledgments

We thank Fred Wolf, Anita Devineni, Karla Kaun and the anonymous reviewers for helpful comments that improved this manuscript.

Author Contributions

Conceived and designed the experiments: ME UH. Performed the experiments: ME ATB. Analyzed the data: ME ATB. Contributed reagents/materials/analysis tools: ME ATB MBS UH. Wrote the paper: ME UH.

References

1. Thompson RF (2009) Habituation: a history. Neurobiol Learn Mem 92: 127–134 .
- View Article
- Google Scholar
2. Rankin CH, Abrams T, Barry RJ, Bhatnagar S, Clayton DF, et al. (2009) Habituation revisited: an updated and revised description of the behavioral characteristics of habituation. Neurobiol Learn Mem 92: 135–138 .
- View Article
- Google Scholar
3. Groves PM, Thompson RF (1970) Habituation: a dual-process theory. Psychol Rev 77: 419–450.
- View Article
- Google Scholar
4. Wilson DA, Linster C (2008) Neurobiology of a simple memory. J Neurophysiol 100: 2–7 .
- View Article
- Google Scholar
5. Geyer MA, Braff DL (1982) Habituation of the Blink reflex in normals and schizophrenic patients. Psychophysiology 19: 1–6.
- View Article
- Google Scholar
6. Ludewig K, Geyer MA, Vollenweider FX (2003) Deficits in prepulse inhibition and habituation in never-medicated, first-episode schizophrenia. Biol Psychiatry 54: 121–128.
- View Article
- Google Scholar
7. Frankland PW, Wang Y, Rosner B, Shimizu T, Balleine BW, et al. (2004) Sensorimotor gating abnormalities in young males with fragile X syndrome and Fmr1-knockout mice. Mol Psychiatry 9: 417–425 .
- View Article
- Google Scholar
8. Ornitz EM, Lane SJ, Sugiyama T, de Traversay J (1993) Startle modulation studies in autism. J Autism Dev Disord 23: 619–637.
- View Article
- Google Scholar
9. Morasch KC, Hunt PS (2009) Persistent deficits in heart rate response habituation following neonatal binge ethanol exposure. Alcohol Clin Exp Res 33: 1596–1604 .
- View Article
- Google Scholar
10. Hunt PS, Morasch KC (2004) Modality-specific impairments in response habituation following postnatal binge ethanol. Neurotoxicol Teratol 26: 451–459 .
- View Article
- Google Scholar
11. Christoffersen GR (1997) Habituation: events in the history of its characterization and linkage to synaptic depression. A new proposed kinetic criterion for its identification. Prog Neurobiol 53: 45–66.
- View Article
- Google Scholar
12. Gover TD, Abrams TW (2009) Insights into a molecular switch that gates sensory neuron synapses during habituation in Aplysia. Neurobiol Learn Mem 92: 155–165 .
- View Article
- Google Scholar
13. Glanzman DL (2009) Habituation in Aplysia: the Cheshire cat of neurobiology. Neurobiol Learn Mem 92: 147–154 .
- View Article
- Google Scholar
14. Pinsker H, Kupfermann I, Castellucci V, Kandel E (1970) Habituation and dishabituation of the gill-withdrawal reflex in Aplysia. Science 167: 1740–1742.
- View Article
- Google Scholar
15. Kupfermann I, Castellucci V, Pinsker H, Kandel E (1970) Neuronal correlates of habituation and dishabituation of the gill-withdrawal reflex in Aplysia. Science 167: 1743–1745.
- View Article
- Google Scholar
16. Castellucci V, Pinsker H, Kupfermann I, Kandel ER (1970) Neuronal mechanisms of habituation and dishabituation of the gill-withdrawal reflex in Aplysia. Science 167: 1745–1748.
- View Article
- Google Scholar
17. Larkin A, Karak S, Priya R, Das A, Ayyub C, et al. (2010) Central synaptic mechanisms underlie short-term olfactory habituation in Drosophila larvae. Learn Mem 17: 645–653 .
- View Article
- Google Scholar
18. Das S, Sadanandappa MK, Dervan A, Larkin A, Lee JA, et al. (2011) Plasticity of local GABAergic interneurons drives olfactory habituation. Proc Natl Acad Sci USA 108: E646–E654 .
- View Article
- Google Scholar
19. Engel JE, Wu C-F (2008) Neurogenetic approaches to habituation and dishabituation in Drosophila. Error: 1–10. doi:10.1016/j.nlm.2008.08.003.
20. Wolf FW, Eddison M, Lee S, Cho W, Heberlein U (2007) GSK-3/Shaggy regulates olfactory habituation in Drosophila. Proc Natl Acad Sci USA 104: 4653–4657 .
- View Article
- Google Scholar
21. Sharma P, Keane J, O’Kane CJ, Asztalos Z (2009) Automated measurement of Drosophila jump reflex habituation and its use for mutant screening. J Neurosci Methods 182: 43–48 .
- View Article
- Google Scholar
22. Cho W, Heberlein U, Wolf FW (2004) Habituation of an odorant-induced startle response in Drosophila. Genes Brain Behav 3: 127–137 .
