Modulation of peripheral olfactory responses to a food odor
The olfactory sensilla of Drosophila were first classified by morphology into basiconic, trichoid, and coeloconic shapes [21]. The largest are the club-shaped basiconic sensilla, which are divided by location, size, and OR expression into 13 subclasses [2]. Among the large antennal basiconics (ab), each sensillum of the ab3 class houses two OSNs designated ab3A and ab3B on the basis of spike amplitude, with ab3A giving the larger spikes [22]. These ab3A neurons express the olfactory co-receptor Orco along with the odorant-specific odorant receptor OR22a [23, 24]. OR22a responds to several fruity-smelling esters including ethyl butyrate, a volatile abundant in apples and many other fruits [25].
Using the single sensillum electrophysiological recording technique, we confirmed that ab3A neurons are robustly activated by fresh apple odor and by ethyl butyrate, one of apple odor’s major components (Fig. 1a, b). Since an animal’s attraction to food odor depends on physiological states like sleep, hunger, and even age, we wondered if the peripheral responses of the ab3A neurons may be modulated. Therefore, we measured the responses of ab3A neurons to ethyl butyrate (EB) after rearing flies under several conditions designed to change their internal physiological states. Under our conditions, we saw no significant changes in the dose-response relationship of the ab3A neurons to EB after rearing on protein-rich or carbohydrate-rich diets, or rearing at high (29 °C) and low (18 °C) temperatures; neither did we see changes with circadian rhythm (data not shown). We found, however, that the responses of the ab3A neurons change as female flies mature post-eclosion. While the ab3A neurons of male w
1118 flies show a consistent response rate from 1–7 days post-eclosion, female ab3A neurons show a consistent age-dependent increase in EB responses reaching male levels by 7 days post-eclosion (Fig. 1c).
NPF-NPFR signaling sensitizes ab3A neurons
We expected that this modulation of the ab3A neurons may be peptidergic. The combination of a GAL4 enhancer trap expressed in many adult peptidergic neurons (386Y-GAL4) [26] with an RNA interference (RNAi) line specific to neuropeptide F (NPF) (UAS-NPF-IR) significantly reduces the responses of ab3A neurons to EB (Fig. 2a). In Drosophila, NPF affects several physiological phenomena including appetite [18] and the modulation of gustatory responses to sugar [19]. We also found that limiting the knock-down of NPF to NPFergic neurons using NPF-GAL4 produces an even larger reduction in ab3A responses to EB (Fig. 2b). NPF > NPF-IR brains show almost no NPF signal compared to the appropriate heterozygous controls (i.e., crossed to w
1118) (Additional file 1: Figure S1A). Using two copies of NPF-GAL4 and two copies of the membrane-tethered Green fluorescent protein (GFP) transgene UAS-myr::GFP, we were able to visualize the projections of these cells into the antennal lobes (Additional file 1: Figure S1B, C). The reduction in peripheral olfactory responses is specific to the odor-evoked activity of the ab3A neurons, as their spontaneous activity is unaffected by NPF knock-down (Fig. 2c). It also seems to be specific to the ab3A neuronal subclass. The responses of both ab1A/B and ab2A to EB and methyl acetate (MA), respectively, are unaffected by NPF knock-down (Fig. 2d, e). Here, we did not sort ab1A spikes from ab1B spikes because the similar amplitudes of both classes of spikes complicate the sorting process. Although both ab1A and ab1B neurons respond to EB, because ab1A responses to EB are much stronger than ab1B responses, ab1A activity likely dominates the results shown in Fig. 2d. Although the ab2B, ab8A/B, and pb1A (palp basiconic 1A) sensilla also respond to EB, NPF knock-down does not affect them (Additional file 2: Figure S2).
Since NPF acts via the G protein-coupled NPF receptor (NPFR) [27], we next asked whether loss of NPFR also affects the responses of ab3A neurons to EB. The ab3A neurons of flies carrying homozygous piggyBac element insertions in the NPFR locus (NPFR
c01896) show significantly lower responses to EB than those of the control w
1118 genetic background (Fig. 2f). Although Krashes et al. verified NPFR
c01896 as an NPFR hypomorph [17], for further evidence of NPFR’s involvement, we combined NPFR
c01896 with a chromosomal deficiency covering the NPFR locus (NPFR
Def). As expected, transheterozygous loss of NPFR (NPFR
c01896
/NPFR
Def) produces the same dramatic reduction in ab3A responses to EB compared to the corresponding heterozygous controls (Fig. 2f). We also found that NPFR
c01896 ab3A neurons show reduced responses to other apple odors including methyl butyrate (Additional file 3: Figure S3).
