Poster annotation

Article

Coordinated and Compartmentalized

Neuromodulation Shapes Sensory Processing in Drosophila

Graphical Abstract

Highlights

d Mushroom body dopaminergic neurons act in concert to

represent contextual cues

d Dopamine bidirectionally modifies synapses in precise

domains along Kenyon cell axons

d Odor signals are differentially conveyed to each

postsynaptic target of a Kenyon cell

d Activity of output pathways depends on an animal’s external

context or internal state

Cohn et al., 2015, Cell 163, 1742–1755 December 17, 2015 ª2015 Elsevier Inc. http://dx.doi.org/10.1016/j.cell.2015.11.019

Authors

Raphael Cohn, Ianessa Morantte,

Vanessa Ruta

Correspondence [email protected]

In Brief

The fruit fly mushroom body functions like

a switchboard in which dopaminergic

inputs reroute the same odor signal to

different behavioral circuits, depending

on the internal state and experience of the

animal.

Article

Coordinated and Compartmentalized Neuromodulation Shapes Sensory Processing in Drosophila Raphael Cohn,1 Ianessa Morantte,1 and Vanessa Ruta1,* 1Laboratory of Neurophysiology and Behavior, The Rockefeller University, New York, NY 10065, USA

*Correspondence: [email protected]

http://dx.doi.org/10.1016/j.cell.2015.11.019

SUMMARY

Learned and adaptive behaviors rely on neural cir- cuits that flexibly couple the same sensory input to alternative output pathways. Here, we show that the Drosophila mushroom body functions like a switchboard in which neuromodulation reroutes the same odor signal to different behavioral circuits, de- pending on the state and experience of the fly. Using functional synaptic imaging and electrophysiology, we reveal that dopaminergic inputs to the mushroom body modulate synaptic transmission with exquisite spatial specificity, allowing individual neurons to differentially convey olfactory signals to each of their postsynaptic targets. Moreover, we show that the dopaminergic neurons function as an interconnected network, encoding information about both an ani- mal’s external context and internal state to coordi- nate synaptic plasticity throughout the mushroom body. Our data suggest a general circuit mechanism for behavioral flexibility in which neuromodulatory networks act with synaptic precision to transform a single sensory input into different patterns of output activity.

INTRODUCTION

Animals must constantly adapt their behavior to meet the de-

mands of their ever-changing external environment and internal

needs. Neuromodulators provide an evolutionarily conserved

mechanism to generate behavioral variability (Bargmann, 2012;

Marder, 2012). By rapidly regulating neuronal excitability and

the strength of synaptic connections between neurons, neuro-

modulators confer functional flexibility to anatomically invariant

circuits. Context- and state-dependent patterns of neuromodu-

lator release can thereby tune neural circuit properties to pro-

duce alternative responses to the same sensory stimulus.

Dopaminergic pathways have been investigated extensively

as part of a reinforcement system that motivates and modifies

many facets of animal behavior (Beninger, 1983; Bromberg-Mar-

tin et al., 2010; Redgrave and Gurney, 2006; Schultz et al., 1997;

Waddell, 2013; Wise, 2004). Dopamine acts through multiple re-

ceptors that couple to distinct intracellular signaling cascades,

enabling this single neuromodulator to have diverse effects on

synaptic function and communication (Tritsch and Sabatini,

1742 Cell 163, 1742–1755, December 17, 2015 ª2015 Elsevier Inc.

2012). Linking mechanisms of synaptic modulation to the gener-

ation of adaptive behaviors, however, requires a circuit-level un-

derstanding of how dopaminergic pathways encode the ongoing

experience of an animal and reinforce appropriate neural circuit

configurations. While mammalian midbrain dopaminergic neu-

rons are known to be important mediators of flexible circuit pro-

cessing, their anatomic and functional heterogeneity and the

intricate wiring of their target neuropils (Beier et al., 2015; Fiorillo

et al., 2013; Lammel et al., 2014; Lerner et al., 2015) have made it

difficult to resolve how they can selectively alter synaptic

signaling between different neural pathways. Moreover, the

dopamine they release has been suggested to act over long dis-

tances, by diffusing through the extracellular space, and at

select synaptic sites (Rice et al., 2011). Consequently, how

dopaminergic pathways sculpt synaptic connections to pre-

cisely shape circuit function remains unclear.

The insect mushroom body is an integrative brain center

whose orderly circuit architecture provides an opportunity to

examine how neuromodulators flexibly regulate the flow of sen-

sory information. In Drosophila, the mushroom body has been

best studied for its essential role in olfactory learning in which

past experience alters the subsequent behavioral response to

an odor (Heisenberg, 2003; Keene and Waddell, 2007). The

convergence of olfactory and dopaminergic reinforcement sig-

nals in the mushroom body also renders it ideally suited to shape

odor processing based on the acute needs of an animal. Indeed,

the mushroom body plays a role in the context-dependent pro-

cessing of odor signals, modulating innate olfactory preferences

in response to ongoing changes to a fly’s environment or internal

state (Lewis et al., 2015; Owald et al., 2015).

In the mushroom body, odors are encoded as sparse ensem-

bles of activated Kenyon cells (KCs) (Campbell et al., 2013), each

of which integrates input from diverse combinations of olfactory

glomeruli in the calyx (Caron et al., 2013; Gruntman and Turner,

2013). The �2,000 KCs propagate their odor responses along parallel axon fibers into the mushroom body’s output lobes.

Here we focus on g KCs, each of which projects a single axon

that traverses across the g lobe (Figures 1A and 1B). Each KC

axon synapses onto a small number of mushroom body output

neurons (MBONs), whose segregated dendrites tile the com-

plete length of the lobe to form five discrete anatomic compart-

ments (g1–g5) (Figures 1A and 1C). The MBONs, as the only

known efferent pathways of the mushroom body, must translate

KC odor representations into adaptive behavioral responses

(Aso et al., 2014a; Tanaka et al., 2008). MBON axons converge

on a small number of target neuropils, where their concerted ac-

tivity has been proposed to bias an animal’s innate and learned

A

B

C

D

E

Figure 1. Compartmentalized Architecture

of the Mushroom Body

(A) Schematic of mushroom body anatomy

focusing on the g lobe. Each g Kenyon cell (KC,

blue) receives olfactory input in the calyx and

projects a single axon into the g lobe (dashed line).

KCs form en passant synapses with mushroom

body output neurons (MBONs, green) and receive

modulatory input from dopaminergic neurons

(DANs, magenta) within discrete anatomic com-

partments (shown for g2–g5).

(B) A single g KC axon photolabeled with PA-GFP

projects across the complete length of the lobe

(dashed line).

(C) Segregated dendritic innervation of MBONs is

revealed by expression of GFP in pairs of MBONs

in each panel using MBON-specific drivers.