- View Article
- Google Scholar
23. Vosshall LB, Stocker RF (2007) Molecular architecture of smell and taste in Drosophila. Annu Rev Neurosci 30: 505–533 .
- View Article
- Google Scholar
24. Masse NY, Turner GC, Jefferis GSXE (2009) Olfactory information processing in Drosophila. Curr Biol 19: R700–R713 .
- View Article
- Google Scholar
25. Olsen SR, Wilson RI (2008) Lateral presynaptic inhibition mediates gain control in an olfactory circuit. Nature 452: 956–960 .
- View Article
- Google Scholar
26. Wilson RI, Laurent G (2005) Role of GABAergic inhibition in shaping odor-evoked spatiotemporal patterns in the Drosophila antennal lobe. Journal of Neuroscience 25: 9069–9079 .
- View Article
- Google Scholar
27. Root CM, Masuyama K, Green DS, Enell LE, Nässel DR, et al. (2008) A presynaptic gain control mechanism fine-tunes olfactory behavior. Neuron 59: 311–321 .
- View Article
- Google Scholar
28. Acevedo SF, Froudarakis EI, Kanellopoulos A, Skoulakis EMC (2007) Protection from premature habituation requires functional mushroom bodies in Drosophila. Learn Mem 14: 376–384 .
- View Article
- Google Scholar
29. Davis RL (2011) Traces of Drosophila memory. Neuron 70: 8–19 .
- View Article
- Google Scholar
30. Martin JR, Ernst R, Heisenberg M (1998) Mushroom bodies suppress locomotor activity in Drosophila melanogaster. Learn Mem 5: 179–191.
- View Article
- Google Scholar
31. McCann C, Holohan EE, Das S, Dervan A, Larkin A, et al. (2011) The Ataxin-2 protein is required for microRNA function and synapse-specific long-term olfactory habituation. Proc Natl Acad Sci USA 108: E655–E662 .
- View Article
- Google Scholar
32. Sudhakaran IP, Holohan EE, Osman S, Rodrigues V, Vijayraghavan K, et al. (2012) Plasticity of recurrent inhibition in the Drosophila antennal lobe. Journal of Neuroscience 32: 7225–7231 .
- View Article
- Google Scholar
33. Lamb RS, Ward RE, Schweizer L, Fehon RG (1998) Drosophila coracle, a member of the protein 4.1 superfamily, has essential structural functions in the septate junctions and developmental functions in embryonic and adult epithelial cells. Mol Biol Cell 9: 3505–3519.
- View Article
- Google Scholar
34. Hoyle G (1952) High blood potassium in insects in relation to nerve conduction. Nature 169: 281–282.
- View Article
- Google Scholar
35. Banerjee S, Bhat MA (2008) Glial ensheathment of peripheral axons in Drosophila. J Neurosci Res 86: 1189–1198 .
- View Article
- Google Scholar
36. Bhat MA (2003) Molecular organization of axo-glial junctions. Curr Opin Neurobiol 13: 552–559.
- View Article
- Google Scholar
37. Poliak S, Peles E (2003) The local differentiation of myelinated axons at nodes of Ranvier. Nat Rev Neurosci 4: 968–980 .
- View Article
- Google Scholar
38. Salzer JL (2003) Polarized domains of myelinated axons. Neuron 40: 297–318.
- View Article
- Google Scholar
39. Auld VJ, Fetter RD, Broadie K, Goodman CS (1995) Gliotactin, a novel transmembrane protein on peripheral glia, is required to form the blood-nerve barrier in Drosophila. Cell 81: 757–767.
- View Article
- Google Scholar
40. Schulte J, Tepass U, Auld VJ (2003) Gliotactin, a novel marker of tricellular junctions, is necessary for septate junction development in Drosophila. The Journal of Cell Biology 161: 991–1000 .
- View Article
- Google Scholar
41. Engel JE, Xie XJ, Sokolowski MB, Wu CF (2000) A cGMP-dependent protein kinase gene, foraging, modifies habituation-like response decrement of the giant fiber escape circuit in Drosophila. Learn Mem 7: 341–352 .
- View Article
- Google Scholar
42. Scheiner R, Sokolowski MB, Erber J (2004) Activity of cGMP-dependent protein kinase (PKG) affects sucrose responsiveness and habituation in Drosophila melanogaster. Learn Mem 11: 303–311 .
- View Article
- Google Scholar
43. Davies S-A (2006) Signalling via cGMP: Lessons from Drosophila. Cellular Signalling 18: 409–421 .
- View Article
- Google Scholar
44. Belay AT, Scheiner R, So AK-C, Douglas SJ, Chakaborty-Chatterjee M, et al. (2007) The foraging gene of Drosophila melanogaster: spatial-expression analysis and sucrose responsiveness. J Comp Neurol 504: 570–582 .
- View Article
- Google Scholar
45. Osborne KA, Robichon A, Burgess E, Butland S, Shaw RA, et al. (1997) Natural behavior polymorphism due to a cGMP-dependent protein kinase of Drosophila. Science 277: 834–836.
