nihms-163146.pdf

Two Different Forms of Arousal in

Drosophila

are Independently

and Oppositely Regulated by the Dopamine D1 Receptor DopR

via Distinct Neural Circuits

Tim J. Lebestky

1,3

Jung-Sook C. Chang

1,3

Heiko Dankert

1,2

Lihi Zelnik

Young-Cho

Kim

Kyung-An Han

Pietro Perona

, and

David J. Anderson

1,3,5

Division of Biology 216-76 California Institute of Technology, Pasadena, CA 91125

Division of Engineering and Applied Science 136-93 California Institute of Technology,

Pasadena, CA 91125

Howard Hughes Medical Institute California Institute of Technology, Pasadena, CA 91125

Department of Biology The Huck Institute Pennsylvania State University, University Park, PA

16802

SUMMARY

Arousal is fundamental to many behaviors, but whether it is unitary, or whether there are different

types of behavior-specific arousal, has not been clear. In

Drosophila

, dopamine promotes sleep-

wake arousal. However there is conflicting evidence regarding its influence on environmentally

stimulated arousal. Here we show that loss-of-function mutations in the D1 dopamine receptor

DopR

enhance repetitive startle-induced arousal, while decreasing nocturnal arousal (i.e.,

increasing sleep). These two types of arousal are also inversely influenced by cocaine, whose

effects in each case are opposite to, and abrogated by, the

DopR

mutation. Selective restoration of

DopR function in the central complex rescues the enhanced stimulated arousal but not the

increased sleep phenotype of

DopR

mutants. These data provide evidence for at least two different

forms of arousal, which are independently regulated by dopamine in opposite directions, via

distinct neural circuits.

INTRODUCTION

“Arousal,” a state characterized by increased activity, sensitivity to sensory stimuli and

certain patterns of brain activity (Coull, 1998), accompanies many different behaviors,

including circadian rhythms, escape, aggression, courtship and emotional responses in

higher vertebrates (Cahill and McGaugh, 1998; van Swinderen and Andretic, 2003; Devidze

et al., 2006). A key unanswered question is whether arousal is a uni-dimensional,

generalized state (Hebb, 1955; Pfaff et al., 2005), or rather multi-dimensional (Robbins,

1997). Biogenic amines, such as dopamine (DA), norepinephrine (NE), serotonin (5-HT)

and histamine, as well as cholinergic systems, have all been implicated in arousal in

numerous behavioral settings (Robbins et al., 1998; Pfaff et al., 2002; Berridge, 2006;

Author for correspondence Tel: (626) 395-6821 FAX: (626) 564-8243 wuwei@caltech.edu.

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Published as:

Neuron

. 2009 November 25; 64(4): 522–536.

HHMI Author Manuscript

Devidze et al., 2006). For several reasons, however, it is not clear whether these

neuromodulators act on a common “generalized arousal” pathway (Pfaff et al., 2005), or

rather control distinct arousal pathways that independently regulate different behaviors. This

is because a single amine typically acts through multiple receptors. Thus different receptors

(or even a single receptor subtype) may act in distinct circuits to control different forms of

arousal. Resolving this issue requires identifying the receptors and circuits on which these

modulators act, in different behavioral settings of arousal.

Most studies of arousal in

Drosophila

have focused on spontaneous locomotor activity

associated with sleep-wake arousal, a form of “endogenously generated” arousal (van

Swinderen and Andretic, 2003). Several lines of evidence point to a role for DA in

enhancing this form of arousal in

Drosophila

(reviewed in (Birman, 2005). Drug-feeding

experiments, as well as genetic silencing of dopaminergic neurons, have indicated that DA

promotes waking during the subjective night phase of the circadian cycle (Andretic et al.,

2005). Similar conclusions were drawn from studying mutations the

Drosophila

transporter (dDAT) (Kume et al., 2005; Wu et al., 2008). Consistent with these data,

overexpression of the vesicular monoamine transporter (dVMAT-A), promoted

hyperactivity in this species (Chang et al., 2006), as did activation of DA neurons in

quiescent flies (Lima and Miesenbock, 2005; Wu et al., 2008).

Evidence regarding the nature of DA effects on “exogenously generated,” or

environmentally stimulated arousal (van Swinderen and Andretic, 2003), such as that licited

by startle, is less consistent. Classical genetic studies and quantitative trait locus (QTL)

analyses have suggested that differences in DA levels may underlie genetic variation in

startle-induced locomotor activity (Connolly, 1967; Tunnicliff et al., 1969; Carbone et al.,

2006; Jordan et al., 2006).

Fmn

(

dDAT

) mutants displayed hyperactivity in response to

mechanical shocks, implying a positive-acting role for DA in controlling environmentally-

induced arousal (Kume et al., 2005). In contrast, other data imply a negative-acting role for

DA in controlling stimulated arousal. Mutants in

Tyr-1

, which exhibit a reduction in

dopamine levels (Burnell and Daly, 1982), show an increased in stimulated but not

spontaneous levels of locomotor activity (Meehan and Wilson, 1987). Genetic inhibition of

tyrosine hydroxylase-expressing neurons caused hyperactivity in response to mechanical

startle (Friggi-Grelin et al., 2003). Finally, transient activation of DA neurons in hyperactive

flies inhibited locomotion (Lima and Miesenbock, 2005). Whether these differing results

reflect differences in behavioral assays, the involvement of different types of DA receptors,

or an “inverted U”-like dosage sensitivity to DA (Birman, 2005), is unclear.

