Odor ligand tuning of individual larval olfactory sensory neurons
To examine the ligand tuning of individual larval OSNs, we developed a preparation to image odor-evoked calcium increases at axon terminals of genetically labeled neurons (Figure ). The Gal4-UAS system [30
] was used to express the genetically encoded calcium sensor, G-CaMP [31
], in identified larval OSNs using Gal4 drivers with promoters from individual larval OR genes [22
] (Figure ). We observed robust odor-evoked fluorescence increases in the axon terminals of larval OSNs. An example of odor-evoked calcium signals from two OSNs expressing Or35a
is shown in Figure . Three different odors differentially activated these two OSNs. Ethyl butyrate activated both neurons, but hexyl acetate and cyclohexanone selectively activated only Or35a
(Figure ). The response duration in a given OSN was odor-dependent. For instance, hexyl acetate induced a prolonged response in the Or35a
-expressing OSN but cyclohexanone elicited a shorter response in the same neuron (Figure ).
Figure 1 Imaging odor-evoked activity in larval olfactory neurons. (a) Schematic of the larval imaging preparation showing head dissection (left) and mounting of inverted sample for G-CaMP imaging (right). (b) Whole-mount immunofluorescence staining of G-CaMP (more ...)
We applied this imaging technique to examine the native responses of 11 larval OSNs to a panel of 22 odors (Figure and Additional data file 1 (Figure S1a)). The ligand selectivity of larval OSNs we tested varied widely. Or35a
-expressing OSNs reliably responded to 15/22 odors, while Or82a
-expressing neurons responded only to geranyl acetate. Our results match the response profile of larval ORs studied by ectopic expression in the adult 'empty neuron' system [23
]. Consistent with previous reports, larval OSNs could be categorized into aromatic odor-sensitive and non-aromatic odor-sensitive classes (Additional data file 1 (Figure S1b,c)) [23
Figure 2 Ligand tuning of larval olfactory neurons in wild-type animals. (a) Odor-response profiles of the three OSNs most sensitive to ethyl butyrate, measured at axon termini of a given OSN in the antennal lobe, against a panel of 22 odorants (10-2 odor dilution) (more ...)
Among the panel of odors tested, we focused on ethyl butyrate, an ester widely found in fruits [32
] and thus likely to be encountered by larvae in their natural habitat. Drosophila
larvae show robust chemotaxis to this ester [22
]. Our calcium-imaging results indicated that ethyl butyrate consistently activated only 3 of the 11 larval OSNs we tested: Or35a
, and Or42b
(Figure and Additional data file 1 (Figure S1a)). None of the other ten remaining larval ORs responded strongly to ethyl butyrate in previous studies [23
]. Thus, we conclude that these three OSNs constitute the primary sensors of ethyl butyrate in the larval olfactory system. Because G-CaMP imaging lacks the sensitivity and temporal resolution of electrophysiology, we cannot exclude the possibility that other neurons are weakly activated by ethyl butyrate, but below the detection threshold of G-CaMP. Hoare et al
] recently reported stochastic ('fuzzy') electrophysiological responses to odor stimulation in various larval OSNs, but did not examine responses of Or35a
-, or Or42b
-expressing neurons to ethyl butyrate. All three OSNs responded reliably to odors in our imaging study. Therefore, we did not find evidence supporting the fuzzy nature of the odor code reported for other larval OSNs.
Concentration-dependent responses in OSNs to ethyl butyrate
To ask whether Or35a, Or42a, and Or42b OSNs show differential sensitivity to ethyl butyrate, we carried out a dose-response analysis of these OSNs by calcium imaging. Whereas all three OSNs responded to high concentrations of ethyl butyrate (10-2 dilution of odor (v:v in paraffin oil), referred to henceforth as 'odor dilution'; Figures and ), the odor concentration threshold at which these OSNs first reliably responded differed greatly (Figure ). Or35a OSNs showed reliable responses only at the 10-2 odor dilution, Or42a OSNs had a response threshold of 10-3 odor dilution, and Or42b OSNs responded initially at 10-4 odor dilution.