- View Article
- Google Scholar
46. Mery F, Belay AT, So AK-C, Sokolowski MB, Kawecki TJ (2007) Natural polymorphism affecting learning and memory in Drosophila. Proc Natl Acad Sci USA 104: 13051–13055 .
- View Article
- Google Scholar
47. Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118: 401–415.
- View Article
- Google Scholar
48. Hofmann F (2006) Function of cGMP-Dependent Protein Kinases as Revealed by Gene Deletion. Physiological Reviews 86: 1–23 .
- View Article
- Google Scholar
49. Sweeney ST, Broadie K, Keane J, Niemann H, O’Kane CJ (1995) Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects. Neuron 14: 341–351.
- View Article
- Google Scholar
50. Larsson MC, Domingos AI, Jones WD, Chiappe ME, Amrein H, et al. (2004) Or83b encodes a broadly expressed odorant receptor essential for Drosophila olfaction. Neuron 43: 703–714 .
- View Article
- Google Scholar
51. Renn SC, Park JH, Rosbash M, Hall JC, Taghert PH (1999) A pdf neuropeptide gene mutation and ablation of PDF neurons each cause severe abnormalities of behavioral circadian rhythms in Drosophila. Cell 99: 791–802.
- View Article
- Google Scholar
52. Renger JJ, Yao WD, Sokolowski MB, Wu CF (1999) Neuronal polymorphism among natural alleles of a cGMP-dependent kinase gene, foraging, in Drosophila. Journal of Neuroscience 19: RC28.
- View Article
- Google Scholar
53. Fitzpatrick MJ, Sokolowski MB (2004) In Search of Food: Exploring the Evolutionary Link Between cGMP-Dependent Protein Kinase (PKG) and Behaviour. Integr Comp Biol 44: 28–36 .
- View Article
- Google Scholar
54. Neale BM, Medland S, Ripke S, Anney RJL, Asherson P, et al. (2010) Case-control genome-wide association study of attention-deficit/hyperactivity disorder. J Am Acad Child Adolesc Psychiatry 49: 906–920 .
- View Article
- Google Scholar
55. Massa J, O’Desky IH (2011) Impaired Visual Habituation in Adults With ADHD. J Atten Disord. doi:10.1177/1087054711423621.
56. Korpi ER (1994) Role of GABAA receptors in the actions of alcohol and in alcoholism: recent advances. Alcohol Alcohol 29: 115–129.
- View Article
- Google Scholar
57. Wang JW (2012) Presynaptic modulation of early olfactory processing in Drosophila. Dev Neurobiol 72: 87–99 .
- View Article
- Google Scholar
58. Kroner C, Boekhoff I, Lohmann SM, Genieser HG, Breer H (1996) Regulation of olfactory signalling via cGMP-dependent protein kinase. Eur J Biochem 236: 632–637.
- View Article
- Google Scholar
59. Moon C, Jaberi P, Otto-Bruc A, Baehr W, Palczewski K, et al. (1998) Calcium-sensitive particulate guanylyl cyclase as a modulator of cAMP in olfactory receptor neurons. J Neurosci 18: 3195–3205.
- View Article
- Google Scholar
60. Yao X (2007) TRPC, cGMP-dependent protein kinases and cytosolic Ca2+. Handb Exp Pharmacol: 527–540. doi:10.1007/978-3-540-34891-7_31.
61. Xue J, Wang X, Malladi CS, Kinoshita M, Milburn PJ, et al. (2000) Phosphorylation of a new brain-specific septin, G-septin, by cGMP-dependent protein kinase. J Biol Chem 275: 10047–10056.
- View Article
- Google Scholar
62. Xue J, Milburn PJ, Hanna BT, Graham ME, Rostas JAP, et al. (2004) Phosphorylation of septin 3 on Ser-91 by cGMP-dependent protein kinase-I in nerve terminals. Biochem J 381: 753–760 Available: http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=15107017&retmode=ref&cmd=prlinks.
- View Article
- Google Scholar
63. Prasad HC, Zhu C-B, McCauley JL, Samuvel DJ, Ramamoorthy S, et al. (2005) Human serotonin transporter variants display altered sensitivity to protein kinase G and p38 mitogen-activated protein kinase. Proc Natl Acad Sci USA 102: 11545–11550 .
- View Article
- Google Scholar
64. Zhang Y-W, Gesmonde J, Ramamoorthy S, Rudnick G (2007) Serotonin transporter phosphorylation by cGMP-dependent protein kinase is altered by a mutation associated with obsessive compulsive disorder. Journal of Neuroscience 27: 10878–10886 .
- View Article
- Google Scholar
65. Best AR (2005) Cortical Metabotropic Glutamate Receptors Contribute to Habituation of a Simple Odor-Evoked Behavior. Journal of Neuroscience 25: 2513–2517 .
- View Article
- Google Scholar
66. Wolf FW, Rodan AR, Tsai LT-Y, Heberlein U (2002) High-resolution analysis of ethanol-induced locomotor stimulation in Drosophila. Journal of Neuroscience 22: 11035–11044.
- View Article
- Google Scholar