(D) Compartmentalized axonal projections of

DANs photolabeled with PA-GFP in alternating

compartments. PA-GFP is expressed under the TH

and DDC promoters.

(E) sytGCaMP expressed in all g KCs with only

a single KC functionally activated. Maximum in-

tensity projection shows peak fluorescence from

multiple T-series in different Z planes. Magnified

view shows individual KC synaptic puncta (right).

See also Figure S1.

olfactory preferences (Aso et al., 2014b; Hige et al., 2015; Owald

et al., 2015).

The mushroom body lobes are also richly innervated by dopa-

minergic neurons (DANs) thought to convey the contextual sig-

nals that impart meaning to an odor (Aso et al., 2012; Burke

et al., 2012; Claridge-Chang et al., 2009; Liu et al., 2012; Mao

and Davis, 2009; Waddell, 2013; Yamagata et al., 2015).

Rewarding and punishing experiences have been shown to acti-

vate distinct subsets of DANs, each of which projects axons into

just one of the lobe compartments (Figures 1A and 1D), mirroring

the segregated innervation pattern of the MBONs. This anatomic

arrangement suggests that DANs may convey positive and

negative contextual information to different synapses along a

KC axon, permitting differential tuning of neurotransmission to

each MBON under different circumstances.

Here we took advantage of the mushroom body’s anatomic

organization to elucidate how dopaminergic pathways instruct

synaptic and circuit plasticity. We developed a synaptic activity

reporter to visualize spatiotemporal patterns of modulation and

demonstrate that DANs modify synapses in discrete sub-cellular

Cell 163, 1742–1755, De

domains along the length of individual KC

axons. Moreover, we show that DANs

form part of a highly interconnected

network that coordinates synaptic plas-

ticity across the mushroom body in

response to both external contextual

cues and the fly’s internal state. Thus,

the concerted action of the dopaminergic

population functions with exquisite

spatial precision to regulate the flow of ol-

factory signals to each mushroom body

output pathway, providing a circuit mechanism to generate flex-

ible responses to an odor.

RESULTS

An Optical Sensor of Presynaptic Activity Presynaptic Ca2+ is a key regulator of neurotransmission and

effector of neuromodulation (Regehr, 2012), suggesting it should

provide a sensitive readout of synaptic function. We therefore

generated sytGCaMP, in which the genetically encoded Ca2+ in-

dicator GCaMP6s is tethered to the C terminus of the vesicular

synaptic protein synaptotagmin (Figure S1A). We initially

confirmed that sytGCaMP co-localizes with endogenous synap-

tic proteins and consequently detects Ca2+ influx specifically at

presynaptic terminals (Figures S1B–S1G). We then expressed

sytGCaMP in all g KCs using a selective promoter (Figure S1H)

and focally stimulated the calyx to activate an individual neuron

in a brain explant. We performed volumetric two-photon imaging

to capture the fluorescently tagged synapses of the KC’s full

axonal arbor as it ramified through multiple imaging planes within

cember 17, 2015 ª2015 Elsevier Inc. 1743

the g lobe. Stimulation of a single KC evoked robust fluores-

cence increases at punctate loci distributed along the length of

its axon (Figures 1E and S1I), consistent with sytGCaMP’s syn-

aptic localization and anatomic evidence that KCs form output

synapses in all compartments of the g lobe (Figures S1J and

S4B). Thus, sytGCaMP facilitates the detection of Ca2+ influx

at individual synaptic sites, providing a technical strategy to

resolve differences in presynaptic function and modulation

across the compartments of the lobe.

DANs Represent Context through Coordinated Patterns of Activity We next used the presynaptic localization of sytGCaMP to

monitor the activity of the DAN population and gain insight into

the patterns of dopamine release across the g lobe in different

contexts. We combined the tyrosine hydroxylase (TH) and

dopa-decarboxylase (DDC) promoters to drive expression of

sytGCaMP in the DANs targeting every compartment of the g

lobe. The axon terminals of DANs innervating the g2–g5 com-

partments could be monitored in a single optical imaging plane,

allowing us to simultaneously record their synaptic responses to

positive and negative reinforcement stimuli (Figure 2A).

Discrete subsets of DANs are sufficient to instruct learned

odor attraction or aversion, suggesting they may autonomously

encode the contextual signals that impart meaning to an olfac-

tory experience. However, while sugar feeding activated the g4

and g5 DANs, in accord with their established role in driving the

formation of appetitive olfactory associations (Liu et al., 2012;

Yamagata et al., 2015; Waddell, 2013), we observed that su-

crose ingestion also inhibited the g2 and g3 DANs (Figure 2B;

Movie S1; Table S1). Conversely, the g2 DAN has been shown

to respond to electric shock and contribute to aversive olfactory

conditioning (Aso et al., 2012; Claridge-Chang et al., 2009; Mao

and Davis, 2009). We confirmed that a brief electric shock

applied to the fly’s abdomen activated the g2 DAN, but found

that it also excited the g3 DANs and inhibited the g4 and g5

DANs (Figure 2C). Thus, the DANs of each compartment repre-

sent reinforcement stimuli through either excitation or inhibition,

analogous to the bidirectional signaling observed in mammalian

midbrain DANs in response to positive and negative cues

(Bromberg-Martin et al., 2010; Lerner et al., 2015). The recip-

rocal patterns of DAN activity evoked by these appetitive and

aversive stimuli suggest that mushroom body reinforcement

pathways may act in concert to regulate olfactory processing

through coordinated patterns of dopamine release across

compartments.

DAN activity fluctuated significantly even in the absence of

overt stimulation. Video monitoring of the fly during DAN imaging

revealed that these fluctuations are highly correlated with motor

output (Figure 2D; Movie S2). Tethered animals generally alter-

nated between two distinct behavioral states—quiescence and

rapid kicking or flailing that resembled escape behavior. Flailing

was strongly correlated with high g2/g3 and low g4/g5 DAN ac-

tivity, similar to the pattern evoked by electric shock. In contrast,

quiescence elicited the reciprocal pattern, resembling the DAN

response to sugar feeding (Figures 2E and S2A). Thus, different

behavioral states induce distinct bidirectional patterns of activity

across the DAN population. Interestingly, the strict correlations

1744 Cell 163, 1742–1755, December 17, 2015 ª2015 Elsevier Inc.

exhibited by DANs during tethered behavior were altered when

the same fly walked on a freely rotating ball (Figure S2B). For

example, during walking, g4 and g5 DANs were no longer strictly

synchronized and g4 DANs instead became transiently en-

trained to either g3 or g5 DAN activity. Odor stimuli, likewise,

disrupted the baseline correlations between DANs (Figure S2C).