We assessed the stability of these differential odor thresholds in wild-type larvae having 21 functional neurons compared with those obtained from larvae that had only a single functional OSN. Larvae with a single functional OSN were constructed by exploiting the Or83b
mutation, which renders animals insensitive to odors by preventing the normal trafficking and functioning of all OR proteins [34
]. By genetically restoring wild-type Or83b
function to individual neurons using the Gal4-UAS system [30
], we restored normal OR trafficking and function only in a given OSN [22
]. Such genetically manipulated animals, which we term 'OrX
-functional', were constructed in this study by restoring Or83b
function either to Or35a
, or Or42b
OSNs in anosmic Or83b-/-
mutants. There was no statistically significant difference between the sensitivity of wild-type and OrX
-functional OSNs to ethyl butyrate (Figure and Additional data file 1 (Figures S2 and S3); see also EC50
values in Materials and methods). This suggests that pre-synaptic inhibition reported for the adult olfactory system in flies and vertebrates is unlikely to play a critical role in larvae [15
Behavioral sensitivity to ethyl butyrate in wild-type and manipulated larvae
The differential sensitivity to ethyl butyrate of Or35a
OSNs prompted us to ask whether these three OSNs mediate concentration-dependent behavioral responses to ethyl butyrate. To investigate this question we used two different experimental paradigms, which measure different aspects of olfactory behavior. A single odor source device [26
] (Figure ) was used to quantify the olfactory sensitivity of individual larvae to a point source of an odor, and a multiple odor source device [26
] (Figure ) was used to assess the ability of larvae to ascend odor gradients.
Figure 3 Behavioral sensitivity to ethyl butyrate in wild-type and manipulated larvae. (a) Schematic of the single odor source assay, with a 0.5 M ethyl butyrate source at position E7 on the lid of a 96-well plate used to generate a radial odor gradient. (b) (more ...)
Figure 4 Chemotaxis to ethyl butyrate in wild-type and manipulated larvae. (a) Schematic of the multiple odor source assay. Source concentrations (M) used to generate ethyl butyrate gradients. (b) Average odor concentrations (mean ± SEM) obtained by FT-IR (more ...)
In the single odor source assay, a drop of ethyl butyrate of desired concentration was introduced into the lid in the center of a rectangular arena (Figure ). Diffusion of odorant molecules generated a Gaussian-like radially symmetric odor distribution centered on the source [26
] (Figure ). Odor concentrations in air were considerably lower than source concentrations (compare 500 mM source with 50 μM peak gradient; Figure ). Single larvae were introduced into the arena under a drop of ethyl butyrate of varying concentrations ('the odor source'), and their position was tracked for 5 minutes. We observed three different responses to odors in this assay, which allowed us to classify the olfactory sensitivity of our larvae. Animals that can detect the odor, and are attracted to it, will remain in close proximity to the odor source. Animals that do not detect the odor, such as the anosmic Or83b
mutants, dispersed in the arena (Figure ). Finally, animals that can detect the odor but are repelled by the high concentration rapidly leave the area under the point source and navigate in isoconcentration circles at a distance from the source.
To quantify odor responses in this assay, the spatial distribution of each animal within a set of concentric 0.25 cm circles was determined. Because anosmic Or83b-/- control larvae dispersed in the arena (tracks in inset in Figure ) and showed a flat occupancy distribution (bar plot histogram, Figure ), we defined dispersion as a failure to detect the odor, and remaining in proximity to the odor as odor detection.
At low source concentrations of ethyl butyrate (0.96 μM or 15 μM), the distribution of wild-type larvae did not differ significantly from that of Or83b-/- control larvae (Figure , green). However, at concentrations of 60 μM and 240 μM, wild-type larvae remained within less than 1 cm of the odor source throughout the 5-minute experiment (Figure , green). The attraction of wild-type larvae to ethyl butyrate was remarkably stable, such that animals remained within approximately 1 cm of even very high source concentrations ranging from 7.5 to 30 mM (Figure , green). We conclude that the olfactory threshold of wild-type larvae to ethyl butyrate is 60 μM and that these animals have a mechanism to remain attracted to this odor over at least a 500-fold concentration range. We propose that this consistent attraction to a point source of odor that varies across a wide range of concentrations is evidence for concentration-invariant behavior by wild-type larvae.
To ask whether concentration-invariant attraction requires combinatorials of functional OSNs, we examined the sensitivities of larvae with olfactory input limited to a single OSN expressing Or35a, Or42a, or Or42b. Consistent with the low ethyl butyrate sensitivity of the Or35a OSN, Or35a-functional animals did not show any behavioral responses to ethyl butyrate between 0.96 μM and 15 mM, but showed weak, yet significant, behavioral responses to a high concentration of ethyl butyrate (30 mM; Figure , orange).
Or42a-functional animals were less sensitive to ethyl butyrate than wild-type larvae, showing a threshold sensitivity of 240 μM (Figure , violet). As odor concentrations increased, Or42a-functional larvae showed a characteristic circling behavior in which they occupied a circle of increasing diameter from the odor source, ranging as odor concentrations increased from 1 cm with a 240 μM odor source to 2.25 cm with a 30 mM odor source (Figure , violet).