[ref1] 1. Thompson RF (2009) Habituation: a history. Neurobiol Learn Mem 92: 127–134 .
View Article
Google Scholar

[2] View Article

[3] Google Scholar

[ref2] 2. Rankin CH, Abrams T, Barry RJ, Bhatnagar S, Clayton DF, et al. (2009) Habituation revisited: an updated and revised description of the behavioral characteristics of habituation. Neurobiol Learn Mem 92: 135–138 .
View Article
Google Scholar

[5] View Article

[6] Google Scholar

[ref3] 3. Groves PM, Thompson RF (1970) Habituation: a dual-process theory. Psychol Rev 77: 419–450.
View Article
Google Scholar

[8] View Article

[9] Google Scholar

[ref4] 4. Wilson DA, Linster C (2008) Neurobiology of a simple memory. J Neurophysiol 100: 2–7 .
View Article
Google Scholar

[11] View Article

[12] Google Scholar

[ref5] 5. Geyer MA, Braff DL (1982) Habituation of the Blink reflex in normals and schizophrenic patients. Psychophysiology 19: 1–6.
View Article
Google Scholar

[14] View Article

[15] Google Scholar

[ref6] 6. Ludewig K, Geyer MA, Vollenweider FX (2003) Deficits in prepulse inhibition and habituation in never-medicated, first-episode schizophrenia. Biol Psychiatry 54: 121–128.
View Article
Google Scholar

[17] View Article

[18] Google Scholar

[ref7] 7. Frankland PW, Wang Y, Rosner B, Shimizu T, Balleine BW, et al. (2004) Sensorimotor gating abnormalities in young males with fragile X syndrome and Fmr1-knockout mice. Mol Psychiatry 9: 417–425 .
View Article
Google Scholar

[20] View Article

[21] Google Scholar

[ref8] 8. Ornitz EM, Lane SJ, Sugiyama T, de Traversay J (1993) Startle modulation studies in autism. J Autism Dev Disord 23: 619–637.
View Article
Google Scholar