These observations imply that the functional relationships be-

tween DANs are not absolute but rather an emergent property,

depending on both salient external sensory signals and a fly’s

internal state.

Functional Communication between Compartments Coordinates DAN Activity Recent anatomic data reveal that DANs and MBONs innervating

different compartments may be functionally interconnected via

overlapping projections in the protocerebrum (Aso et al.,

2014a). We therefore asked whether the correlated, partially

antagonistic DAN activity patterns we observed are shaped by

circuit interactions between compartments. We used the

58E02 promoter fragment (Liu et al., 2012) to selectively express

the ATP-gated P2X2 channel in a subset of DANs, including

those innervating the g4 and g5 compartments. Activation of

58E02+ DANs by application of ATP to their dendrites evoked

robust inhibition of the g2 DAN (Figure 3A). The g3 DANs were

also frequently inhibited but occasionally activated, due to vari-

able labeling of this compartment by the 58E02 driver. Therefore,

excitation of a subset of DANs is sufficient to suppress those tar-

geting other compartments, yielding a bidirectional pattern of

activity similar to that evoked by a sugar reward. Direct or indi-

rect communication between the DANs innervating different

compartments may, therefore, underlie their concerted repre-

sentation of reinforcement signaling.

To investigate whether feedback from MBONs contributes to

the functional coordination of DANs, we stimulated individual g

MBONs using sparse driver lines to express P2X2 and focally in-

jected ATP onto their axons. Activation of each g lobe MBON

triggered excitation or inhibition of the DANs in every compart-

ment (Figures 3B–3E), similar to the distributed activity patterns

evoked by physiological reinforcement experiences. The bidi-

rectional nature of DAN activity elicited by excitation of single

MBONs indicates that multisynaptic interactions functionally

link extrinsic neurons innervating different compartments.

Thus, MBONs and DANs comprise a complex interconnected

network, providing a potential substrate for the diverse func-

tional relationships between DANs that emerge in different

behavioral contexts (Figures 2 and S2). Together, these data

suggest that DANs do not act autonomously to convey the

valence of a reinforcement stimulus to just a single compart-

ment, but rather they function as a dynamic ensemble, inte-

grating information about environmental cues and internal state

to convey the moment-by-moment experience of the fly to all

compartments of the lobe.

Compartmentalized Synaptic Domains along KC Axons We next asked how coordinated patterns of DAN activity might

shape the flow of olfactory signals along KC axons as they tra-

verse through the different compartments of the lobe. We ex-

pressed sytGCaMP in g KCs (Figure 4A) and used volumetric

A

D E

B C

Figure 2. DAN Network Activity Reflects Both External Sensory Stimuli and Internal Behavioral State

(A) sytGCaMP was expressed in DANs of all g-lobe compartments, driven by the combination of TH and DDC promoters.

(B and C) Schematic of stimulus (top) with representative heatmap (DF/F0) and normalized intensity trace of DAN sytGCaMP response to the stimulus (B, sucrose;

C, shock) below. (Bottom) Stimulus-triggered averages ± SEM for DANs of each compartment are shown. (B, n = 10 traces in nine flies; C, n = 21 traces in 11 flies).

Fluorescence in other lobes is masked for clarity. Black scale bar indicates 1 s throughout figures unless otherwise noted.

(D) Representative normalized fluorescence traces of g lobe DANs aligned to fly’s motion (top). Dashed lines delineate start and end of a single

representative bout of flailing. Cross-correlations between motion trace and activity in DANs of each compartment are shown (bottom, n = 12 traces in

six flies).

(E) Schematic and still image from video showing the fly in flailing (right) and quiescent (left) behavioral states (top). Representative heatmap (DF/F0) of DAN

activity in response to start and stop of flailing (middle). Average DAN fluorescence ± SEM in each compartment aligned to the start and stop of flailing (bottom,

n = 14 traces in six flies).

See also Figure S2, Table S1, and Movies S1 and S2.

two-photon imaging to monitor their complement of synapses

within the g lobe in vivo. Unexpectedly, the distribution of

odor-evoked presynaptic Ca2+ along KC axons was highly

non-uniform and displayed a discrete modular organization in

each of the 12–18 imaging planes (Figures 4B, 4C, S3A, S3B,

and S4C; Movie S3). The asymmetry in presynaptic Ca2+ was

often apparent basally, prior to odor stimulation (Figure S3D),

suggesting that persistent differences in synaptic function along

KC axons could influence the processing of all incoming odor

stimuli. Consistent with this idea, the same modular pattern of

presynaptic Ca2+ was evoked in response to every odor tested

and over a range of concentrations (Figures S3E and S4H).

C

Alignment of sytGCaMP responses with the projections of

MBONs and DANs in the g lobe indicated that the discrete

Ca2+ domains apparent in KC axons map to the different com-

partments of the lobe (Figure S3F). To confirm this, we imaged

KC synaptic responses in animals that also expressed a red flu-

orophore in a subset of DANs and observed that the sharp bor-

ders separating regions of high and low synaptic Ca2+ aligned

with the compartmental boundaries (Figure 4E). Odor-evoked

synaptic responses in KCs were significantly more robust in

the g2 and g3 compartments relative to those in the g4 compart-

ment, with even weaker responses evident in the g5 compart-

ment (Figure 4C). Thus, the distribution of presynaptic Ca2+

ell 163, 1742–1755, December 17, 2015 ª2015 Elsevier Inc. 1745

A

B

C

D

E

Figure 3. Functional Communication between Compartments

Coordinates DAN Network Activity

(A–E) DAN sytGCaMP activity patterns evoked by activation of P2X2 ex-

pressed in the (A) 58E02+ DANs innervating g4-5, (B) g2 MBON, (C) g3 MBON,

(D) g4 MBON, and (E) g5 MBON. sytGCaMP was expressed in DANs of all g-

lobe compartments using the TH and DDC promoters. Schematic of stimulus

(top left), representative heatmap (bottom left, DF/F0), normalized intensity

trace for representative experiment shown (top right), and stimulus-triggered

averages ± SEM for DANs of each compartment (bottom right) are shown. ATP

stimulation is shown as pink bar (58E02, n = 8; g2, n = 8; g3, n = 8; g4, n = 8; g5,

n = 12).

See also Table S1.

1746 Cell 163, 1742–1755, December 17, 2015 ª2015 Elsevier Inc.

along KC axons adheres to the modular architecture of the

lobes, demonstrating that the anatomic compartments repre-

sent functionally distinct units. Moreover, KC classes innervating

other lobes also exhibited modular sytGCaMP signals (Fig-

ure S3G), indicating that compartmentalized synaptic Ca2+ is a

general feature of odor representations in the mushroom body

lobes.

