Background: Activity-dependent modulation of sensory systems has been documented in many organisms and is likely to be essential for appropriate processing of information during different behavioral states. However, the mechanisms underlying these phenomena remain poorly characterized.

Results: We investigated the role of octopamine neurons in the flight-dependent modulation observed in visual interneurons in Drosophila. The vertical system (VS) cells exhibit a boost in their response to visual motion during flight compared to quiescence. Pharmacological application of octopamine evokes responses in quiescent flies that mimic those observed during flight, and octopamine cells that project to the optic lobes increase in activity during flight. Using genetic tools to manipulate the activity of octopamine neurons, we find that they are both necessary and sufficient for the flight-induced visual boost.

Conclusions: This study provides the first evidence that endogenous release of octopamine is involved in state-dependent modulation of visual interneurons in flies

Dickinson, Michael H.

Mamiya, Akira

Suver, Marie P.

SummaryBackgroundActivity-dependent modulation of sensory systems has been documented in many organisms and is likely to be essential for appropriate processing of information during different behavioral states. However, the mechanisms underlying these phenomena remain poorly characterized.ResultsWe investigated the role of octopamine neurons in the flight-dependent modulation observed in visual interneurons in Drosophila. The vertical system (VS) cells exhibit a boost in their response to visual motion during flight compared to quiescence. Pharmacological application of octopamine evokes responses in quiescent flies that mimic those observed during flight, and octopamine cells that project to the optic lobes increase in activity during flight. Using genetic tools to manipulate the activity of octopamine neurons, we find that they are both necessary and sufficient for the flight-induced visual boost.ConclusionsThis study provides the first evidence that endogenous release of octopamine is involved in state-dependent modulation of visual interneurons in flies

Suver, Marie P.

Dickinson, Michael H.

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Octopamine Neurons Mediate Flight-Induced Modulation of Visual Processing in Drosophila 

Caltech Authors - Main

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Current Biology, Volume 22 
Supplemental Information 
 
Octopamine Neurons Mediate 
Flight-Induced Modulation 
of Visual Processing in Drosophila 
 
Marie P. Suver, Akira Mamiya, and Michael H. Dickinson 
 
 
 
 
Supplemental Inventory  
 
Supplemental Figures 
Figure S1: Control for length/design of octopamine pharmacology experiment outlined in 
Figure 2. This figure supports that the effect we attribute to octopamine pharmacology in 
Figure 2 is not due to the sequence or length of the experiment. 
 
Figure S2: Shows effect of flight during octopamine application, related to Figure 2. 
Figure 2 outlines the effect of octopamine pharmacology, but the additional effect of 
flight shown here supports the notion that the baseline shift is not mediated by 
octopamine neurons, as described in the Discussion.  
 
Figure S3: Shows effect of flight during activation of octopamine neurons, related to 
Figure 4 and Figure S2. Figure 4 outlines the effect of octopamine neuron activation, 
and is relevant to our discussion of the origin of the baseline shift. 
 
Supplemental Experimental Procedures 
  
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Figure S1, Related to Figure 2. Saline Control for Octopamine Pharmacology  
(A) Average visual response to 8 Hz downward motion during saline application (green 
trace) and corresponding quiescent responses (‘quie’, black traces). The grey shaded 
region indicates when the visual stimulus was in motion. The average baseline 
membrane potential during the 1 s period immediately before motion onset is shown for 
quiescence and saline.  
(B) Temporal frequency tuning curve for downward motion responses during 
quiescence and saline application. Abscissa is plotted on a log scale.  
(C) Difference between motion responses during quiescence and saline application. 
Abscissa is plotted on a log scale. In B and C, NS indicates speeds at which the 
difference between saline and quiescent responses (computed for each fly) was not 
significantly greater than zero (paired Student’s t-test, alpha = 0.05).  
  