[23] View Article

[24] Google Scholar

[ref9] 9. Morasch KC, Hunt PS (2009) Persistent deficits in heart rate response habituation following neonatal binge ethanol exposure. Alcohol Clin Exp Res 33: 1596–1604 .
View Article
Google Scholar

[26] View Article

[27] Google Scholar

[ref10] 10. Hunt PS, Morasch KC (2004) Modality-specific impairments in response habituation following postnatal binge ethanol. Neurotoxicol Teratol 26: 451–459 .
View Article
Google Scholar

[29] View Article

[30] Google Scholar

[ref11] 11. Christoffersen GR (1997) Habituation: events in the history of its characterization and linkage to synaptic depression. A new proposed kinetic criterion for its identification. Prog Neurobiol 53: 45–66.
View Article
Google Scholar

[32] View Article

[33] Google Scholar

[ref12] 12. Gover TD, Abrams TW (2009) Insights into a molecular switch that gates sensory neuron synapses during habituation in Aplysia. Neurobiol Learn Mem 92: 155–165 .
View Article
Google Scholar

[35] View Article

[36] Google Scholar

[ref13] 13. Glanzman DL (2009) Habituation in Aplysia: the Cheshire cat of neurobiology. Neurobiol Learn Mem 92: 147–154 .
View Article
Google Scholar

[38] View Article

[39] Google Scholar

[ref14] 14. Pinsker H, Kupfermann I, Castellucci V, Kandel E (1970) Habituation and dishabituation of the gill-withdrawal reflex in Aplysia. Science 167: 1740–1742.
View Article
Google Scholar

[41] View Article

[42] Google Scholar

[ref15] 15. Kupfermann I, Castellucci V, Pinsker H, Kandel E (1970) Neuronal correlates of habituation and dishabituation of the gill-withdrawal reflex in Aplysia. Science 167: 1743–1745.
View Article
Google Scholar

[44] View Article

[45] Google Scholar

[ref16] 16. Castellucci V, Pinsker H, Kupfermann I, Kandel ER (1970) Neuronal mechanisms of habituation and dishabituation of the gill-withdrawal reflex in Aplysia. Science 167: 1745–1748.
View Article
Google Scholar

[47] View Article

[48] Google Scholar

[ref17] 17. Larkin A, Karak S, Priya R, Das A, Ayyub C, et al. (2010) Central synaptic mechanisms underlie short-term olfactory habituation in Drosophila larvae. Learn Mem 17: 645–653 .
View Article
Google Scholar

[50] View Article

[51] Google Scholar

[ref18] 18. Das S, Sadanandappa MK, Dervan A, Larkin A, Lee JA, et al. (2011) Plasticity of local GABAergic interneurons drives olfactory habituation. Proc Natl Acad Sci USA 108: E646–E654 .
View Article
Google Scholar

[53] View Article

[54] Google Scholar

[ref19] 19. Engel JE, Wu C-F (2008) Neurogenetic approaches to habituation and dishabituation in Drosophila. Error: 1–10. doi:10.1016/j.nlm.2008.08.003.

[ref20] 20. Wolf FW, Eddison M, Lee S, Cho W, Heberlein U (2007) GSK-3/Shaggy regulates olfactory habituation in Drosophila. Proc Natl Acad Sci USA 104: 4653–4657 .
View Article
Google Scholar

[57] View Article

[58] Google Scholar

[ref21] 21. Sharma P, Keane J, O’Kane CJ, Asztalos Z (2009) Automated measurement of Drosophila jump reflex habituation and its use for mutant screening. J Neurosci Methods 182: 43–48 .
View Article
Google Scholar

[60] View Article

[61] Google Scholar

[ref22] 22. Cho W, Heberlein U, Wolf FW (2004) Habituation of an odorant-induced startle response in Drosophila. Genes Brain Behav 3: 127–137 .
View Article
Google Scholar

[63] View Article

[64] Google Scholar

[ref23] 23. Vosshall LB, Stocker RF (2007) Molecular architecture of smell and taste in Drosophila. Annu Rev Neurosci 30: 505–533 .
View Article
Google Scholar

[66] View Article

[67] Google Scholar

[ref24] 24. Masse NY, Turner GC, Jefferis GSXE (2009) Olfactory information processing in Drosophila. Curr Biol 19: R700–R713 .
View Article
Google Scholar

[69] View Article

[70] Google Scholar

[ref25] 25. Olsen SR, Wilson RI (2008) Lateral presynaptic inhibition mediates gain control in an olfactory circuit. Nature 452: 956–960 .
View Article
Google Scholar