Asymmetric presynaptic Ca2+ domains could arise from differ-

ences in KC innervation along the g lobe or from functional vari-

ation along individual KC axons. Single-cell labeling of >80 g

KCs verified they invariantly traverse the entire lobe (Figure S4A)

and are poised to carry the same odor signals to each compart-

ment. However, functional synaptic heterogeneity was evident

along sparsely labeled KC axons (Figures 4F and S3C) co-ex-

pressing sytGCaMP and a red fluorophore to delineate their

anatomic projections. Synaptic boutons decorating the same

KC axons exhibited differential responses to odor, with more

robust activity evoked in the individual synapses in the g2 and

g3 compartments relative to those in the g4 and g5 compart-

ments. Although we did not routinely image the g1 compartment,

presynaptic Ca2+ was often lower there in comparison to more

distal portions of the lobe (Figure 4C), indicating it is unlikely

that action potentials simply fail to propagate the length of KC

axons. Together, these data suggest that synapses along indi-

vidual KC axons are functionally diverse, such that the same

olfactory signal is differentially represented by each axonal

segment of a neuron.

Dopamine Modulates Synapses along KC Axons The asymmetry of KC synaptic responses points to the possibil-

ity that active modulation by the DANs that tile the g lobe may

regulate synaptic signaling within each compartment. Interest-

ingly, while DAN activity patterns rapidly fluctuated in tethered

animals, the high g2/g3 and low g4/g5 DAN network state

significantly predominated (Figure S2D), potentially reflecting

a fly’s frequent struggle to escape. The similarity between this

DAN network state (Figure 2E) and the pattern of KC presynap-

tic Ca2+ we consistently observed (high g2/g3 and low g4/g5,

Figure 4C) led us to ask if the behavioral state of the fly can

directly modulate olfactory signaling along the lobe. We there-

fore examined KC responses in a brain explant where basal

fluctuations in DAN activity were greatly reduced (data not

shown) and found that direct activation of KCs evoked a uniform

sytGCaMP signal across the length of the lobe in this ex vivo

preparation (Figures 4D, S4D, and S4E). In contrast, direct

KC stimulation in vivo elicited a modular response pattern

A

C D

F

B E

Figure 4. Compartmentalized Ca2+ Domains along KC Axons In Vivo (A) Schematic (top) and representative basal fluorescence of sytGCaMP expressed in g KCs labeled with approximate compartmental borders

(bottom).

(B) Volumetric two-photon resonant imaging of odor-evoked sytGCaMP reveals asymmetric presynaptic Ca2+ in g KCs in each imaging plane.

(C) Maximum-intensity Z-projection of all 15 imaging planes sampled through the g lobe in the example shown in (B) (top). Average normalized odor-evoked profile

of sytGCaMP fluorescence intensity along the g lobe (gray line, n = 21 flies) and peak intensity for each compartment (black dots, n = 21) with mean ± SEM in red

(middle). Odor-evoked time courses were imaged in each compartment for representative experiment shown above (bottom, blue lines indicate 1-s odor stimulus).

(D) Representative image of sytGCaMP signal in g KCs in response to direct stimulation of KCs by acetylcholine iontophoresis into the mushroom body calyx in a

brain explant (top). Normalized intensity profiles for ex vivo stimulation across a range of iontophoretic voltages (1–10 V) with average profile for each voltage in a

different colored line (n = 6). Stimulation-evoked time courses were imaged in each compartment for representative experiment shown above (bottom, blue lines

indicate stimulation).

(E) tdTomato expressed in g4 and g5 DANs using 58E02-LexA (top, middle). Compartmentalized KC sytGCaMP responses in the same fly shows synaptic Ca2+

domains have sharp boundaries that align to the border between g3 and g4 compartments.

(F) Odor response in a sparse subset of g KCs expressing sytGCaMP (heatmap, top) and tdTomato (grayscale, middle). Odor-evoked time courses were

measured at individual synaptic boutons (bottom).

All KC heatmaps in this figure represent peak fluorescence. Values marked with different lowercase letters represent significant differences (p < 0.05 by t test with

correction for multiple comparisons).

See also Figures S3 and S4 and Movie S3.

(Figures S4F and S4G) resembling that triggered by odor. We

therefore conclude that, while KC axons have the anatomic po-

tential to transmit equivalent neural signals to all compartments

of the g lobe, in vivo modulation induces functional heterogene-

ity along their length.

We next asked whether acute alterations to the state or cir-

cumstances of an animal can modify the pattern of presynaptic

Ca2+ along KCs. Given that sucrose ingestion elicits the recip-

rocal pattern of DAN activity (high g4/g5 and low g2/g3, Fig-

ure 2B) as that associated with flailing behavior, we reasoned

this appetitive reward might alter the distribution of presynaptic

Ca2+ across lobe compartments. Overnight fasting did not

change the profile of presynaptic Ca2+ along g KCs (Figure S3E).

However, after sugar feeding the odor-evoked synaptic re-

C

sponses in the g4 and g5 compartments relatively increased,

while the responses in the g2 compartment relatively decreased

(Figure 5A). Sucrose ingestion, therefore, differentially modu-

lates the olfactory responses of KC synapses along the g lobe,

paralleling the bidirectional pattern of DAN activity evoked by

this appetitive reward.

To confirm that DAN activation is sufficient to modify odor-

evoked synaptic responses in KCs, we used the 58E02 promoter

to drive expression of P2X2 in the g4 and g5 DANs excited by

sugar feeding (Figure 2B). Stimulation of 58E02+ DANs with

ATP altered the profile of odor-evoked Ca2+ along g KC axons,

relatively increasing the signal in the distal lobe compartments

while decreasing it in the proximal compartments (Figures 5B

and 5C), resembling the changes induced by sucrose ingestion.

ell 163, 1742–1755, December 17, 2015 ª2015 Elsevier Inc. 1747

A

B

E

C

D Figure 5. Dopaminergic Signaling Shapes the Distribution of KC Presynaptic Ca2+

(A) Representative odor-evoked KC sytGCaMP

response before and after sucrose ingestion (bot-

tom left). Normalized intensity profiles pre- and

post-sugar ingestion and the change due to sugar

feeding (post-pre) for the representative images

are shown (top right). Average change in normal-

ized intensity profile induced by sugar ingestion

(bottom right, n = 11 flies).

(B) Schematic of g lobe P2X2 expression under

the 58E02 promoter (top left) and representative

odor-evoked responses in g KCs expressing

sytGCaMP, pre- and post-activation of 58E02+

DANs with ATP (bottom left). Normalized intensity

profiles and change due to DAN activation for the

representative images (top right). Average change

in normalized intensity profile induced by DAN

activation (bottom right, n = 10 flies).

(C) As in (B), but in control flies lacking P2X2 expression (n = 6 flies).