Larvae with the high-sensitivity Or42b OSN were more sensitive to odors than wild-type larvae, showing a significant response to 15 μM ethyl butyrate (Figure , blue), a source concentration at which wild-type larvae show no odor responses (Figure , green). Like Or42a-functional larvae, Or42b-functional larvae showed concentration-dependent circling behavior and increased their distance from the source as ethyl butyrate concentrations increased.
The effect of summed OSN input on concentration-dependent olfactory behavior was measured in Or42a+Or42b 'double' OSN functional larvae. Their odor sensitivity threshold was 60 μM, intermediate between that of Or42a-functional larvae and Or42b-functional larvae. Or42a+Or42b-functional larvae also showed the circling behavior characteristic of the single functional strains (Figure , cyan).
From an examination of the temporal evolution of the mean distance to odor over the 5-minute experiment (Additional data file 1 (Figure S4)), we can confirm that larvae with one or two functional OSNs are circling at a distance because they are actively repelled by high odor concentrations under the odor source. At the same time, we can exclude the alternative explanation that these manipulated larvae fail to detect an increase in the odor concentration because of sensory neuron saturation. With a 15 mM ethyl butyrate source, Or83b mutants left the source of the odor immediately and spent the rest of the 5-minute period exploring the plate. In contrast, wild-type larvae initially moved away from this odor stimulus but within 60 s of exploration at up to 1 cm away from the point source, these animals returned and stayed within about 0.5 cm of the odor source for the balance of the 5-minute experiment. Or42a-functional animals showed the same departure and return behavior. However, they overshot their preferred distance (approximately 2 cm from the odor source) and returned to it afterwards without visiting the region under the source. They never returned to their original location under the odor source. This strongly argues that single OSN-functional larvae are repelled by high concentrations of odor located close to the point source.
Genetic manipulation of the larval olfactory system to reduce input to one or two OSNs thus dramatically changes the animal's behavior to ethyl butyrate across a large concentration range. Single-OSN- and double-OSN-functional larvae lost the ability to maintain consistent attraction to ethyl butyrate across the concentrations tested and instead showed increasing avoidance of the odorant as concentrations increased. For technical reasons, we were unable to compare the absolute odor concentrations used in calcium imaging with those used in behavior, but in both experimental paradigms Or42b was about 10 times more sensitive than Or42a and 100 times more sensitive than Or35a.
Chemotaxis to ethyl butyrate in wild-type and manipulated larvae
To test further the ability of individual ethyl butyrate-sensitive OSNs to detect subtle changes in odor concentrations, we challenged single-OSN-functional animals in a multiple odor source assay [26
] (Figure ). This assay differs from that in Figure because animals start at the low concentration end of the gradient rather than being placed directly under the highest odor concentration as in the single odor source assay. The assay tests the ability of larvae to detect and ascend odor gradients. An exponential gradient of ethyl butyrate was created based on six odor sources aligned in the middle of the arena (Figure ) and validated by infrared spectroscopy (Figure ). We arbitrarily divided the arena into three zones of low (Z1), medium (Z2), and high (Z3) ethyl butyrate concentrations (Figure ) defined on the basis of concentration isoclines of the gradient. Single larvae were introduced into the assay at the low end of the gradient and their movement tracked as described elsewhere [26
]. The percentage time that each animal spent in zones Z1–Z3 was calculated (Figure ). The ability of individual larvae to follow the odorant line was quantified with a combined chemotaxis index [26
] (Figure ). Chemotaxis was studied in ethyl butyrate gradients of varying amplitude.
Or83b-/- mutant larvae did not chemotax in the highest concentration range of ethyl butyrate gradient (3.75–120 mM; Figure , gray boxplot). Or35a-functional larvae did not chemotax in response to any gradients tested (Figure , orange boxplots). The failure of Or35a-functional larvae to chemotax may be because the starting concentration of all gradients tested here was below the high detection threshold of these low-sensitivity animals.
In gradients ranging from low (0.2–7.5 mM) to high concentrations (3.75–120 mM), wild-type larvae showed consistently strong chemotaxis, characterized by spending significantly more time in medium to high concentration zones (Z2–Z3; Figure , green) and by a high combined chemo-taxis score (Figure , right). Thus, the same concentration-invariant olfactory behavior of wild-type larvae seen in the single odor source assay (Figure ) was obtained in the multiple odor source chemotaxis assay.