We have developed a novel behavioral paradigm for environmentally induced arousal, using

repetitive mechanical startle as a stimulus, and have carried out a screen for mutations that

potentiate this response. One such mutation is an hypomorphic allele of the D1 receptor

ortholog,

DopR

. This same mutation caused decreased spontaneous activity during the night

phase of the circadian cycle due to increased sleep. In both assays, cocaine influenced

behavior in the opposite direction as the

DopR

mutation, and the effect of cocaine was

abolished in

DopR

mutant flies, supporting the idea that DA inversely regulates these two

forms of arousal. Genetic rescue experiments, using Gal4 drivers with restricted CNS

expression, indicate that these independent and opposite influences of DopR are exerted in

different neural circuits. These data suggest the existence of different types of arousal states

mediated by distinct neural circuits in

Drosophila

, which can be inversely regulated by DA

acting via the same receptor subtype.

Lebestky et al.

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RESULTS

Repetitive stress induces an extended state of locomotor hyperactivity

In an effort to develop a

Drosophila

model of cumulative stress-induced arousal, we tested

whether closely spaced repetitive startle stimuli could produce an extended period of

hyperactivity. We delivered a succession of brief air puffs (200 msec duration at 5 sec

intervals, 35 psi), to adult flies placed in horizontal plastic tubes (10 flies/tube) (Fig. 1A), in

an 8-tube manifold (the “puff-o-mat”) based on a device developed by Heberlein and

colleagues (Wolf et al., 2002; Rothenfluh et al., 2006). These airpuffs, while relatively

gentle, were strong enough to blow the flies against the mesh at the back of the tube, from

which they immediately rebounded (Supplemental Movie SM1). Application of 6 successive

puffs produced an extended period of hyperactivity, which lasted 7-10 minutes (Fig. 1B).

We call this behavioral response Repetitive Startle-induced Hyperactivity (ReSH).

To characterize ReSH behavior more quantitatively, we developed custom software to

record the position, velocity, acceleration and trajectories of the flies in response to the

airpuffs (see Supplementary Information). The acceleration of the flies, in the 5-second

period immediately following each puff, increased steeply during the presentation of the first

three puffs (Fig. 1C), suggesting a cumulative effect of the stimuli. Following a 6-puff

exposure, the average velocity of the flies was elevated almost 10-fold, relative to pre-

stimulus baseline, and gradually declined thereafter (Fig. 1B).

Flies walk intermittently in bouts of activity interrupted by periods of immobility (Martin et

al., 1999a; Wolf et al., 2002). An increase in average velocity could, in principle, reflect a

change in bout duration, bout frequency or walking speed during the bout. Our analysis

indicated that the airpuffs caused little change in the average duration of walking bouts

(Supplemental Figure S1A), but instead transiently increased both the bout frequency

(Figure 1D), and average speed during the bouts (Supplemental Figure S1C).

The gradual decline in locomotor activity during the post-puff period appeared to follow

exponential decay kinetics. Indeed, the response profile was fit well by a modified

exponential function (Fig. 1E; see Experimental Procedures). This model permitted us to

extract a number of parameters, including the peak height, decay constant tau (

), and the

total distance traveled following the puffs (see Experimental Procedures), and to determine

the effect of varying different stimulus properties on these response parameters. As the

number of puffs was systematically varied from 1-6 (Fig. 1F), the net increase in peak

velocity, and the total distance traveled after the puffs, increased up to an apparent saturation

point at 4 puffs (Fig. 1H,J). The magnitude of

also increased with increasing puff number,

although this was more variable between experiments (Fig. 1I). Peak velocity and distance

traveled also increased as a function of stimulus intensity (psi/puff) (Supplemental Fig. S7G,

J). These data suggest that the ReSH response scales in proportion to the amount of

stimulation, and therefore that it indeed reflects a response to repetitive, cumulative stress.

The ReSH response also exhibited sensitization. Flies were exposed to 6 puffs, and allowed

10 minutes to recover. Subsequently, they were exposed to a single puff, and their responses

compared to those of naïve flies exposed to a single puff. Both

and the total distance

traveled during the post-puff period were significantly higher in flies that had previously

been exposed to 6 puffs (Supplemental Figure S2). However, if flies were allowed to rest for

30 minutes after the 6 puffs, there was no statistically significant difference in their

subsequent response to a single air puff (data not shown), implying that the sensitization

state undergoes time-dependent extinction.

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The sensitization induced by repeated air-puff exposure generalized to at least one other

sensory modality, olfaction. When flies are briefly exposed to a high concentration of an

odor, they exhibit a transient increase in locomotor activity, a response termed olfactory

startle (Wolf et al., 2002; Cho et al., 2004). Following recovery from ReSH, the olfactory

startle response to methyl cyclohexanol (MCH) was significantly enhanced (Supplemental

Figure S3). These data suggest that repetitive startle induces an extended state of elevated

arousal, which is manifested by: 1) increased acceleration following each successive

stimulus; 2) a protracted period of locomotor hyperactivity in the post-stimulus period; and

3) sensitization to a subsequent low-intensity stimulus, a state which persists even after the

overt locomoter hyperactivity phase has subsided, and which extends to at least one other

sensory modality.