(D) Representative odor-evoked response of g

KCs expressing sytGCaMP in DopR2 mutant and

wild-type flies (top). Fluorescence in other lobes is

masked for clarity. Average normalized odor-

evoked profile across the g lobe and compart-

mental averages (bottom) in flies mutant for DopR2

(red, n = 8) and wild-type (black, n = 8) are shown.

(E) As in (D), but comparing g KC-specific knock-

down of DopR2 using RNAi (red, n = 14) to wild-

type flies (black, n = 5).

All KC heatmaps in this figure represent peak

fluorescence to odor stimulation. Error bars in all

panels are SEM. Significant differences in relative

compartment intensity compared to wild-type are

indicated as follows: *p < 0.05, **p < 0.005, and

***p < 0.0005.

See also Figure S5.

Thus, both exogenous and physiological activation of DAN rein-

forcement pathways can modulate KC synapses with striking

spatial precision. Changes in odor-evoked presynaptic Ca2+

persisted for the duration of an experiment (up to �1 hr, Fig- ure S5A), indicating that intensely salient experiences, like teth-

ering or sugar ingestion, can alter the state of KC synapses

with enduring consequences for how all subsequent olfactory

signals are processed.

Dopamine receptors DopR1 and DopR2 are both highly ex-

pressed within the mushroom body lobes and comprise essen-

tial molecular pathways regulating the formation and mainte-

nance of learned olfactory associations (Berry et al., 2012; Kim

et al., 2007; Qin et al., 2012). To verify that dopaminergic

signaling directly contributes to compartmentalized patterns of

synaptic modulation along KCs, we examined mutants for these

receptors and found that the profile of odor-evoked sytGCaMP

fluorescence was inverted in DopR2 mutants (Figure 5D). Selec-

tive knockdown of DopR2 in g KCs using RNAi similarly altered

1748 Cell 163, 1742–1755, December 17, 2015 ª2015 Elsevier Inc.

the pattern of synaptic Ca2+ along KC axons (Figures 5E and

S5B), demonstrating that dopamine acts presynaptically to

shape odor processing.

DopR1 mutants exhibited a subtler phenotype (Figure S5C),

while the distribution of synaptic Ca2+ in DopR1/DopR2 double

mutants was still asymmetric (Figure S5D), implying that addi-

tional dopamine receptors or neuromodulatory pathways (Aso

et al., 2014a; Tanaka et al., 2008) may influence the patterning

of KC presynaptic Ca2+. We therefore examined synaptic re-

sponses in mutants for the dopamine reuptake transporter

(DAT), which mediates clearance of dopamine from the synaptic

cleft (Kume et al., 2005) and regulates dopamine signaling inde-

pendent of any specific receptor. The profile of odor-evoked pre-

synaptic Ca2+ in DAT mutants was significantly altered, resem-

bling the phenotype of the DopR1/DopR2 mutant (Figure S5E).

These manipulations of dopamine detection and handling

confirm that dopaminergic signaling, and not simply DAN activ-

ity, contributes to the precise spatial topography of presynaptic

A

B C

D E

Figure 6. DANs Selectively Potentiate KC-

MBON Synaptic Transmission in Individual

Compartments

(A) Schematic of experimental setup. Synaptic cur-

rents were measured in the g4 MBON (green) by

voltage-clamp recordings in response to direct KC

stimulation by acetylcholine iontophoresis in the calyx

(Stim). P2X2-expressing 58E02+ DANs (magenta)

were activated by local ATP injection (left). Repre-

sentative g4 MBON recordings (center) show overlay

of ten KC stimulations pre- (grayscale) and post-

(redscale) activation of 58E02+ DANs by ATP injec-

tion. Note the potentiation evident in both sponta-

neous and evoked EPSCs. Vertical line denotes 2-ms

KC stimulation. Amplitude of evoked currents in the g4

MBON pre- and post-ATP injection (right, average of

ten stimulations each in n = 5 recordings).

(B–E) MBON responses to KC stimulation are poten-

tiated by activation of DANs within the same

compartment, but not in other compartments. Sche-

matic (top), time courses (bottom left), and quantifi-

cation of responses to KC stimulation (bottom right)

before and after ATP injection were recorded in (B) the

g4 MBON with activation of the g4-g5 (58E02+) DANs

(n = 6), (C) the g2 MBON with activation of the g2 DAN

(n = 6), (D) the g2 MBON with activation of the g4-5

DANs (n = 6), and (E) the g4 MBON with activation of

the g2 DAN (n = 6). All pairwise comparisons plot

mean ± SEM. Significance of change after activation

is indicated as follows: *p < 0.05 and **p < 0.005.

See also Figure S6.

Ca2+ along KC axons, providing a functional link between molec-

ular and neural mechanisms.

Dopaminergic Modulation of KC-MBON Neurotransmission Together, our experiments indicate that dopaminergic modula-

tion can acutely modify synaptic responses in discrete subcellu-

lar domains along individual KC axons. If this presynaptic mod-

ulation resulted in altered neurotransmission to the MBONs, our

data would suggest that the state of the DAN network could

Cell 163, 1742–1755,

dynamically regulate the flow of olfactory in-

formation to each output pathway. We

therefore assessed how DAN activity mod-

ifies KC-MBON signaling using electrophys-

iology to monitor neurotransmission at the

resolution of individual synaptic events.

We targeted the g4 MBON for voltage-

clamp recordings as it innervates the

compartment exhibiting the most robust

dopamine-dependent modulation of KC

presynaptic Ca2+ (Figure 5B). Recordings

were performed in a brain explant where

reduced basal activity allowed for the mea-

surement of well-isolated synaptic currents

and provided precise control over the neu-

romodulatory state of synapses. We stimu-

lated KC dendrites in the calyx to evoke

excitatory postsynaptic currents (EPSCs)

in the g4 MBON and observed that the strength of these synap-

tic inputs markedly increased following 58E02+ DAN activation

(Figure 6A). Spontaneous synaptic events were also potenti-

ated whether DANs were activated using P2X2 or a red-shifted

channelrhodopsin (Figure S6A). The average latency of EPSCs

after KC stimulation was 3.8 ± 0.1 ms, consistent with mono-

synaptic transmission (Kazama and Wilson, 2008), identifying

KC-MBON synapses as the site of dopaminergic modulation.

Focal application of the inhibitory neurotransmitter GABA

onto KC dendrites resulted in the loss of synaptic events,

December 17, 2015 ª2015 Elsevier Inc. 1749

further substantiating KCs as the source of this potentiated syn-

aptic input (Figure S6A). In contrast to the prominent modula-

tion of synaptic currents, activation of 58E02+ DANs had no

apparent effect on the baseline membrane voltage or evoked

spiking of g KCs (Figure S6B). Dopaminergic modulation, there-

fore, potentiates neurotransmission at KC-MBON synapses

without appearing to change the intrinsic excitability of KCs,

providing a mechanism to alter the propagation of olfactory sig-

nals to each MBON without modifying the underlying KC odor

representation.

