The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.
Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.
Animals rely on olfactory and gustatory information to mediate decisions essential for survival and reproduction. Understanding how chemosensory cues are detected and processed by the nervous system requires identification of the sensory receptor(s) and the corresponding chemical ligands. Drosophila detect a staggering variety of volatile and non-volatile compounds and are an excellent model in which to study the physiological mechanisms underlying chemosensation. While the olfactory organs perceive volatile molecules, the gustatory organs are specialized to detect low volatility compounds. Here, we present a method to directly measure neuronal responses from the taste organs of Drosophila melanogaster to low-volatility, lipophilic ligands.
Gustatory organs of the fly include the forelegs, proboscis, and wings. Distributed on the surface of the taste organs are hair-like structures known as sensilla that respond to sugars, bitters, salts, water and pheromones8. The sensilla have been classified morphologically into taste bristles and taste pegs9. There are about 31 taste bristles on the labellum that are classified into the long (l-type), short (s-type) and intermediate (i-type) morphologies. The ‘l’ and ‘s’ sensilla house 4 sensory neurons that respond physiologically to sugar (the S cell), low salt (the L1 cell), high salt and bitter compounds (the L2 cell) and water (the W cell)10,11. The ‘i’ sensilla house 2 sensory neurons, one of which responds both to low salt and sugar, while the other responds to high salt12. There are approximately 41 taste sensilla in males and 26 sensilla in females distributed on each of the forelegs. For both males and females, there are 21 sensilla on the midleg, and about 22 sensilla on the hindleg13. The gustatory neurons enclosed by the taste sensilla on the legs are also classified into L1, L2, W and S types.
One standard method for measuring electrical activity from single neurons uses extracellular or intracellular electrodes to record ion flow. Electrode measurements allow neuronal function to be studied in non-model organisms such as Drosophila species, moths, and bees which lack extensive genetic tools for neural labeling. However, while electrophysiological methods have been routinely used to measure activity from Drosophila taste sensilla4,13,14, applying this approach presents several technical challenges. First, taste bristles need to be identified based on morphology and spatial location. Upon identification, electrophysiological measurements can be hindered by the small size of the sensilla, limited accessibility due to position, and difficulty in applying controlled volumes of a chemical stimulus to a taste bristle. Also, stimulation of sensilla may generate signals from more than one neuronal type15. Second, detection of electrical signals can be confounded by background noise resulting from mechanical vibrations and noise from electronic equipment. Third, the use of a sharpened electrode can damage the fly preparation, if used incorrectly14. Finally, assembling an electrophysiology rig requires specialized electronic components for stimulus delivery, signal recording, and data analysis and can be costly.
In D. melanogaster, the availability of genetically-encoded tools has facilitated the development of imaging techniques that allow the responses of small populations of neurons to be studied. One such approach is the use of the CaLexA reporter16. In this method, gene sequences encoding the LexA-VP16 transcription factor and a calcium sensitive protein NFAT are fused together. Calcium activation of the protein phosphatase calcineurin catalyzes the de-phosphorylation of NFAT and, in turn, facilitates its import into the nucleus. Inside the nucleus, the LexA domain binds to a LexAop-DNA binding motif which directs the expression of a green fluorescent protein (GFP) reporter, thus allowing sustained identification of functionally activated neurons. The approach has been used successfully to measure the response of specific olfactory glomeruli in the antennal lobe following exposure of live flies to an odorant16. Recently, physiological responses of IR52c gustatory receptor neurons were measured from live flies using CaLexA7. However, for this study, it was necessary to age flies for up to 6 weeks to enhance GFP transcription. While immunostaining with an anti-GFP antibody can be used to boost the CaLexA signal, this method would require tissue fixation, thus precluding possibility for live-cell imaging.
The genetically encoded calcium indicator protein GCaMP has also been extensively used to study neuronal responses in a number of species17. The protein GCaMP fluoresces with low intensity prior to neuronal stimulation. Application of a stimulus triggers an action potential in the neuron, resulting in the influx of Ca2+. GCaMP, bound to Ca2+, undergoes a conformational change, causing it to fluoresce with brighter intensity (Figure 1). A recently developed single–neuron calcium imaging approach was used to identify glucose as the ligand for the foreleg specific Gr61a gustatory neurons18. In this study, dissected forelegs from transgenic Drosophila expressing GCaMP in Gr61a gustatory neurons were covered with a layer of agarose prior to imaging. However, the use of dissected tissue can cause eventual rundown of GCaMP expression, thus limiting measurement time and detection sensitivity. In addition, agarose can limit sensitivity due to high background levels of fluorescence and its light scattering properties.