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Figure S2, Related to Figure 2. Effect of Flight during Octopamine Application  
(A) Average visual response to 8 Hz downward motion during quiescence (‘quie’, black 
traces), octopamine application (OA; green trace) and flight during octopamine 
application (OA flight; purple trace). The grey shaded region indicates when the visual 
stimulus was in motion. The average baseline membrane potential during the 1 s 
immediately before motion onset is shown for quiescence, OA and OA flight.  
(B) Temporal frequency tuning curve for downward motion responses during 
quiescence, octopamine application, and flight during octopamine application. Abscissa 
is plotted on a log scale.  
(C) Difference between motion responses during octopamine application and 
quiescence, and flight during octopamine application and quiescence. Abscissa is 
plotted on a log scale. In B and C, NS indicates speeds at which the difference between 
OA flight responses and OA responses (computed for each fly) was not significantly 
greater than zero (paired Student’s t-test, alpha = 0.05).  
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Figure S3, Related to Figure 4. Effect of Flight during Activation of Octopamine Neurons  
(A) Average visual response to 8 Hz downward motion before (19°C, black trace), 
during (28°C, red trace), during while flying (28°C flight, purple trace), and after (19°C 
post, grey trace) dTrpA1 channels were activated in octopamine cells (Tdc2-Gal4, UAS-
dTrpA1). Shaded light grey region indicates when the visual stimulus was in motion. 
Average baseline membrane potential during the 1 s immediately before motion onset is 
shown for each of these four conditions. 
(B) Temporal frequency tuning curve for downward motion responses before, during, 
during while flying, and after dTrpA1 activation.  
(C) Average response difference between during (28°C) and before (19°C) dTrpA1 
activation, and between during while flying (28°C flight) and before (19°C) dTrpA1 
activation. In B and C, NS indicates speeds at which the difference between 28°C flight 
responses and 28°C responses (computed for each fly) was not significantly greater 
than zero (paired Student’s t-test, alpha = 0.05). 
  
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SUPPLEMENTAL EXPERIMENTAL PROCEDURES 
 
Animals 
We used 1-3 day old female Drosophila melanogaster raised on standard cornmeal 
medium at 25°C with a 14:10 light/dark schedule. To encourage long flight bouts, we 
removed the pro- and meso-thoracic legs. 
 
Whole Cell Patch Clamp Recordings 
We used electrodes with resistance of 4.8-7.4 MΩ. Our intracellular, external, and 
collagenase solutions were identical to those used in Maimon, et al. [6]. We added 
20µM Alexa 568 (Invitrogen #A-10437) and 13mM biocytin (Invitrogen #B1093) to the 
intracellular solution for cell visualization. For 13 cells, we omitted biocytin and observed 
no obvious effect in the physiological responses. The average resting potential of cells 
after compensation for an experimentally-measured junction potential (-13mV) was -
46.4mV. We injected 20-30pA constant hyperpolarizing current into the cells prior to 
presentation of visual stimuli to aid with dye fills, which decreased the membrane 
potential by an average of 3.6mV (to -50.0 mV). The access resistance (Racc) for all 
recordings was 31.8 +/- 6.8 MΩ S.D., which is in the typical range for Drosophila whole-
cell patch clamp recordings [43]. Any cells with Racc greater than 50MΩ were excluded 
from our analysis.  
We controlled the temperature of the bath with a bipolar temperature controller 
and an in-line heater/cooler (CL-100 and SC-20, Warner Instruments). For all 
experiments with the exception of dTrpA1-activation and parental controls, we raised 
the bath temperature to 30°C during the initial desheathing step, and then lowered the 
bath to temperature 19°C for the remainder of the experiment. We performed the 
desheathing without any applied heat in all dTrpA1 activation and parental control 
experiments to avoid contaminating results with pre-exposure to heat. For these dTrpA1 
activation experiments, we held the external saline at 19°C, increased it to 28°C over a 
time course of approximately 120 seconds, and then lowered it back to 19°C. 
 
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Pharmacology 
We dissolved octopamine (DL-Octopamine hydrochloride, Fluka) in extracellular saline 
at a concentration of 100µM on the day of each experiment. For comparison, this 
concentration of octopamine, the lowest level at which VS cell responses were 
noticeably and reliably affected, lies at or near concentrations used in previous studies 
in locusts [45, 46], crustaceans [47], and crickets [48]. We modified the holder from 
Maimon, et al. [6] to more rapidly apply octopamine by aiming the perfusion input 
directly towards the exposed neuropil. The cells never fully recovered to pre-octopamine 
levels of activity during a washout of octopamine, so we do not present these 
responses. 
 