In contrast, Or42a-functional animals showed robust chemotaxis over a narrower concentration range of 0.06–1.88 mM to 0.2–7.5 mM and only showed significant accumulation in the high-concentration Z3 zone in the 0.2–7.5 mM gradient (Figure , magenta). As gradient concentrations increased, these animals showed a characteristic avoidance of the high-concentration Z3 zone and instead accumulated in the intermediate Z2 zone (Figure , magenta). When odor concentrations increased further, these animals lost all ability to chemotax and did not differ from Or83b-/- mutants in their combined chemotaxis score (Figure ).
Or42b-functional larvae showed strong chemotaxis behavior at considerably lower concentrations than wild-type larvae (3.75–120 μM gradient; Figure , blue). Like Or42a-functional larvae, they avoided the high-concentration Z3 zone as gradient amplitudes increased and, unlike wild-type larvae, they failed to chemotax in gradients with the two highest amplitudes (Figure ).
Odor-evoked responses at projection neuron terminals in the mushroom body
To examine how input from three ethyl butyrate-sensitive OSNs – Or35a
, and Or42b
– is relayed to higher olfactory centers, we imaged odor-evoked responses at projection neuron (PN) axon terminals in the mushroom body (Figure ). GH146, a Gal4 driver that labels the majority of larval PNs [37
], was used to drive G-CaMP for calcium imaging in the axon terminals of PNs in the mushroom body (Figure ). Larval GH146-expressing PNs are cholinergic (Figure ), confirming previous analysis of adult PNs [38
]. In initial experiments, we attempted to image PN responses in wild-type larvae having 21 functional OSNs. Unfortunately, insufficient spatial resolution and the absence of PN-specific genetic markers produced inconclusive results (data not shown). Imaging signals can be obtained in response to odor stimulation, but we have no means of mapping the resulting data onto a coordinate system for a given PN. To solve this registration problem, olfactory input was genetically restricted to a single olfactory neuron by carrying out imaging in Or35a
-, or Or42b
-functional larvae. A representative subset of eight odors was used to probe responses in PNs.
Figure 5 Odor responses of larval projection neurons. (a) Schematic for measuring functional activation of larval PNs in Or35a-, Or42a-, or Or42b-functional larvae at axon terminals in the mushroom body (blue box). Intrinsic G-CaMP fluorescence of the mushroom (more ...)
Odors activated distinct single and positionally conserved mushroom body glomeruli in both Or35a
- and Or42a
-functional animals (Figure ). In Or42b
-functional animals, odors reliably activated two mushroom body glomeruli (Figure ). This observation could be due to terminal axonal branching of a single PN innervating the Or42a
OSN or to two PNs innervating the Or42b
OSN, but was not investigated further here. In some cases there was faint activation outside of the primary glomeruli analyzed here (Additional data file 1 (Figure S5)), but we focused our analysis on the most reliably and strongly activated regions in the mushroom body. These data comprise the first report of odor-evoked responses in the larval mushroom body. Importantly, our results provide functional confirmation of previous anatomical analysis showing that the larval mushroom body is organized into discrete glomeruli representing a 1:1 synaptic relationship between OSNs and PNs in the olfactory circuit [37
Analysis of PN responses to a panel of eight odors in the engineered configuration of input from only a single OSN revealed a good qualitative correspondence between the response profile of the primary olfactory neurons and second-order PNs (compare Figures and ). The only exception was cyclohexanol, which did not significantly activate the Or42a
OSN, but did elicit a weak response in PN terminals in the mushroom body of Or42a
functional animals. Consistent with previous observations of Or42a
receptor tuning made in the empty neuron system [28
], the PN response to ethyl acetate was strongly concentration dependent for Or42a
- and Or42b
-functional animals. Whereas both Or42a
PNs showed responses at a 10-2
dilution of ethyl acetate, only Or42b
responded to a 10-4
dilution of ethyl acetate (Figure ). Direct quantitative comparisons of the thresholds of PNs and OSNs are not possible because different versions of G-CaMP were used to image these cells, but we note that for both cell types, Or42b
was about 10 times more sensitive than Or42a
and 100 times more sensitive than Or35a
(Figure ; see EC50
values of PNs in Materials and methods).
High-concentration threshold for activation of inhibitory local interneurons
Inhibitory LNs in the adult insect antennal lobe have been implicated as modulators of olfactory information processing [17
], but no functional analysis of larval Drosophila
LNs has been described. To image odor-evoked activation of larval LNs, we characterized the expression patterns of Gal4 lines known to be expressed in LNs in the adult antennal lobe (Additional data file 1 (Figure S6)). Of the four Gal4 lines tested, only LN2-Gal4 [40
] selectively labeled LNs that were positive for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) and negative for choline acetyltransferase, a marker of cholinergic neurons (Figure and Additional data file 1 (Figure S6a)). The LNs labeled by LN2-Gal4 extended processes throughout all glomeruli in the larval antennal lobe (Figure ), consistent with previous descriptions of larval LN connectivity [37
Figure 6 Threshold response properties of larval local interneurons. (a) LN2 cells in the antennal lobe stained to reveal G-CaMP (left; anti-GFP antibody, green) and gamma-aminobutyric acid (middle; anti-GABA, magenta). Arrows in the merged image (right) indicate (more ...)