To address some of these drawbacks, we describe the use of GCaMP-mediated calcium imaging to record physiological responses from foreleg and proboscis gustatory neurons of intact animals. We show the physiological responses of Gr68a and ppk23 neurons expressing genetically encoded GCaMP5G19 to a Drosophila lipid pheromone, (3R, 11Z, 19Z)-3-acetoxy-11,19-octacosadien-1-ol (CH503)20,21. Neuronal responses are measured by quantifying the increase in fluorescence of the GCaMP5G signal during pheromone stimulation. In this protocol, neurons are imaged for a total duration of 120 sec, which is sufficient to differentiate patterns of neuronal activation in individual cells.
1. Sample Preparation
2. Preparation of Stimulus Solution
3. Stimulation of Gustatory Neurons and Image Acquisition
4. Image Processing and Analysis: Quantification of the Response
The GCaMP calcium indicator was genetically expressed using Gr68a-Gal4 or ppk23-Gal4 drivers. Distinct populations of foreleg neurons and non-neural support cells are labeled by each driver (Figure 3A-C). Ligand-specific responses to the lipophilic pheromone CH503 were observed in Gr68a-Gal4 and ppk23-Gal4 cells expressing GCaMP (Figure 3D). The relative change in fluorescence intensity of the GCaMP5G signal (ΔF/F) and increased with higher concentrations of CH503 (Figure 3E). Interestingly, the 5 ng dose of CH503 may suppress the neural response.
Gr68a and ppk23 neurons exhibited different response patterns. Gr68a neurons showed a tonic pattern of neuronal response (Figure 3F), whereas ppk23 neurons showed a phasic, oscillatory response (Figure 3G). Responses from ppk23 neurons in the proboscis were also measured using this preparation and exhibited a combination of phasic and oscillatory responses (Figure 3H).
Figure 1: Basic principle of the GCaMP calcium imaging technique. GCaMP5G is genetically expressed in Gal4-labeled cells and fluoresces with low intensity prior to neuronal stimulation. Application of a stimulus triggers an action potential in the neuron and causes Ca2+ influx. GCaMP5G, once bound to Ca2+, undergoes a conformational change, resulting in increased fluorescence (ΔF), which changes over time. The baseline fluorescence (F) refers to fluorescent signal intensity prior to stimulation.
Figure 2: Mounting a live fly for imaging of the foreleg or proboscis neurons. (A) Mounting the foreleg: a 3.2X image of a live male fly, showing the forelegs fastened to the coverslip with tape. (B) A 40X image of the fly, showing each of the tarsal segments (T1-5) and the placement of tape above the first tarsal segment and on the fifth tarsal segments. (C) Mounting the proboscis: a piece of tape is placed over the rostrum to expose the tip of the proboscis. (D) The pipette tip for stimulus addition is held slightly above the fly and does not make direct contact with the preparation.