DopR negatively regulates repetitive startle-induced hyperactivity

To identify genes controlling ReSH behavior, we screened several hundred lines from a

collection of transposon insertion mutants (Artavanis-Tsakonas, 2004), focusing on genes

with neurobiological relevance. The response of each mutant to a sub-saturating (2-puff)

stimulus was analyzed using our software, and compared to the average of the entire

collection. Candidates were identified by >2 standard deviations from the mean population

values of parameters such as

(Fig. 1E), or as outliers in Principal Component space (Fig.

2A). Lines exhibiting both diminished and exaggerated responses were identified; we

focused on those lines exhibiting hyperactive responses. One such mutant was a piggyBac

transposon insertion in the

dDA1/DopR1

locus (Gotzes et al., 1994; Sugamori et al., 1995),

DopR

f02676

(Fig. 2A; hereafter referred to as DopR). The

of this line was almost ten-fold

higher than that of the mean of the collection of lines screened.

The

DopR

insertion was back-crossed into a Canton-S (CS) background for six generations

for further analysis.

DopR/+

and

DopR/DopR

flies exhibited both elevated pre-puff baseline

and post-puff velocities (Fig. 2C), reflecting an increase in locomotor bout frequency and

bout velocity (Supplemental Figure S1B). When the pre-puff baseline velocity of the mutant

was normalized to that of wild-type CS flies, both

DopR/+

and

DopR/DopR

flies still

showed an extended period of post-puff hyperactivity (Fig. 2C, inset; D, E). This suggested

that the

DopR

mutation does not cause simply a “shift-up” in the puff-response curve, due to

an increase in spontaneous locomotor activity, but rather that the flies take longer to “calm

down” following the repetitive startle stimulus. This enhanced reactivity to mechanical

startle is also reflected in the elevated pre-puff activity of the flies immediately following

introduction into the apparatus (Connolly, 1967; Burnell and Daly, 1982). This

interpretation was confirmed by additional controls in which the flies were anaesthetized

prior to introduction to the puff-o-mat (Supplemental Footnote S1 and Supplemental Figure

S6).

To confirm that the phenotype of

DopR

flies was indeed due to a disruption of the

DopR

gene, we first measured the levels of

DopR

mRNA in different genotypes by quantitative

RT-PCR. There was an approximately 50% reduction in the amount of

DopR

mRNA in

DopR/+

flies, and an ~95% reduction in

DopR/DopR

files (Fig. 2B). These data confirm

prior analysis of the

DopR

f02676

/dumb

allele (Kim et al., 2007), as well as independent

studies by Wolf et al. (submitted), and indicate that this allele is a strong molecular

hypomorph. We further confirmed that this insertion caused the ReSH phenotype by

isolating revertant flies bearing a precise excision of the piggyBac transposon (Thibault et

al., 2004) (see Supplemental Methods). When crossed into a

background, the puff-

response of these excision flies was indistinguishable from that of

CS controls, by all

parameters measured (Fig. 2F-H, orange vs. blue curves, Supplemental Figure S4 D-F).

Lebestky et al.

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Further confirmation that the phenotype is due to disruption of the

DopR

gene was provided

by analyzing trans-heterozygous flies bearing an independent transposon insertion in the

DopR

locus (

DopR

PL00420

)

over a deficiency spanning the locus (

Df(3R)ED5364

) (Figure

S5), as well as by genetic rescue experiments (see below). Taken together, these data

indicate that the ReSH phenotype is due to a reduction in

DopR

function. The similar ReSH

phenotypes of

DopR/+

and

DopR/DopR

flies (Fig. 2C-E) suggests that this behavior is

sensitive to

DopR

gene dosage in this genetic background. However, it should be noted that

the magnitude of the kinetic parameters characterizing both the wild-type response, and the

DopR

mutant phenotype, varied with genetic background, consistent with evidence that

startle-induced locomotor activity is controlled by complex genetic networks (Jordan et al.,

2007; Yamamoto et al., 2008).

The exaggerated ReSH response in

DopR

flies exposed to 2 puffs (Fig. 2D, E) suggested

that the mutants might be hypersensitive to the airpuff stimulus. To investigate this in more

detail, we systematically varied the puff number and intensity. In response to small numbers

of puffs, or to low puff intensities,

DopR

flies showed much stronger increases in

and

post-puff distance traveled, than did wild-type flies (Fig. 3A-D, and Supplemental Figure

S7). Furthermore,

DopR/DopR

flies appeared to reach saturation in their post-stimulus

activity after a smaller number of puffs, in comparison to CS controls (Fig. 3B). These data

support the idea that the

DopR

mutation causes hyper-reactivity to the puff stimulus, as well

as an increased time to recover from repetitive puffs.

If an hypomorphic mutation in

DopR

enhances ReSH behavior, then one might predict that

elevating DA should, conversely, suppress ReSH behavior. To test this, we examined the

effect of cocaine, which elevates synaptic DA. Previous studies have indicated that cocaine

can cause hyperactivity in

Drosophila

(McClung and Hirsh, 1998; Bainton et al., 2000; Li et

al., 2000). While cocaine indeed promoted spontaneous activity measured in a circadian

monitor (see below), it suppressed the ReSH response at the same doses (Fig. 3E).