[72] View Article

[73] Google Scholar

[ref26] 26. Wilson RI, Laurent G (2005) Role of GABAergic inhibition in shaping odor-evoked spatiotemporal patterns in the Drosophila antennal lobe. Journal of Neuroscience 25: 9069–9079 .
View Article
Google Scholar

[75] View Article

[76] Google Scholar

[ref27] 27. Root CM, Masuyama K, Green DS, Enell LE, Nässel DR, et al. (2008) A presynaptic gain control mechanism fine-tunes olfactory behavior. Neuron 59: 311–321 .
View Article
Google Scholar

[78] View Article

[79] Google Scholar

[ref28] 28. Acevedo SF, Froudarakis EI, Kanellopoulos A, Skoulakis EMC (2007) Protection from premature habituation requires functional mushroom bodies in Drosophila. Learn Mem 14: 376–384 .
View Article
Google Scholar

[81] View Article

[82] Google Scholar

[ref29] 29. Davis RL (2011) Traces of Drosophila memory. Neuron 70: 8–19 .
View Article
Google Scholar

[84] View Article

[85] Google Scholar

[ref30] 30. Martin JR, Ernst R, Heisenberg M (1998) Mushroom bodies suppress locomotor activity in Drosophila melanogaster. Learn Mem 5: 179–191.
View Article
Google Scholar

[87] View Article

[88] Google Scholar

[ref31] 31. McCann C, Holohan EE, Das S, Dervan A, Larkin A, et al. (2011) The Ataxin-2 protein is required for microRNA function and synapse-specific long-term olfactory habituation. Proc Natl Acad Sci USA 108: E655–E662 .
View Article
Google Scholar

[90] View Article

[91] Google Scholar

[ref32] 32. Sudhakaran IP, Holohan EE, Osman S, Rodrigues V, Vijayraghavan K, et al. (2012) Plasticity of recurrent inhibition in the Drosophila antennal lobe. Journal of Neuroscience 32: 7225–7231 .
View Article
Google Scholar

[93] View Article

[94] Google Scholar

[ref33] 33. Lamb RS, Ward RE, Schweizer L, Fehon RG (1998) Drosophila coracle, a member of the protein 4.1 superfamily, has essential structural functions in the septate junctions and developmental functions in embryonic and adult epithelial cells. Mol Biol Cell 9: 3505–3519.
View Article
Google Scholar

[96] View Article

[97] Google Scholar

[ref34] 34. Hoyle G (1952) High blood potassium in insects in relation to nerve conduction. Nature 169: 281–282.
View Article
Google Scholar

[99] View Article

[100] Google Scholar

[ref35] 35. Banerjee S, Bhat MA (2008) Glial ensheathment of peripheral axons in Drosophila. J Neurosci Res 86: 1189–1198 .
View Article
Google Scholar

[102] View Article

[103] Google Scholar

[ref36] 36. Bhat MA (2003) Molecular organization of axo-glial junctions. Curr Opin Neurobiol 13: 552–559.
View Article
Google Scholar

[105] View Article

[106] Google Scholar

[ref37] 37. Poliak S, Peles E (2003) The local differentiation of myelinated axons at nodes of Ranvier. Nat Rev Neurosci 4: 968–980 .
View Article
Google Scholar

[108] View Article

[109] Google Scholar

[ref38] 38. Salzer JL (2003) Polarized domains of myelinated axons. Neuron 40: 297–318.
View Article
Google Scholar

[111] View Article

[112] Google Scholar

[ref39] 39. Auld VJ, Fetter RD, Broadie K, Goodman CS (1995) Gliotactin, a novel transmembrane protein on peripheral glia, is required to form the blood-nerve barrier in Drosophila. Cell 81: 757–767.
View Article
Google Scholar

[114] View Article

[115] Google Scholar

[ref40] 40. Schulte J, Tepass U, Auld VJ (2003) Gliotactin, a novel marker of tricellular junctions, is necessary for septate junction development in Drosophila. The Journal of Cell Biology 161: 991–1000 .
View Article
Google Scholar

[117] View Article

[118] Google Scholar

[ref41] 41. Engel JE, Xie XJ, Sokolowski MB, Wu CF (2000) A cGMP-dependent protein kinase gene, foraging, modifies habituation-like response decrement of the giant fiber escape circuit in Drosophila. Learn Mem 7: 341–352 .
View Article
Google Scholar