Figure 3: Pheromone-induced neural responses in GCaMP-expressing neurons. (A) Schematic of the male foreleg of Drosophila showing the spatial positions of Gr68a-Gal4 labeled cells on tarsal segments T2-4. Non-neural cells (identified based on size and shape) are colored yellow. (B) Raw fluorescent image of the male foreleg showing neural and support cells labeled by the Gr68a-Gal4 driver. Scale bar = 50 µm. (C) Schematic of the male foreleg of Drosophila showing the spatial positions of ppk23-Gal4 labeled cells on tarsal segments T2-4. (D) Response of Gr68a- and ppk23-expressing T4 neurons to CH503 (chemical structure shown above). The average ΔF/F in response to CH503 was compared to the average ΔF/F following application of PBST solvent alone; Student’s t-test with unequal variance (for Gr68a) or Mann-Whitney test (for ppk23): *p <0.05, ***p <0.001. Error bars depict s.e.m. Statistical power = 0.93. (E) A dose-dependent change in ΔF/F is observed in Gr68a neurons in T4. Student’s t-test with unequal variance: **p <0.01, ***p <0.001. Statistical power = 0.86-0.96. (F) A color-coded time course showing the tonic response in GCaMP fluorescence following CH503 stimulation (500 ng) of Gr68a-expressing neurons. The positions of the neurons on the foreleg are shown in the raw fluorescent image (left). Scale bar = 10 µm; time scale = 0-96 sec. The accompanying line graph shows the tonic response from a Gr68a neuron to stimulation by 500 ng of CH503. The red arrow indicates the time point at which the stimulus was added. The peak response occurs at 120 sec. (G) A color-coded time course showing an oscillatory, phasic response in GCaMP fluorescence following CH503 stimulation (500 ng) of ppk23-expressing neurons. The positions of the neurons on the foreleg are shown in the raw fluorescent image (left). Scale bar: 10 µm; time scale: 0-96 sec. The accompanying line graph shows a bursting response from a ppk23 neuron to stimulation by 500 ng of CH503. Red arrow indicates the time at which the stimulus was added. The peak response occurs at 76 sec. (H) A color-coded time course of ppk23-expressing neurons on the proboscis showing a late-onset tonic response from the cell body and an oscillatory response from an axonal projection following CH503 stimulation (500 ng). The positions of the cells are shown in the raw fluorescent image (left). Scale bar = 10 µm; time scale: 0-96 sec. Reproduced with permission from Shankar, S., et al. The neuropeptide tachykinin is essential for pheromone detection in a gustatory neural circuit. doi: 10.7554/eLife.06914 (2015). Please click here to view a larger version of this figure.
Solvent1,2 | Solubility of CH5033 | Does the solvent alone trigger neuronal activity?4 |
0.1% PBST | YES | NO |
100% Ethanol | YES | YES |
100% Hexane | YES | YES |
10% Ethanol | NO | (not tested) |
10% Hexane | NO | (not tested) |
100% DMSO | YES | YES |
10% DMSO | YES | YES |
0.4% DMSO | YES | YES |
0.2% DMSO | YES | YES |
Table 1: Solubility of CH503 in various solvents.1 DMSO: Dimethyl sulfoxide.2 Ethanol, hexane, and DMSO solutions were in diluted with dH2O (v/v).3 Solubility of CH503 in 0.2% DMSO was limited only to 250 ng/ml.4 Increase in ΔF/F with solvent alone was equivalent to that observed upon stimulation with pheromone.
We describe here a method to perform live calcium imaging of Drosophila peripheral neurons in 2 different sensory organs. The Ca2+-evoked GCaMP fluorescent responses in Gr68a-neurons induced by the pheromone ligand CH503 were dose-dependent and quantitative. It was also possible to discern different neural response patterns such as phasic and tonic responses.
Neurons showing phasic responses are believed to allow rapid adaptation to continuous stimuli. This type of response has been associated with odor detection25 and the generation of rhythmic motor activity patterns26. In contrast, neural oscillations, which have been described in many sensory systems, are thought to synchronize multiple inputs onto a single postsynaptic site and to convey specific features about the stimulus27. The molecular basis of oscillations associated with ppk23 neurons has not yet been reported, to our knowledge. In other types of excitable cells showing oscillatory responses, the frequency of the oscillations has been shown to be regulated by a process called calcium-induced calcium release28. In this process, calcium is released from internal stores under the control of the secondary messenger inositol triphosphate, and the frequency of its release is dependent on the strength of the external stimulus. Insights into the molecular basis of tonic neuronal responses have been gained from studies on oxygen sensing receptors of C. elegans29. A prolonged increase in the calcium levels within neurons was found to be mediated by L-type voltage gated calcium channels, ryanodine, and inositol triphosphate signaling pathways29. It will be important to ascertain whether similar mechanisms play a role in the responses of ppk23 neurons.
Live calcium imaging can be adapted in several ways for the study of other populations of Drosophila sensory neurons or other chemical ligands. First, the responses from gustatory neurons located on the proboscis, legs, or wings can in a screen for ligands for orphan gustatory receptors. Second, modification of the solvent will allow testing neural responses to more polar molecules. Third, an onstage incubator can be coupled with the spinning disc microscope. This setup would be ideal for activating or silencing neuronal activity using temperature-sensitive transgenes like UAS-TrpA1 or UAS-Shibirets, and measuring the corresponding changes in fluorescence in these neurons upon stimulation4. Fourth, the use of a continuous time series for image collection can facilitate detection of fast responding cells. Finally, with the addition of targeted laser illumination, this method could be expanded for use with other advanced live-cell imaging methods such as FRAP and FRET.