Unlike the glomerulus-specific activation patterns evoked by activation of OSNs, odors induced global activation of LN processes throughout the antennal lobe (Figure ). To standardize our analysis of LN responses, we restricted the area of interest to genetically labeled terminals of the Or42a OSN in the antennal lobe (Figure , left panel) and used eight representative odors to probe LN activation in wild-type larvae and single- and double-OSN-functional larvae (Figure ). LNs in wild-type larvae responded strongly and reliably to only four of the eight odors: ethyl butyrate, 1-Hexanol, 2-Heptanone, and acetophenone. Weak responses were found for a 10-2 dilution of ethyl acetate, pentyl acetate, cyclohexanol, and methyl salicylate. No responses were detected after application of a 10-4 dilution of ethyl acetate. When we restricted olfactory input to the Or42a or Or42b neurons only, the LNs did not respond to any of the odors tested. Larvae in which both the Or42a and Or42b neurons were functional showed weak responses to a 10-2 dilution of ethyl acetate, ethyl butyrate, and 1-Hexanol and no responses to the remaining five odors (Figure ).
These results suggest that the LNs may have a higher odor-activation threshold than OSNs or PNs, and further that summation of OSN input modulates LN responses. To explore this idea, we asked how LNs respond to ethyl butyrate in a range of odor dilutions from 10-1 to 10-7(Figure ). In wild-type larvae, LNs showed reliable responses only at 10-2 and 10-1 dilutions of ethyl butyrate, with partial activation at 10-3 odor dilution. LNs of Or42a or Or42b single functional animals did not respond to any concentration of ethyl butyrate, but the summed input of Or42a and Or42b neurons in Or42a+Or42b-functional neurons induced modest responses of LNs from 10-1 to 10-3 dilutions of ethyl butyrate only (Figure ).
Inhibition of PN odor responsivity by summed OSN input
The recruitment of LN activation by summation of OSN input prompted us to ask if PN output is modulated according to the magnitude of OSN input. Or42a+Or42b-functional larvae were constructed to express G-CaMP under the control of GH146 and odor-evoked calcium activation was measured at PN terminals in the mushroom body as described in Figure . This was technically demanding because our CCD-based imaging system lacks the three-dimensional resolution to image odor-evoked calcium responses simultaneously at multiple Z planes. Thus, only samples in which the three activated mushroom body glomeruli in Or42a+Or42b-functional animals were fortuitously located in the same focal plane could be analyzed (Figure ). Between five and six samples with such an orientation were analyzed for responses to ethyl acetate, 2-Heptanone, and a concentration series of ethyl butyrate (Figure ). Responses in the Or42a-specific subdomain were compared with data obtained from the same subdomain in Or42a-functional animals in Figure . For optimal comparisons across these genotypes, strains were designed such that the same insertion of Or42a-Or83b was used and G-CaMP dosage was kept constant. Therefore, we are confident that any functional differences are a product of the biology of the circuit.
Figure 7 Modulation of odor-evoked signals in the mushroom body by addition of a second functional OSN. (a) Representative G-CaMP activity in PN terminals in mushroom body elicited by three odorants (10-2 dilution except ethyl acetate, 10-4 dilution) and paraffin (more ...)
PN responses to 10-3
, and 10-1
dilutions of ethyl butyrate were significantly weaker in Or42a
-functional animals compared to responses in Or42a
-functional animals (Figure ). To ask if this reduction in response was specific to the Or42a
activation subdomain, we tested 2-Heptanone, which selectively activates Or42a
but not Or42b
OSNs (Figure ) [28
]. Unexpectedly, responses to 2-Heptanone were reduced in the two OSN-functional backgrounds, even though we did not detect an increase in LN function in Or42a
-functional larvae compared to Or42a
-functional larvae (Figure ). It is plausible that spontaneous activity or weak evoked responses from the Or42b
-functional neuron can modulate the LNs and thus the circuit dynamics, but that this was below the detection threshold of G-CaMP in Figure . Future work examining the synaptic physiology of these PNs in relation to OSN and LN input will be crucial for understanding the functional relationships within this circuit, as has recently been accomplished in the adult antennal lobe [20