Compartmental Specificity of Dopaminergic Modulation Dopamine can act diffusely, regulating circuit properties at a dis-

tance from its site of release (Rice et al., 2011). We therefore

asked whether DANs modulate synaptic signaling only in the

compartments they innervate or more broadly along the lobe.

Functional imaging revealed that the response of the g4 MBON

to direct KC stimulation was enhanced after 58E02+ DAN activa-

tion (Figures 6B and S6C), consistent with the potentiation

measured by electrophysiology. In contrast, activation of the

g4 DAN had no effect on the response of the g2 MBON (Fig-

ure 6D). Likewise, activation of the g2 DAN strengthened the

g2 MBON response to KC stimulation (Figures 6C and S6C),

but resulted in a small but significant depression of activity in

the g4 MBON (Figure 6E). Together, these experiments indicate

that the segregated axonal innervation by DANs permits spatially

restricted potentiation of KC-MBON neurotransmission, local-

ized to the synapses within a compartment.

State-Dependent Changes in MBON Activity Patterns Heterogeneous neurotransmission from the synapses along a

single axon has been described in the cortex and hippocampus

as a possible substrate for independent plasticity between a

neuron and its myriad of postsynaptic targets (Markram et al.,

1998; Pelkey and McBain, 2007). However, rarely has it been

possible to trace the propagation of neural signals from nearby

synapses on the same axons to distinct postsynaptic pathways.

We took advantage of the compartmentalized architecture of the

mushroom body to examine whether the localized synaptic

modulation along KC axons results in differential olfactory re-

sponses across the MBONs that tile the lobe. We expressed

GCaMP6s in pairs of g MBONs (g2/g4 or g3/g5, Figure 7A)

and simultaneously measured dendritic Ca2+ responses to

odor stimuli in their segregated projections. Every odor evoked

significantly more robust responses in the g2 and g3 MBONs

compared to the g4 and g5 MBONs (Figure 7B), paralleling the

compartmental differences exhibited by KCs to olfactory stimuli

(Figure 4C). In contrast, direct stimulation of KCs in a brain

explant elicited comparable responses across MBONs (Fig-

ure 7C), confirming that, in the absence of in vivo modulation,

KCs have the inherent capacity to transmit equivalent signals

to the different output pathways of the lobe (Figures 4D, S4D,

and S4E).

Together, these observations indicate the odor responses of

MBONs are differentially tuned by the activity of their cognate

DANs, allowing contextual cues to shape olfactory signaling

through the parallel outputs of the lobe. In support of this conclu-

sion, we found that exogenous activation of the g4 DANs via

1750 Cell 163, 1742–1755, December 17, 2015 ª2015 Elsevier Inc.

P2X2 resulted in robust potentiation of the g4 MBON responses

to all odors tested (Figures 7D, S7A, and S7B), while olfactory re-

sponses in the g2 MBON were unaffected (Figure 7E). Similarly,

sugar feeding, an appetitive stimulus that activates the g4/g5

DANs and inhibits the g2/g3 DANs (Figure 2B), enhanced the

g4 MBON odor response relative to the g2 MBON response (Fig-

ure 7F). Thus, acute changes to the state of an animal can rapidly

gate the transmission of olfactory signals to the MBONs of the

lobe, permitting the same odor stimulus to drive distinct patterns

of output activity in different contexts.

DAN Activity Bidirectionally Modulates KC-MBON Signaling Dopamine can modulate synaptic communication in diverse

ways—including potentiation or depression of neurotransmis-

sion and modifications to both short- and long-term plasticity

(Tritsch and Sabatini, 2012). Our data indicate that DAN activa-

tion by salient reinforcement experiences can modify the state

of KC synapses with enduring consequences to how all subse-

quent odor signals are processed. In contrast, during associative

learning, the contingent pairing of olfactory and dopaminergic

reinforcement pathways is thought to alter neurotransmission

from odor-specific KC ensembles to allow formation of select ol-

factory memories (Heisenberg, 2003; Keene and Waddell, 2007).

We therefore asked whether coincident activation of KCs and

DANs elicits a distinct form of synaptic modulation in compari-

son to when DANs are activated independently. Remarkably,

temporally pairing 58E02+ DAN activation with KC stimulation

resulted in depression of KC-evoked g4 MBON responses, in

contrast to the robust potentiation induced by activation of

DANs alone (Figures 7G–7I). Interleaving temporally paired and

unpaired stimulation protocols produced depression and poten-

tiation within the same preparation, indicating that KC-MBON

synapses are capable of rapid and reversible bidirectional plas-

ticity (Figures 7H, S7C, and S7I). Depression of KC-MBON

signaling was restricted to the compartment innervated by the

activated DANs, suggesting a similar spatial specificity for these

opposing forms of dopaminergic modulation (Figure S7J).

If depression of KC-MBON signaling were limited to only KCs

activated during an olfactory experience, our observations

would provide a mechanistic basis for the odor-specific modula-

tion thought to underlie learned olfactory associations within the

mushroom body. We therefore monitored the responses of the

g4 MBON to two different odors and then paired the presentation

of one odor with 58E02+ DAN stimulation. Following DAN activa-

tion, the MBON’s response to the paired odor was significantly

reduced relative to the unpaired odor (Figure 7J). Thus, DAN ac-

tivity can bidirectionally modulate KC-MBON signaling, allowing

for both odor-independent synaptic potentiation as well as odor-

specific depression.

DISCUSSION

In this study, we took advantage of the mushroom body’s orderly

architecture to gain insight into the circuit mechanisms through

which neuromodulation mediates flexible sensory processing.

Compartmentalized dopaminergic signaling permits indepen-

dent tuning of synaptic transmission between an individual KC

A

D

G

H I

J

E F

B C

Figure 7. State-Dependent and Bidirectional Modulation of KC-MBON Signaling

(A) Schematic shows pairs of MBONs expressing soluble GCaMP6s used for functional imaging in (B) and (C).

(B and C) Representative heatmaps of evoked fluorescence (top left in each panel, DF/F0), time courses (bottom left), and scatterplots (right) of responses to odor

stimuli (blue line) in pairs of MBONs in vivo (B, n = 8 for g2 versus g4, n = 11 for g3 versus g5) and evoked by calycal stimulation in a brain explant (C, n = 8 for each

pair). Values marked with different lowercase letters represent significant differences (p < 0.05 by t test with correction for multiple comparisons).