[120] View Article

[121] Google Scholar

[ref42] 42. Scheiner R, Sokolowski MB, Erber J (2004) Activity of cGMP-dependent protein kinase (PKG) affects sucrose responsiveness and habituation in Drosophila melanogaster. Learn Mem 11: 303–311 .
View Article
Google Scholar

[123] View Article

[124] Google Scholar

[ref43] 43. Davies S-A (2006) Signalling via cGMP: Lessons from Drosophila. Cellular Signalling 18: 409–421 .
View Article
Google Scholar

[126] View Article

[127] Google Scholar

[ref44] 44. Belay AT, Scheiner R, So AK-C, Douglas SJ, Chakaborty-Chatterjee M, et al. (2007) The foraging gene of Drosophila melanogaster: spatial-expression analysis and sucrose responsiveness. J Comp Neurol 504: 570–582 .
View Article
Google Scholar

[129] View Article

[130] Google Scholar

[ref45] 45. Osborne KA, Robichon A, Burgess E, Butland S, Shaw RA, et al. (1997) Natural behavior polymorphism due to a cGMP-dependent protein kinase of Drosophila. Science 277: 834–836.
View Article
Google Scholar

[132] View Article

[133] Google Scholar

[ref46] 46. Mery F, Belay AT, So AK-C, Sokolowski MB, Kawecki TJ (2007) Natural polymorphism affecting learning and memory in Drosophila. Proc Natl Acad Sci USA 104: 13051–13055 .
View Article
Google Scholar

[135] View Article

[136] Google Scholar

[ref47] 47. Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118: 401–415.
View Article
Google Scholar

[138] View Article

[139] Google Scholar

[ref48] 48. Hofmann F (2006) Function of cGMP-Dependent Protein Kinases as Revealed by Gene Deletion. Physiological Reviews 86: 1–23 .
View Article
Google Scholar

[141] View Article

[142] Google Scholar

[ref49] 49. Sweeney ST, Broadie K, Keane J, Niemann H, O’Kane CJ (1995) Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects. Neuron 14: 341–351.
View Article
Google Scholar

[144] View Article

[145] Google Scholar

[ref50] 50. Larsson MC, Domingos AI, Jones WD, Chiappe ME, Amrein H, et al. (2004) Or83b encodes a broadly expressed odorant receptor essential for Drosophila olfaction. Neuron 43: 703–714 .
View Article
Google Scholar

[147] View Article

[148] Google Scholar

[ref51] 51. Renn SC, Park JH, Rosbash M, Hall JC, Taghert PH (1999) A pdf neuropeptide gene mutation and ablation of PDF neurons each cause severe abnormalities of behavioral circadian rhythms in Drosophila. Cell 99: 791–802.
View Article
Google Scholar

[150] View Article

[151] Google Scholar

[ref52] 52. Renger JJ, Yao WD, Sokolowski MB, Wu CF (1999) Neuronal polymorphism among natural alleles of a cGMP-dependent kinase gene, foraging, in Drosophila. Journal of Neuroscience 19: RC28.
View Article
Google Scholar

[153] View Article

[154] Google Scholar

[ref53] 53. Fitzpatrick MJ, Sokolowski MB (2004) In Search of Food: Exploring the Evolutionary Link Between cGMP-Dependent Protein Kinase (PKG) and Behaviour. Integr Comp Biol 44: 28–36 .
View Article
Google Scholar

[156] View Article

[157] Google Scholar

[ref54] 54. Neale BM, Medland S, Ripke S, Anney RJL, Asherson P, et al. (2010) Case-control genome-wide association study of attention-deficit/hyperactivity disorder. J Am Acad Child Adolesc Psychiatry 49: 906–920 .
View Article
Google Scholar

[159] View Article

[160] Google Scholar

[ref55] 55. Massa J, O’Desky IH (2011) Impaired Visual Habituation in Adults With ADHD. J Atten Disord. doi:10.1177/1087054711423621.