NPF-NPFR signaling sensitizes ab3A neurons as flies mature
Since the ab3A neurons of male and female flies (w
1118) show differences in their responses to ethyl butyrate 1 day after eclosion (Fig. 1c), and adult male brains have more NPF neurons than female brains of unspecified ages [28], we wondered whether the olfactory sexual dimorphism we identified can be attributed to NPF signaling. After dissecting brains from w
1118 males and females 1 and 7 days after eclosion, we stained them with an NPF-specific antiserum (Fig. 3a, b). Then, by processing similar stacks of confocal images of the brains from each sex with ImageJ [29], we found higher NPF staining per pixel in male brains than in female brains (Fig. 3c). We next compared the electrophysiological responses of ab3A neurons from w
1118 flies with those of NPFR
c01896 flies over the first week of adult life. We found that the ab3A neurons of both male (♂) and female (♀) NPFR
c01896 flies show low responses to ethyl butyrate (EB, 10−5 v/v) steadily through the first week post-eclosion (Fig. 3d). By comparison, the responses of w
1118 males are significantly higher from days 1–7. The responses of female w
1118 ab3A neurons, in contrast, are as low as those of NPFR
c01896 ab3A neurons on day 1, but rise to the level of male w
1118 ab3A neurons by day 7 (Fig. 3d). Note that the w
1118 data presented in Fig. 3d are identical to those in Fig. 1c and are shown again for comparison’s sake. Together, these results suggest that sexual dimorphisms in NPF signaling may account for the differential responsiveness of female ab3A neurons as they mature.
ab3A neurons require NPFR to produce wild-type responses
We next asked where NPFR is required for determining ab3A responses. Since the most obvious candidate is the OSNs themselves, we first used Orco-GAL4 to knock down NPFR in all the OSNs that express the olfactory co-receptor Orco. We found that ab3A neurons from Orco > NPFR-IR flies show significantly lower responses to ethyl butyrate (EB, 10−6 and 10−5 v/v) than their heterozygous controls (crossed to w
1118) (Fig. 4a). While Orco-GAL4 labels most OSNs in the antennae and all OSNs in the maxillary palps, Or22a-GAL4 labels only ab3A neurons. As with Orco > NPFR-IR flies, the ab3A neurons of Or22a > NPFR-IR flies show lower ab3A responses to EB than their heterozygous controls (Fig. 4b). Interestingly, we also found that knock-down of NPF in most OSNs with Orco-GAL4 and ab3A neurons with Or22a-GAL4 also produces a mild reduction in the responses of ab3A neurons to EB (Additional file 4: Figure S4). This suggests that the OSNs themselves may produce small amounts of NPF.
We next isolated total RNA from w
1118 heads for cDNA synthesis. From the resulting head cDNAs, we cloned versions of two of the NPFR transcripts annotated in Flybase, NPFR-RB and NPFR-RD. We used both to generate transgenic UAS-NPFR lines and then used them to perform an ab3A neuron-specific rescue of the NPFR
c01896 mutation. From the sample traces for this experiment, it is clear that NPFR-RD fully rescues the responses of ab3A neurons to EB (10−6 v/v) (Fig. 4c). In Fig. 4d, we present a quantification of recordings comparing the w^1118 genetic background control to the NPFR
c01896 mutant and the ab3A neuron-specific rescue (Or22a-GAL4 > UAS-NPFR-RD, NPFR
c01896) compared to its appropriate heterozygous controls (Fig. 4d). Interestingly, although the NPFR-RB isoform appeared far more often among the w
1118 cDNAs we used to generate the UAS-NPFR lines than the NPFR-RD isoform, it fails to rescue the ab3A response phenotype (Fig. 4d). We speculate that the 23 C-terminal amino acid residues (382–404) of the NPFR-RD isoform absent in the NPFR-RB isoform may affect receptor trafficking, post-translational modifications, or receptor membrane topology (Additional file 5: Figure S5).