(legend continued on next page)

Cell 163, 1742–1755, December 17, 2015 ª2015 Elsevier Inc. 1751

and its repertoire of postsynaptic MBON targets. As a conse-

quence, the same KC odor representation can evoke different

patterns of output activity, depending on the state of the animal

and the dopaminergic network. Recent data indicate that the

ensemble of MBONs acts in concert to bias an animal’s behav-

ioral response to an odor such that altering the balance of their

activity can modify the olfactory preferences of both naive and

trained animals (Aso et al., 2014a; Lewis et al., 2015; Owald

et al., 2015). In accord with such a model, we reveal how a

distributed neuromodulatory network is poised to orchestrate

plasticity across all 15 compartments of the mushroom body

and reweight the net output of the MBONs, allowing for adaptive

behavioral responses based on the immediate needs or past

experience of the animal.

A Dynamic Neuromodulatory Network Distinct subsets of DANs are sufficient to drive learned olfactory

associations (Aso et al., 2012; Claridge-Chang et al., 2009; Liu

et al., 2012; Waddell, 2013; Yamagata et al., 2015), leading to

the suggestion they may act autonomously to encode the

rewarding or punishing contextual stimuli that assign meaning

to an odor. Our data, however, suggest a more complex circuit

architecture, in which rich functional interconnectivity between

compartments contributes to coordinated and bidirectional pat-

terns of activity across the DAN population. This raises the pos-

sibility that reinforcement experiences may be represented

by combinatorial patterns of DAN excitation and inhibition in

different compartments, endowing the dopaminergic population

with a greater capacity to instruct behavior via the limited reper-

toire of mushroom body outputs. Intriguingly, midbrain dopami-

nergic neurons responsive to punishment and reward also proj-

ect to distinct targets in the mammalian brain and display a

similar functional opponency as a consequence of reciprocal

network interactions (Cohen et al., 2012; Lammel et al., 2012,

2014; Lerner et al., 2015). Thus, the concerted and partially

antagonistic action of neuromodulatory pathways may represent

a general and conserved circuit principle for generating adaptive

behavioral responses.

Distinct DAN network activity states are evoked by electric

shock and sugar ingestion, reinforcers classically used in asso-

ciative olfactory conditioning paradigms because of their strong

inherent valence. However, similarly distributed patterns of

DAN activity are correlated with the fly’s motor output, implying

that an animal’s behavioral state might serve as a reinforce-

ment stimulus that itself drives synaptic plasticity to shape

(D and E) MBON olfactory responses are potentiated by activation of DANs within

quantification of g4 MBON odor responses before and after stimulation of the

activation of the g4-5 DANs was quantified (n = 6).

(F) Ratio between odor-evoked responses in the g4 MBON and g2 MBON before

(G) Schematic (left) and experimental design (right) for (H)–(J). The g4 MBON respo

and after 58E02+ DAN activation that was either temporally paired or unpaired w

(H) Representative g4 MBON GCaMP responses to KC stimulation showing bid

depending upon whether DAN and KC activation were temporally paired or unpa

(I) Changes in g4 MBON responses to KC stimulation following activation of 58E0

unpaired (right, n = 12, starting from a depressed state) with KC stimulation (see

(J) Change in g4 MBON response to an odor that was paired with 58E02+ DAN

All pairwise comparisons in this figure represent the mean (±SEM) with significan

See also Figure S7.

1752 Cell 163, 1742–1755, December 17, 2015 ª2015 Elsevier Inc.

odor processing. Metabolic states, such as thirst and hunger,

have been shown to gate appetitive reinforcement by water

and sugar rewards (Burke et al., 2012; Huetteroth et al.,

2015; Lin et al., 2014), permitting state-dependent formation

of olfactory associations only in motivated animals. Our data

highlight an additional facet of how an animal’s internal state

can regulate dopamine release to adjust the salience of contex-

tual cues. Together, these observations indicate that the

distributed DAN network integrates information about external

context and internal state with MBON feedback to represent

the moment-by-moment experience of an animal and dynami-

cally regulate the flow of olfactory signals through the mush-

room body.

Spatially Precise Synaptic Modulation The independent regulation of synapses along an axon is thought

to permit a single neuron to convey specialized information to

different downstream targets, providing additional flexibility and

computational power to neural circuits (Markram et al., 1998; Pel-

key and McBain, 2007). In the mushroom body, synapse-specific

plasticity is achieved through spatially restricted patterns of

dopaminergic modulation that divide a KC axon into functionally

distinct segments. Thus, the ensemble of synapses within a

compartment, as the site of convergence for sensory and contex-

tual signals, represents the elementary functional unit that under-

lies experience-dependent mushroom body output.

Within a compartment, multiple neuromodulatory mecha-

nisms appear to shape synaptic signaling. We observed broad

potentiation of KC-MBON synapses after DAN activation but

odor-specific depression if DANs were coincidently activated

with KCs, consistent with the synaptic changes previously pro-

posed to occur after learning (Aso et al., 2014b; Owald et al.,

2015; Séjourné et al., 2011). Taken together, these findings indi-

cate that neuromodulation in the mushroom body instructs

opposing forms of synaptic plasticity, analogous to the bidirec-

tional tuning of synaptic strength by dopamine in mammalian

brain centers (Huang et al., 2004; Shen et al., 2008; Tritsch and

Sabatini, 2012). The molecular mechanisms through which

dopamine can direct diverse synaptic changes within a compart-

ment remain to be elucidated, but they may depend on signaling

through different dopamine receptors or downstream signaling

cascades that function as coincidence detectors. Indeed, while

DopR1 in KCs is essential to the formation of learned olfactory

associations (Kim et al., 2007; Qin et al., 2012), we find this re-

ceptor plays only a subtle role in the context-dependent

the same compartment, but not in other compartments. (D) Schematic (left) and

g4-5 (58E02+) DANs (n = 6, right). (E) As in (D), but g2 MBON response with

and after sugar feeding (n = 10).

nses to direct KC stimulation (in H–I) or odor stimuli (in J) were recorded before

ith KC stimulation. Dashed lines here and below represent >45-s delays.

irectional modulation of KC-MBON signaling by activation of 58E02+ DANs

ired. Blue lines indicate time of KC stimulation.

2+ DANs that was either paired (left, n = 6, starting from a potentiated state) or

Figure S7).

activation using P2X2 relative to a second odor that was unpaired (n = 10).

t changes indicated as follows: *p < 0.05, **p < 0.005, and ***p < 0.0005.

patterning of Ca2+ along their axons. Conversely, DopR2

strongly influences the topography of presynaptic Ca2+ along

KC axons, in accord with evidence that tonic release of dopa-

mine during ongoing behavior acts through this receptor to inter-

fere with the maintenance of specific learned olfactory associa-

tions (Berry et al., 2012, 2015). Thus, distinct molecular

pathways may transform the same dopaminergic reinforcement

signals into synaptic changes of opposite polarity to shape olfac-

tory processing based on both the present context and prior ex-

periences of an individual.