Several technical considerations are imperative to obtain stable images and to accurately measure the time course of physiological responses. First, the foreleg must be securely positioned since the fly exhibits vigorous movement upon addition of the solution, which can be a source of variation between preparations. Although bath application of pheromone solution allows the ligand to reach all of the gustatory sensilla on the exposed tarsal segments, some of the solution can flow towards other parts of the mounted preparation. Also, the foreleg could potentially drift away when solution is added. Second, to accurately image near-instantaneous increases in fluorescence, it is important to resume imaging immediately after addition of the stimulus since some neurons show a rapid and maximal response within 4 seconds of stimulation. Alternatively, a continuous time series can be initiated prior to adding the stimulus solution, and the precise time point at which the stimulus is administered can be marked.
Two sources of false positive responses were observed. First, the solvent itself can induce fluorescent responses even in the absence of a ligand. The PBST solvent caused such a response in some ppk23-Gal4 labeled cells. In addition, DMSO, a commonly used solvent for polar and apolar molecules, also induced a response in some Gr68a-Gal4 labeled neurons. It is therefore essential to determine whether the solvent itself induces a fluorescent signal and to select a solvent system with low background activity. Second, a strong phasic-type response in non-neural support cells was observed to the solvent PBST alone. The Gr68a-Gal4 driver is expressed in both neurons and support cells that appear to surround some of the neurons and in general, are larger, amorphously shaped, and lack visible projections. It is therefore important to differentiate neural from non-neuronal responses for each Gal4 line when choosing ROIs.
One major limitation of this method is that the same fly preparation cannot be used for successive experiments to test different concentrations or types of ligands, unless the applied stimulus is first thoroughly removed from the surface of the coverslip. In addition, this method cannot be used for high throughput imaging of several flies at once, due to the high magnification needed for visualization of Drosophila neurons, and the need for fast acquisition and high time resolution. Lastly, this method requires the availability of genetic tools for transgenic gene expression, thus limiting application primarily to major model organisms.
Altogether, this method is an easier alternative to cellular recordings using electrodes and does not require specialized instrumentation. In addition, unlike other genetically encoded reporters such as CaLexA, the animal is kept alive throughout imaging allowing physiological responses to be measured in real-time with high sensitivity in flies of all ages. This feature could potentially allow for screening of age-dependent responses to ligands or comparison of neural responses following changes in social conditions or reproductive state. Moreover, sustained recordings can be performed for at least 10 min without obvious photobleaching or rundown of GCaMP signal.
The authors have nothing to disclose.
This work was supported by the Singapore National Research Foundation (grant NRF-RF2010-06 to J.Y.Y.).
Gr68a-Gal4 | Gift from H. Amrein (Texas A&M Health Science Center, TX, USA) and J. Carlson (Yale University, CT, USA) | ||
ppk23-Gal4 | Gift from K. Scott (Univ. of California, Berkeley, CA, USA) | ||
UAS-GCaMP5 | 42037 | Bloomington Drosophila Stock Center | |
0.17 mm coverslip (Gold-Seal coverslip) | Electron Microscopy Services | 63790-10 | |
Nail polish, "Hard as Nails Clear" | Sally Hansen | ||
PAP pen | Sigma-Aldrich | Z377821 | |
Paint brush | fine-tipped brush | ||
Tape | Scotch brand | ||
Triton X-100 | Sigma-Aldrich | 13021 | |
Ethanol, lab grade | Merck | 10094 | |
Hexane, HPLC grade | Sigma-Aldrich | H303SK-4 | |
DMSO | Sigma-Aldrich | 472301 | |
PBST | Recipe described in the protocol section | ||
CH503 | Synthesis described in Mori et al., 2010 | ||
sCMOS Camera (ORCA Flash4.0) | Hamamatsu | C11578-22U | |
Microscope (Ti-Eclipse) | Nikon | Ni-E | |
Spinning Disk Scan head | Yokogawa | CSU-X1-A1 | |
Aquistion Software (MetaMorph Premier) | Molecular Devices | 40002 | |
Fiji software | open source | http://fiji.sc/Fiji |