Parameter analysis indicated a significant depression of

and post-puff distance traveled in

wild-type flies (Supplemental Figure S7N-O; white bars). Strikingly this effect was

eliminated in

DopR/DopR

mutants (Fig. 3F, Supplemental Figure S7N-O; black bars), as

well as in

DopR/+

heterozygotes (data not shown). A similar result was obtained using

Df(3R)ED5364/DopR

PL00420

transheterozygotes (data not shown). Taken together, these

data indicate both that the effect of cocaine in this assay is opposite to that of the

DopR

mutation, and that DopR is the major receptor mediating this effect.

DopR

flies exhibit decreased spontaneous activity during the night

To examine directly the effect of the

DopR

mutation on spontaneous (unstimulated)

locomotion, we measured circadian activity using a standard

Drosophila

Activity

Monitoring System (DAMS) (TriKinetics, Inc.). Under these conditions, both

DopR/+

and

DopR/DopR

flies showed substantially decreased activity, in comparison to wild-type

controls, during the night phase (Fig. 4A, F). This phenotype is consistent with the results of

previous pharmacological and genetic studies indicating that DA positively regulates

spontaneous locomotor activity (Bainton et al., 2000; Andretic et al., 2005; Kume et al.,

2005; Chang et al., 2006).

DopR/+

and

DopR/DopR

flies showed a modest increase in

activity during the morning phase (Fig. 4D), an effect that declined as the day progressed

(Fig. 4A). This morning hyperactivity, however, cannot account for the

DopR

ReSH

phenotype, since this phenotype was observed even when the puff-o-mat assays were

performed at midnight (Fig. 4B), a time when spontaneous locomotor activity is strongly

decreased in the mutant (Fig. 4A, asterisk). These data provide further evidence that the

ReSH phenotype of the

DopR

mutation does not simply reflect a general elevation of

Lebestky et al.

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HHMI Author Manuscript

spontaneous locomotor activity. To the contrary, they suggest that DA regulates ReSH and

spontaneous nocturnal activity in opposite directions, via DopR.

We examined in more detail the behavioral basis of the decreased spontaneous nocturnal

activity in

DopR

flies, by measuring various sleep parameters. Sleep in

Drosophila

has been

defined as discrete periods of inactivity lasting 5 minutes or longer, during which time the

flies show increased arousal thresholds (Shaw et al., 2000; Nitz et al., 2002; Andretic and

Shaw, 2005). Strikingly, both

DopR/+

and

DopR/DopR

flies showed increased overall sleep

in comparison to wild-type (Fig. 4G). Analysis of sleep bout structure (Andretic and Shaw,

2005) indicated that the average sleep bout duration, and the length of the longest bout, were

both significantly greater in

DopR

heterozygous and homozygous mutant flies, than in wild-

type (Fig. 4I, J) . Thus,

DopR

flies are less active at night and sleep more, supporting a

positive-acting role for DA in controlling sleep-wake arousal (Andretic et al., 2005).

We next investigated whether cocaine inversely influenced ReSH and sleep, but in the

opposite direction as the

DopR

mutation. Cocaine increased activity in CS flies in the

circadian monitor during both the day and night phases (Fig. 4K), but its effect was much

more pronounced at night (Fig. 4M). Surprisingly, the effect of cocaine was entirely

abolished in

DopR/DopR

flies (Fig. 4L, N), indicating that none of the other three

Drosophila

DA receptors (Feng et al., 1996; Han et al., 1996; Hearn et al., 2002; Srivastava

et al., 2005) mediates the influence of the drug in this assay. The opposite effects of cocaine

on spontaneous vs. environmentally stimulated locomotor activity (Fig. 3E), taken together

with the fact that both effects of the drug are abolished by the

DopR

mutation, provides

further evidence that the phenotype of the mutation in both assays is due to alterations in DA

sensitivity.

Selective rescue of the DopR ReSH phenotype in subsets of CNS neurons

We next sought to determine the neural substrates of DopR action in controlling ReSH

behavior. To do this, we tested various Gal4 enhancer trap lines for their ability to rescue the

ReSH phenotype, taking advantage of the Gal4 UAS element in the first intron of the

DopR

f02676

allele (Fig. 2B). Transcription from this site is predicted to produce a truncated

protein(s) with a shortened extracellular domain, presumably translated from one of several

internal methionines; this N-terminal domain is non-essential for DopR function, at least in

cell culture (Gotzes and Baumann, 1996). This strategy has also been used successfully to

rescue the learning and memory deficit observed in homozygous

DopR/DopR

flies (Kim et

al., 2007), as well as the reduced ethanol sensitivity of these flies (Wolf et al., submitted).

The fact that

DopR/+

heterozygotes show dominant phenotypes in both the ReSH and sleep

assays (Fig. 2C-E), afforded the opportunity to test the ability of different Gal4 lines to

rescue the phenotype in

Gal4/+; DopR/+

flies. To control for genetic background effects,

Gal4/+; DopR/+

flies were always compared to controls (

Gal4/+

DopR/+

) in an F1 hybrid

background derived from the same parental

DopR

and Gal4 strains (see Experimental

Procedures).