A Common Integrative Circuit Architecture for Adaptive Responses The mushroom body has been most extensively studied as a site

for associative learning (Heisenberg, 2003; Keene and Waddell,

2007) in which the temporal pairing of an odor with a reinforce-

ment experience selectively alters subsequent behavioral re-

sponses to that odor. Our data suggest that the convergence

of DAN network activity and KC olfactory representations within

the mushroom body lobes may drive associative plasticity in

each compartment, allowing the odor tuning of the MBON reper-

toire to reflect the unique experiences of an individual (Hige et al.,

2015). However, our observations also provide insight into the

mushroom body’s broader role in the context-dependent regula-

tion of innate behaviors (Lewis et al., 2015; Owald et al., 2015).

The ongoing activity of the distributed DAN network, encoding

information about a fly’s current environmental context and

behavioral state, is poised to continuously reconfigure the

activity patterns of the MBON population to allow for adaptive

responses based on the acute needs of the animal. This

context-dependent synaptic modulation could potentially erode

odor-specific learned associations within the mushroom body,

permitting the immediate circumstances of an animal to domi-

nate over previously learned olfactory associations that may no

longer be predictive or relevant. The axons of MBONs ultimately

converge with output pathways from the lateral horn (Aso et al.,

2014a, 2014b), a Drosophila brain center thought to mediate ste-

reotyped responses to odors, providing a potential substrate for

learned and context-dependent output from the mushroom

body to influence inherent olfactory preferences.

Thus, the dual role of neuromodulation in the mushroom

body—to select among alternative circuit states that regulate

both innate and learned behaviors—is reminiscent of its function

in other higher integrative brain centers. In the basal ganglia, for

example, different temporal patterns of dopamine release are

thought to select the relevant circuit configurations that control

inherently motivated behaviors as well as reinforcement learning

(Graybiel et al., 1994; Grillner et al., 2005; Kreitzer and Malenka,

2008; Yin and Knowlton, 2006). The generation of flexible behav-

ioral responses based on experience, whether past or present,

may therefore rely on common integrative brain structures in

which neuromodulatory networks act with exquisite spatial pre-

cision to shape sensory processing.

EXPERIMENTAL PROCEDURES

Detailed methods associated with all procedures below are available in the

Supplemental Experimental Procedures.

C

Fly Stocks

A detailed list of fly genotypes can be found in the Supplemental Experimental

Procedures. sytGCaMP was generated by linking the GCaMP6s and

Drosophila synaptotagmin 1 coding sequences through a 33GS linker. The

resulting construct (sytGCaMP) was used to generate transgenic flies by

PhiC31-based integration into attp40, attp5, and VK00005 (BestGene).

Functional Imaging

All functional imaging experiments were performed on an Ultima two-photon

laser-scanning microscope (Bruker Nanosystems) as previously described

(Ruta et al., 2010). For volumetric imaging, the laser was directed through an

8-kHz resonant scanning galvonometer and the objective was controlled by

a piezo-electric Z-focus. Z planes (12–18 planes), spaced �2 mm apart, were defined to encompass the entire volume of the g lobe and imaged at

�1.5 Hz. Odor stimulation was achieved by directing a continuous stream (400–500 ml/min) of air through a 2-mm-diameter teflon tube at the fly. At a

trigger, 5%–10% of the total airstream was diverted from a 10-ml glass vial

containing paraffin oil to to a vial containing odorants diluted in paraffin oil.

Electrophysiology

g4 MBON and KC soma were targeted for patch recording using GCaMP or

GFP fluorescence. Recordings were carried out as previously described

(Ruta et al., 2010). The membrane potential during voltage clamp was nomi-

nally �70 mV. At this voltage, unclamped action potentials rarely broke through and were readily detected by their large amplitude.

Exogenous Activation of KCs and DANs

Glass stimulating electrodes (resistance of 7–10 MU) were filled with 2 mM ATP

or 10 mM acetylcholine. To stimulate DANs and MBONs expressing the P2X2 receptor, electrodes were positioned dorsal to the mushroom body’s medial

lobes at the site of DAN dendritic and MBON axonal innervation and ATP

was applied by a positive pressure pulse. KCs were stimulated by iontophor-

esing acetylcholine into the calyx using voltage pulses. For paired trials, KC

and DAN stimulation were carried out within <500 ms of each other. For

unpaired trials, KC and DAN stimulation were separated by >45 s. All DAN

activation experiments were unpaired unless noted.

Tethered Fly Behavior

A Point Grey Firefly Camera with Infinity Lens (94-mm focal length) was

focused on a fly illuminated by infrared light-emitting diodes (LEDs). Video

was captured at 30 frames/s. Fly motion traces were extracted using a custom

MATLAB script. For sugar feeding, flies were fasted for 20–26 hr and then

offered a paper wick soaked in 0.2–1 M sucrose using a motorized microma-

nipulator (Scientifica). For punitive shock, steel electrode leads were posi-

tioned on a fly’s abdomen and 0.5-s pulses of 60–150 V were applied from a

stimulator (Grass Technologies).

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures,

seven figures, one table, and three movies and can be found with this article

online at http://dx.doi.org/10.1016/j.cell.2015.11.019.

AUTHOR CONTRIBUTIONS

R.C. and V.R. conceived the project and wrote the paper. R.C. performed all

experiments and data analysis, except Figures 5E, S1G, S4B, S5B, and

S5E, which were carried out by I.M.

ACKNOWLEDGMENTS

We thank Leslie Vosshall, Cori Bargmann, Richard Axel, Larry Abbott, San-

deep Datta, Barbara Noro, Daisuke Hattori, Meg Younger, and members of

the V.R. lab for valuable discussion. We thank Donovan Ventimiglia, Tim

Ryan, Ari Zolin, Eliezer Pickholtz, and Josh Salvi for technical advice. R.C.

was supported by a David Rockefeller Graduate Student Fellowship. V.R. is

ell 163, 1742–1755, December 17, 2015 ª2015 Elsevier Inc. 1753

a New York Stem Cell Foundation Robertson Neuroscience Investigator. This

work was supported by the New York Stem Cell Foundation, a Pew Biomedical

Scholar Award, a McKnight Scholar Award, the Alfred P. Sloan Foundation, an

Irma T. Hirschl Award, a Sinsheimer Foundation Award, and an NIH New Inno-

vator Award (DP2 NS0879422013).

Received: August 14, 2015

Revised: October 12, 2015

Accepted: November 9, 2015

Published: December 17, 2015

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