[ref56] 56. Korpi ER (1994) Role of GABAA receptors in the actions of alcohol and in alcoholism: recent advances. Alcohol Alcohol 29: 115–129.
View Article
Google Scholar

[163] View Article

[164] Google Scholar

[ref57] 57. Wang JW (2012) Presynaptic modulation of early olfactory processing in Drosophila. Dev Neurobiol 72: 87–99 .
View Article
Google Scholar

[166] View Article

[167] Google Scholar

[ref58] 58. Kroner C, Boekhoff I, Lohmann SM, Genieser HG, Breer H (1996) Regulation of olfactory signalling via cGMP-dependent protein kinase. Eur J Biochem 236: 632–637.
View Article
Google Scholar

[169] View Article

[170] Google Scholar

[ref59] 59. Moon C, Jaberi P, Otto-Bruc A, Baehr W, Palczewski K, et al. (1998) Calcium-sensitive particulate guanylyl cyclase as a modulator of cAMP in olfactory receptor neurons. J Neurosci 18: 3195–3205.
View Article
Google Scholar

[172] View Article

[173] Google Scholar

[ref60] 60. Yao X (2007) TRPC, cGMP-dependent protein kinases and cytosolic Ca2+. Handb Exp Pharmacol: 527–540. doi:10.1007/978-3-540-34891-7_31.

[ref61] 61. Xue J, Wang X, Malladi CS, Kinoshita M, Milburn PJ, et al. (2000) Phosphorylation of a new brain-specific septin, G-septin, by cGMP-dependent protein kinase. J Biol Chem 275: 10047–10056.
View Article
Google Scholar

[176] View Article

[177] Google Scholar

[ref62] 62. Xue J, Milburn PJ, Hanna BT, Graham ME, Rostas JAP, et al. (2004) Phosphorylation of septin 3 on Ser-91 by cGMP-dependent protein kinase-I in nerve terminals. Biochem J 381: 753–760 Available: http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=15107017&retmode=ref&cmd=prlinks.
View Article
Google Scholar

[179] View Article

[180] Google Scholar

[ref63] 63. Prasad HC, Zhu C-B, McCauley JL, Samuvel DJ, Ramamoorthy S, et al. (2005) Human serotonin transporter variants display altered sensitivity to protein kinase G and p38 mitogen-activated protein kinase. Proc Natl Acad Sci USA 102: 11545–11550 .
View Article
Google Scholar

[182] View Article

[183] Google Scholar

[ref64] 64. Zhang Y-W, Gesmonde J, Ramamoorthy S, Rudnick G (2007) Serotonin transporter phosphorylation by cGMP-dependent protein kinase is altered by a mutation associated with obsessive compulsive disorder. Journal of Neuroscience 27: 10878–10886 .
View Article
Google Scholar

[185] View Article

[186] Google Scholar

[ref65] 65. Best AR (2005) Cortical Metabotropic Glutamate Receptors Contribute to Habituation of a Simple Odor-Evoked Behavior. Journal of Neuroscience 25: 2513–2517 .
View Article
Google Scholar

[188] View Article

[189] Google Scholar

[ref66] 66. Wolf FW, Rodan AR, Tsai LT-Y, Heberlein U (2002) High-resolution analysis of ethanol-induced locomotor stimulation in Drosophila. Journal of Neuroscience 22: 11035–11044.
View Article
Google Scholar

[191] View Article

[192] Google Scholar

Figures

Abstract

Introduction

Materials and Methods

Fly Strains

Habituation Assay

Calculations and Statistics

Genetic Screen

Molecular Characterization of for Alleles

RNA Analysis

PKG Enzymatic Activity Assay and Immunohistochemistry

Results

A Behavioral Screen for Mutations Affecting Olfactory Startle Habituation

Several Olfactory Startle Habituation Mutations are Associated with Septate Junctions

Foraging Regulates Olfactory Startle Habituation

Molecular Characterization of for Alleles that Disrupt Olfactory Startle Habituation

for11.247-GAL4 Expression Partially Recapitulates the FOR Expression Pattern

for-T1 Functions in for11.247-GAL4 Neurons to Inhibit Olfactory Startle Habituation

Synaptic Silencing of ORNs Inhibits Olfactory Startle Habituation

FOR-T1 Overexpression in the ORNs Inhibits Olfactory Startle Habituation

Discussion

Supporting Information

Figure S1.

Figure S2.

Figure S3.

Figure S4.

Table S1.

Acknowledgments

Author Contributions

References

for^11.247-GAL4 Expression Partially Recapitulates the FOR Expression Pattern

for-T1 Functions in for^11.247-GAL4 Neurons to Inhibit Olfactory Startle Habituation