Initial experiments indicated that the

DopR

ReSH phenotype could be rescued by Elav-Gal4

(Supplemental Figure S8), a pan-neuronal driver (Robinow and White, 1988), suggesting

that this phenotype reflects a requirement for DopR function in the nervous system. All

behavioral parameters were rescued, including pre-puff baseline and post-puff peak

velocities,

, and distance traveled (Fig. S8B-F). Importantly, Elav-Gal4 also restored (and

even enhanced) the ability of cocaine to suppress the ReSH response (data not shown). The

observations that Gal4-driven expression of DopR rescued both the ReSH phenotype, and

the sensitivity of ReSH behavior to cocaine, provide additional evidence that the ReSH

phenotype is indeed due to a reduction in DopR function.

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In order to localize further the site of DopR function in the nervous system, we sought to

rescue the phenotype using Gal4 lines with more restricted CNS expression (Manseau et al.,

1997). DopR has previously been reported to be expressed at highest levels in two major

brain structures: the mushroom body (MB) and central complex (CC) (Kim et al., 2003).

Twenty-four different Gal4 lines were tested for their ability to rescue the ReSH phenotype

DopR

flies. Of these, 8 lines expressing in the CC rescued the phenotype, while none of

the lines with MB expression yielded rescue (Table I).

Detailed analysis of the rescue obtained with several lines is shown in Fig. 5 and

Supplemental Figure S9. Line c547, which (like lines 11.148 and 5.30) expresses in R4m/R2

neurons of the ellipsoid body (EB), a CC substructure (Fig. 6A, A2), (Renn et al., 1999),

rescued the

DopR

mutant phenotype as effectively as Elav-Gal4 (Fig. 5A-A2).

Immunostaining with an anti-DopR antibody (see Supplemental Fig. S10 for specificity)

confirmed that endogenous DopR expression overlaps that of c547 in the EB (Fig. 6A1, A3).

Line c547 also rescued the phenotype of

DopR/DopR

homozygous flies (Supplemental

Figure S11). Rescue in these homozygous flies was associated with re-expression of DopR

protein in the EB (Supplemental Figure S12). Rescue was also obtained when expression of

Gal4 in c547 was restricted to the adult phase, using a temperature-sensitive version of the

Gal4 inhibitor, Gal80

(McGuire et al., 2003) (Fig. 5D-D2; E-E2). The partial rescue at

18°C likely reflects incomplete suppression of Gal4 by Gal80

, due to leaky Gal80

inactivation (Kamikouchi et al., 2009); therefore we cannot completely exclude some

developmental contribution of DopR function in mediating the puff response. Nevertheless,

these data indicate that full rescue of the

DopR

ReSH phenotype requires expression of the

receptor in the adult CNS, and also confirm that rescue requires Gal4 activity (McGuire et

al., 2003).

Several other Gal4 lines whose expression overlaps with that of DopR in the EB, including

189y, c761 and 30y, also rescued the

DopR

ReSH phenotype (Fig. 5B-B2; C-C2 and Table

I). Interestingly, lines 189y and c761 express in R3 neurons of the EB, rather than in R2/

R4m neurons (Renn et al., 1999). However, they also overlap DopR expression within this

structure (Fig. 6B1-B3, C1-C3). Thus, DopR expression within the EB is widespread.

Moreover, it is juxtaposed with dense varicosities of TH

fibers (Fig. 6D1-D3), supporting

the idea that the EB receives dopaminergic innervation (see also Wolf et al., submitted). The

Gal4 line c232, which expresses very strongly in R4d neurons (Renn et al. 1999), was also

tested but was hyperactive on its own, and therefore uninformative.

Most of the Gal4 lines that rescue are expressed in other sites besides the CC. While many

of these extra-CC sites do not overlap, some of them do. For example, lines c547 and 189y

also express in median bundle neurons of the pars intercerebralis (PI; Fig. 6A, B). However,

seven other Gal4 lines that express in the PI, but not in R2/R3/R4m neurons of the EB, did

not rescue the phenotype (Table I). Furthermore, immunocytochemical double-labeling

experiments indicated that DopR is normally not expressed by PI neurons (Supplemental

Figure S14). These data argue that rescue is unlikely due to expression in the PI. Lines c547,

189y, c761, and 11-3f also expressed, to different extents, in the antennal lobe (AL).

However, line c739, which expresses in the AL but not the CC, failed to rescue, as did

nanchung-Gal4, a line that expresses in ntennal mechanosensory neurons (Table I). These

data argue against the AL as a site of rescue. Finally, several of the rescuing Gal4 lines also

express in the thoracic ganglia (TG). One such line, 189y, overlaps with GABA in EB

neurons but not in the TG (Supplemental Figure S15). Importantly, rescue of the ReSH

phenotype was obtained with a GAD1-Gal4 line, (Supplemental Figure S13), but not with a

Cha-Gal4 line (Table I). Since rescue requires

DopR

expression in GABAergic neurons, and

since the thoracic neurons that express 189y are not GABAergic, it is unlikely that 189y-

driven expression of

DopR

in the TG is responsible for rescue.

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DA is involved in learning and memory (Neckameyer, 1998; Schwaerzel et al., 2003;

Riemensperger et al., 2005; Schroll et al., 2006). Recent studies indicate that mutants

homozygous for

dumb

, a

DopR

allele, exhibit deficits in olfactory associative learning that

reflect a requirement for this receptor in the MB (Kim et al., 2007). A MB requirement for

DopR also underlies the effect of DA to promote learning in sleep-deprived flies (Seugnet et

al., 2008). Interestingly, Gal4 line c547 (which rescued the ReSH phenotype of

DopR/DopR

flies) did not rescue the olfactory learning deficit of

DopR

mutants (Fig. 5G, red vs. orange

bars). In contrast, line MB247 (Fig. 5H), which rescued the learning phenotype of

DopR/

DopR

mutants (Fig. 5G, green bar and (Kim et al., 2007)), failed to rescue the ReSH

phenotype of

DopR/+

and

DopR/DopR

flies (Fig. 5F). These data demonstrate a double-

dissociation between the requirement for DopR in associative learning vs. ReSH behavior,

in the MB and EB, respectively.