NPFR in ab3A neurons modulates olfactory-guided behavior
We next asked whether NPF-NPFR signaling in the ab3A neurons modulates olfactory-guided behaviors. Trap assays test olfactory function at the behavioral level [30], but they sometimes produce noisy data (not shown). Since social cues modulate olfactory behavior in flies [31], we designed a new trap assay for individual flies (Fig. 5a), hoping to measure olfactory attraction at a higher signal-to-noise ratio. The single fly olfactory trap assay comprises an origin chamber and a bait chamber connected by a short piece of plastic tubing only slightly wider than an individual fly. Although the origin chambers contain wet tissue as a water source, they do not contain food. At first, the individual flies in the origin chamber resist traveling through the narrow tube, but as they starve over the course of the assay, the odor source lures them into the adjoining bait chamber. To perform the assay, we habituate individual 7- to 10-day-old male flies in vials containing a tissue soaked with fresh apple juice. After assembling the traps, we place them in a dark chamber at room temperature for 40 h. From 40–50 h, we check the traps every 2 h, removing and counting “successes” in which the individual flies travel to the bait chamber. We chose fresh apple juice (10−1 v/v) as a bait because it provides a source of sugar and induces a similar olfactory response in the OSNs of large basiconic sensilla as ethyl butyrate (10−4 v/v) (Fig. 5b).
We began by performing initial control experiments to confirm that our single fly trap assay measures olfactory attraction. We found that more than 70% of w
1118 flies successfully reach bait chambers containing apple juice, but they never leave the origin chamber when it contains the apple juice rather than the bait chamber (Fig. 5c). If both chambers contain water, fewer than 20% of w
1118 flies cross to the bait chamber within 50 h. Since Orco
1 flies lack the olfactory co-receptor Orco, which is required for OR trafficking, their OR-expressing OSNs are non-functional [23]. We found that fewer than 30% of Orco
1 flies locate the apple juice bait. These results validate the single fly trap assay for measuring olfactory-guided attraction to fruit odor.
Since ethyl butyrate is one of the major components of apple odor, we next asked whether OR22a—the odorant receptor expressed in the ethyl butyrate-sensing ab3A neurons—is required for behavioral attraction to apple juice. We found that ab3A-specific knock-down of OR22a using Or22a-GAL4 dramatically reduces attraction to apple juice in the trap assay (Fig. 5c). This result suggested that NPF-NPFR signaling may also modulate behavioral attraction to apple odor. Since knock-down of NPF in the NPF neurons could alter the internal physiological state of the flies, making them feel more satiated and less likely to move to the odor bait chamber, we decided to limit our behavioral experiments to genetic manipulations of the OSNs. Knock-down of NPFR in most olfactory neurons using Orco-GAL4 and knock-down in the ab3A neurons alone using Or22a-GAL4 both significantly reduce success in locating the apple juice bait (Fig. 5d). Together, these results suggest that the role of NPF-NPFR signaling in modulating the peripheral responses of the ab3A neurons is behaviorally relevant for olfactory-guided foraging.
Exploring the mechanism of NPFR-mediated olfactory modulation
A small swelling known as the ciliary dilation divides OSN dendrites into inner and outer segments (Fig. 6a). OSNs can only respond to odor by expressing and trafficking ORs to their outer dendritic segments where they can encounter odorant molecules [23, 32]. We hypothesized that NPF-NPFR signaling may modulate the responses of ab3A neurons to ethyl butyrate by altering either the expression or trafficking of the OR expressed in ab3A neurons that responds to ethyl butyrate, OR22a [24]. We, therefore, used an OR22a-specific antiserum to compare the levels of OR22a in the outer dendrites of ab3A neurons from w
1118 and NPFR
c01896 antennae. In w
1118 antennae, the ab3A neurons show almost no OR22a staining in the soma, little in the ciliary dilations, and a strong signal in the outer dendritic segments (Fig. 6b, Additional file 6: Figure S6). NPFR
c01896 mutant ab3A neurons show slightly stronger OR22a staining in their soma, strong punctate staining in their inner dendritic segments and ciliary dilations, and strong staining in their outer dendritic segments (Fig. 6c, Additional file 6: Figure S6). Except for the OR22a signal in the outer dendrites, this staining pattern in NPFR
c01896 ab3A neurons resembles that of Orco
1 mutant ab3A neurons, which lack the olfactory co-receptor Orco and hence show no OR22a in the outer dendrites [33]. Although this increase in intracellular OR22a in the absence of NPFR suggests a role for NPFR in modulating the dendritic trafficking or internalization of OR22a, we could not detect a difference when we quantified the OR22a-stained pixels in the outer dendrites of w
1118 and NPFR
c01896 antennae using ImageJ (Fig. 6d). Using a similar method, we did observe increased punctate staining near the ciliary dilations in NPFR
c01896 antennae compared to w
1118 antennae (Fig. 6e). Importantly, we were unable to detect any difference in Or22a mRNA levels between w
1118 and NPFR
c01896 antennae via qPCR (Fig. 6f), suggesting that the difference in OR22a protein staining in these puncta cannot be attributed to a change in Or22a expression.