Immunohistochemistry 
We dissected brains in 4% paraformaldehyde in PBS and fixed for a total of 30 min. We 
then incubated them overnight at 4°C in a primary antibody solution containing 5% 
normal goat serum in PBS-Tx, mouse anti-nc82 (1:10, DSHB) and rabbit anti-GFP 
(1:1000, Invitrogen). Brains were then incubated overnight at 4°C in a secondary 
antibody solution containing 5% normal goat serum in PBS-Tx, goat anti-mouse Alexa 
Fluor 633 (1:250, Invitrogen) and goat anti-rabbit Alexa Fluor 488 (1:250, Invitrogen). 
We then mounted the brains in Vectashield and imaged them on a Leica SP5 II confocal 
microscope under 40x magnification and scanned at 1µm section intervals. We adjusted 
intensity and contrast for single channels for the entire image using ImageJ 1.45s.  
 
Calcium Imaging 
We imaged the brain using the Prairie Ultima IV two-photon excitation microscope 
controlled by Prairie View Acquisition software (Prairie Technologies). We used a mode 
locked Ti:Sapphire laser (Chameleon Ultra; Coherent) tuned to 930 nm as an excitation 
light source and adjusted the laser power to be 20 mW at the rear aperture of the 
objective lens (Nikon NIR Ap, 40x water-immersion lens, 0.8 NA). We collected 
fluorescence using a multi-alkali photomultiplier tube (Hamamatsu) after bandpass 
filtering it with an HQ525/70m-2p emission filter (Chroma Technologies). We acquired 
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images in a frame scan mode (152x150 pixels, 0.125 s/frame) to record activity of 
octopamine neurons. For each trial, we acquired images for 30 s, starting from 10 s 
before the flight onset. For each fly, we acquired a z-stack image (z step = 1 µm) 
covering the entire dendritic branch of the octopamine neurons near the esophagus 
foramen to confirm the location of each recording within the brain.  
 
Data Analysis and Statistics 
For whole cell patch clamp recordings, we acquired data at 10 kHz using Axoscope 
software. All data analyses were done using Matlab R2010b. We calculated peak visual 
responses by first down-sampling the data to 1 kHz. We then calculated a moving 
average of the membrane potential over a window of 10 points (10 ms) and selected the 
peak during the first cycle of stimulus motion.  
For two-photon imaging experiments, we applied a brief puff of air to the head of 
the fly to initiate flight, as in the electrophysiology experiments. If a fly was still flying 
after the end of two-photon image acquisition (approximately 20 s after the onset of 
flight), we terminated the flight by manually delivering a second puff of air. We waited 4 
min between initiations of flight in the same animal. Only flies that flew for at least five 
bouts lasting 12 s or more were included in the analysis. Throughout the experiment, we 
illuminated the fly from behind with a high-intensity infrared diode (880nm; Golden 
Dragon; Osram) and used a Basler A602f camera with a fixed-focus lens (Infinistix 90, 
94 mm working distance, 1.0x magnification) to record the behavior of the fly from below 
at 100 frames/s. We used FView [49], an open source program written in Python, to 
record images of flies simultaneously with a signal that indicates the timing of two-
photon image acquisition. We analyzed images using custom software written in Matlab 
2011b. We first smoothed the acquired images with a Gaussian filter (3 x 3 pixel, σ= 
0.5) and corrected for small movements of the brain in the x-y direction during the 
image acquisition using a previously published algorithm [50]. We then averaged the 
pixel intensity in the ROI to estimate the fluorescence from this region. For each trial, we 
reviewed the images of flight behavior and determined the flight onset time by finding 
the first frame after the application of air puff where a fly moves its wing forward. We 
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then used the simultaneously recorded signal that indicates the timing of two-photon 
image acquisition to find the frame in the calcium imaging that corresponds to flight 
onset time. We used average fluorescence during the five s period before the onset of 
the flight as baseline fluorescence (F0) and used this value to calculate the ΔF/F signal 
(defined as (F-F0)/F0). We calculated mean ΔF/F signal for each fly using 5 to 6 trials.  


Octopamine Neurons Mediate Flight-Induced Modulation of Visual Processing in Drosophila

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Octopamine Neurons Mediate Flight-Induced Modulation of Visual Processing in Drosophila

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