Independent requirements for DopR in spontaneous vs. stimulated activity

The opposite effects of the

DopR

mutation on spontaneous activity in the circadian monitor

vs. ReSH behavior raised the question of whether these functions are exerted in distinct

neural circuits. To address this question, we examined the ability of different Gal4 lines to

rescue the

DopR

sleep phenotype. Restoration of

DopR

expression throughout the CNS,

using the pan-neuronal driver Elav-Gal4, resulted in a strong rescue of the nocturnal

hypoactivity phenotype (Fig. 7A, F). Analysis of sleep parameters indicated that Elav-Gal4

restored their values to levels close to those of control

Elav-Gal4/+

flies (Fig. 7D-E, green

vs. blue bars). Thus, DopR controls sleep by acting in the nervous system.

In contrast to Elav-Gal4, line c547, which effectively rescued the ReSH phenotype of the

DopR

mutant, did not rescue the nocturnal hypoactivity phenotype (Fig. 7B, F). There was

no statistically significant difference between

DopR/+

and

c547; DopR/+

flies for any sleep

parameters measured (Fig. 7D, E; red vs. green bars). These data suggest that DopR is

unlikely to control sleep-wake arousal by acting in the EB. We therefore sought other Gal4

lines that might rescue the sleep phenotype of

DopR

mutants. Pigment-Dispersing Factor

(PDF)-expressing neurons are circadian pacemaker neurons that regulate the pre-dawn

activity phase of the circadian cycle (Stoleru et al., 2004; Parisky et al., 2008), and arousal

during the night phase (Shang et al., 2008).

pdf-Gal4/DopR

flies showed a significant, albeit

partial, rescue of both average sleep bout duration, and the length of the longest sleep bout

(Fig. 7C-E, green vs. blue bars). Similar results were obtained with line c929, an

independent Gal4 driver that also expresses in circadian pacemaker neurons (Shang et al.,

2008) (data not shown). Importantly, the

pdf-Gal4

driver failed to rescue the ReSH

phenotype of

DopR

flies (Supplementary Figure S16). Taken together, these data provide a

double-dissociation suggesting that the inverse effects of the

DopR

mutation on ReSH

behavior, and on sleep-wake transitions, reflect independent functions for the receptor in

distinct neuronal circuits.

DISCUSSION

Previous studies of arousal in

Drosophila

have focused on sleep-wake transitions (Nitz et al.,

2002; van Swinderen et al., 2004; Andretic et al., 2005; Kume et al., 2005; Sheeba et al.,

2008), a form of “endogenous” arousal (van Swinderen and Andretic, 2003). Here we

introduce a quantitative behavioral assay for repetitive startle-induced hyperactivity, which

displays properties of an environmentally induced (“exogenous”) arousal state. We have

conducted a screen for mutations affecting this behavior, characterized the phenotype of one

such mutation (

DopR

), and mapped the neural substrates of its action by cell-specific

genetic rescue experiments. Our results reveal that DopR independently regulates ReSH and

sleep in opposite directions, by acting on distinct neural substrates. Negative regulation of

the ReSH response requires DopR function in the EB of the CC, while positive regulation of

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waking reflects a function in other populations of neurons, including PDF-expressing

circadian pacemaker cells (Stoleru et al., 2004; Parisky et al., 2008; Shang et al., 2008).

Both of these functions, moreover, are independent of the function of DopR in learning and

memory, which is required in the mushroom body (Kim et al., 2007) (Fig. 8A). Our data

suggest that ReSH behavior and sleep-wake transitions reflect distinct forms of arousal that

are genetically, anatomically and behaviorally separable.

ReSH behavior expresses an environmentally induced arousal state

Early genetic studies of

Drosophila

locomotion have suggested that spontaneous and

environmentally stimulated locomotor activity reflect “distinct behavioral systems”

(Connolly, 1967; Burnell and Daly, 1982). Several lines of evidence suggest that ReSH

behavior represents a form of environmentally stimulated arousal. First, hyperactivity is an

evolutionarily conserved expression of increased arousal (van Swinderen et al., 2004;

Devidze et al., 2006). Although not all arousal is necessarily expressed as hyperactivity,

electrophysiological studies indicate that mechanical startle evokes increases in 20-30 Hz

and 80-90 Hz brain activity, which have been suggested to reflect a neural correlate of

arousal in flies (Nitz et al., 2002; van Swinderen et al., 2004). Second, ReSH does not

immediately dissipate following termination of the stimulus, as would be expected for a

simple reflexive stimulus-response behavior, but rather persists for an extended period of

time, suggesting that it reflects a change in internal state. Third, this state, like arousal, is

scalable: more puffs, or more intense puffs, produce a stronger and/or longer-lasting state of

hyperactivity. Fourth, this state exhibits sensitization: even after overt locomotor activity has

recovered to pre-puff levels, flies remain hypersensitive to a single puff for several minutes.

Fifth, this sensitization state generalizes to a startle stimulus of at least one other sensory

modality (olfactory). In

Aplysia

, sensitization of the gill/siphon withdrawal reflex has been

likened to behavioral arousal (Kandel and Schwartz, 1982). Taken together, these features

strongly suggest that ReSH represents an example of environmentally stimulated

(“exogenous”) arousal in

Drosophila

(Fig. 8A).

DA inversely and independently regulates environmentally stimulated and sleep-wake

arousal

DopR

mutant flies exhibited longer sleep bouts during their subjective night phase,

suggesting that DopR normally promotes waking. These data are consistent with earlier

studies indicating that DA promotes sleep-wake arousal (Andretic et al., 2005; Kume et al.,

2005; Wu et al., 2008). In contrast, prior evidence regarding the role of DA in startle-

induced arousal is conflicting. Some studies have suggested that DA negatively regulates

locomotor reactivity to environmental stimuli (Burnell and Daly, 1982; Friggi-Grelin et al.,

2003), consistent with our observations, while others have suggested that it positively

regulates this response (McClung and Hirsh, 1998; Bainton et al., 2000; Kume et al., 2005).

Even within the same study, light-stimulated activation of TH

neurons produced opposite

effects on locomotor activity, depending on the pre-stimulus level of locomotor activity

(Lima and Miesenbock, 2005).

We find that DA and DopR negatively regulate environmentally stimulated arousal: the

DopR

mutation enhanced ReSH, while cocaine suppressed it. Furthermore, the effect of

cocaine in the ReSH assay was eliminated in the

DopR

mutant but could be rescued by

Gal4-driven DopR expression, confirming that the effect of the drug is mediated by DA.

Taken together, our results reconcile apparently conflicting data on the role of DA in

“arousal” in

Drosophila

, by identifying two different forms of arousal—repetitive startle-

induced arousal and sleep-wake arousal--that are regulated by DA in an inverse manner.

Whether these two forms of arousal are controlled by the same subset of DA neurons is an

interesting question for future study.

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Neural substrates of DA action in repetitive startle-induced arousal

Several lines of evidence suggest that endogenous DopR likely acts in the ellipsoid body

(EB) of the central complex (CC) to regulate repetitive startle-induced arousal. First,

multiple Gal4 lines that drive expression in the EB rescued the ReSH phenotype of

DopR

mutants. Second, endogenous DopR is expressed in EB neurons, including those in which

the rescuing Gal4 drivers are expressed (Fig. 6). Third, the domain of DopR expression in

the EB overlaps the varicosities of TH

fibers. In the accompanying manuscript, Wolf et al.

(2009) identify TH

neurons that are a likely source of these projections to the EB. Fourth,

rescue of the ReSH phenotype is associated with re-expression of DopR in EB neurons.

Finally, rescue is observed using conditional DopR expression in adults. Taken together,

these data argue that rescue of the ReSH phenotype by the Gal4 lines tested reflects their

common expression in the EB, and that this is a normal site of DopR action in adult flies.

A requirement for DopR in the EB in regulating ReSH behavior is consistent with the fact

that the CC is involved in the control of walking activity (Strauss and Heisenberg, 1993;

Martin et al., 1999b; Strauss, 2002; Neuser et al., 2008). However, the mushroom body has

also been implicated in the control of locomotor behavior (Martin et al., 1998; Helfrich-

Forster et al., 2002), and DopR is strongly expressed in this structure as well (Kim et al.,

2003). Our rescue data argue against the MB and in favor of the CC as a neural substrate for

the ReSH phenotype of

DopR

mutants. Unexpectedly, the nocturnal hypoactivity phenotype

DopR

mutants was not rescued by restoration of DopR expression to the CC. Thus, not all

locomotor activity phenotypes of the

DopR

mutant necessarily reflect a function for the gene

in the CC.

Interestingly, Gal4 line c547 expresses in R2/R4m neurons of the EB, while lines 189y and

c761 express in R3 neurons (Renn et al., 1999), yet both rescued the ReSH phenotype of

DopR

mutants. Similar results have been obtained in experiments to rescue the deficit in

ethanol-induced behavior exhibited by the

DopR

mutant (Wolf et al., submitted; see below).

Double-labeling experiments suggest that endogenous DopR is expressed in all of these EB

neuronal subpopulations (Fig. 6 and Wolf et al., submitted). Perhaps the receptor functions

in parallel or in series in R4m and R3 neurons, so that restoration of DopR expression in

either population can rescue the ReSH phenotype. Whether these DopR-expressing EB

subpopulations are synaptically interconnected is an interesting question for future

investigation.

In the accompanying manuscript, Wolf et al. show that DopR acts in the EB to

positively

regulate locomotor hyperactivity induced by chronic ethanol exposure (Wolf et al.,

submitted). There are several explanations for this difference in the “polarity” of the

influence of the

DopR

mutation in the EB. First, the two behavioral paradigms are very

different: hyperactivity in the ReSH paradigm is expressed immediately upon stimulus

exposure and persists following stimulus cessation, while ethanol-induced hyperactivity

develops more slowly and requires chronic ethanol exposure (Wolf et al., 2002; Cho et al.,

2004). Second, the polarity of DA/DopR regulation of activity in the EB may itself be

influenced by ethanol. Finally, DopR activation in the CC in response to different types of

stimuli may either potentiate or suppress activity, depending on the initial state of the circuit

and ambient levels of DA (Birman, 2005; Lima and Miesenbock, 2005). Whatever the

explanation, the role of DopR and DA in regulating locomotor activity in the EB is clearly

complex, and a deeper understanding will require more detailed anatomical mapping of EB

circuitry and functional analysis.

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Modeling “emotional” behaviors in

Drosophila

Despite its power as a system for studying neural development, function and behavior,

Drosophila

has not been extensively used in affective neuroscience (Iliadi, 2009), in part

because of uncertainty as to whether this model organism exhibits states related to emotion

or affect. Increased arousal is a key component of many emotional or affective behaviors

(Russell, 1980). The data presented here establish a quantitative behavioral paradigm for

studying the genetic and neural circuit basis of a persistent arousal state elicited by repetitive

traumatic startle. ReSH behavior exhibits several features that distinguish it from reflexive

stimulus-response paradigms: scalability, persistence following stimulus termination, and

sensitization. In addition, the observation that mechanical trauma promotes release from

Drosophila

of an odorant that repels other flies (Suh et al., 2004) suggests that the arousal

state underlying ReSH behavior may have a negative “ affective valence” as well (Robbins

et al., 1998; Calder et al., 2001). These considerations, together with the fact that ReSH is

influenced by genetic and pharmacologic manipulations of DA, a biogenic amine implicated

in emotional behavior in humans, support the idea that ReSH behavior may represent an

evolutionary precursor of emotional responses in higher organisms.

The phenotype of

DopR

flies, which includes hyper-reactivity to environmental stimuli, is

reminiscent of some symptoms of Attention-Deficit Hyperactivity Disorder (ADHD), an

affective disorder linked to dopamine (Levy, 1991; Solanto, 2002; Bobb et al., 2005). If

humans, like flies, have distinct brain circuits mediating environmentally stimulated and

endogenous arousal, then it is possible given our data that ADHD may specifically involve

dopaminergic dysfunction in circuits mediating the former rather than the latter type of

arousal. The fact that DA can negatively regulate stimulated arousal circuits could then

explain why treatment with DA reuptake inhibitors such as methylphenidate (Ritalin), which

increases synaptic levels of DA, can ameliorate symptoms of ADHD (Arnsten, 2006).

Consistent with this view, in vertebrates DA is thought to promote waking activity via D1

receptors in the nucleus accumbens (Monti and Monti, 2007), while D1 receptors in the pre-

frontal cortex (PFC) have been proposed to negatively regulate activity (Vezina et al., 1991;

Heijtz et al., 2007). Numerous studies have linked dopaminergic dysfunction in the PFC to

ADHD (reviewed in (Brennan and Arnsten, 2008)). While most research has focused on the

role of the PFC in attention and cognition, rather than in environmentally stimulated arousal

per se

, dysfunction of PFC circuits mediating phasic DA release has been invoked to explain

behavioral hypersensitivity to environmental stimuli in ADHD (Sikstrom and Soderlund,

2007). This view of ADHD as a specific disorder of stimulated arousal circuits suggests that

further elucidation of such circuits in humans and vertebrate animal models, as well as in

Drosophila

, may deepen our understanding of this disorder and potentially lead to more

targeted therapies.

EXPERIMENTAL PROCEDURES

Genetics

Homozygous viable insertional piggyBac alleles from the Exelixis collection were acquired

from the Harvard Stock Center and tested in a pilot screen. The

DopR

f02676

allele was

backcrossed to a standardized CS background for 6 generations prior to behavioral testing.

Details of the excision of the piggyBac

DopR

f02676

allele are described in Supplementary

Methods. Rescue experiments using

DopR

f02676

/+ flies were performed in an F1 hybrid

background that reflects equal contribution of the Gal4 genetic background and the

DopR

f02676

allele.

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Behavioral Assays

Flies assayed were males (2-4 days old) CO

anaesthetized and allowed to recover for 2

days prior to testing. Flies were reared on a 12 hr day-night cycle at 25°. Temperature for

behavioral experiments was maintained at 23-25°. For the standard ReSH assay, 10 flies

were manually loaded into tubes and allowed to acclimatize for 10 min prior to filming.

Activity was recorded beginning at one minute before delivery of the puff stimuli, until 3.5

minutes after stimulus termination. Each air puff (35 psi unless otherwise indicated) lasted

200ms with a 5s inter-puff interval. Movies were analyzed using custom locomotor tracking

software (described in Supplemental methods). Cocaine feeding and learning and memory

protocols are described in Supplemental Methods.

Trikinetics Individual Drosophila Activity Monitors and Trikinetics software were used for

all circadian/sleep observations (see Supplemental Methods for details). A period of sleep

was defined as 5 minutes of inactivity (Shaw et al., 2000; Andretic and Shaw, 2005).

For Gal80

experiments, flies were crossed and kept at 18° until eclosion. Flies (2-4 days

old) were collected and maintained at 18° or shifted to 30° for 48 hrs prior to testing.

Animals from both rearing conditions were acclimated to a 25° behavioral room for one hr

prior to testing.

Immunohistochemistry

A polyclonal DopR antibody was raised in guinea pigs against the peptide

CIKAVTRPGEVAEKQRYKSIR, derived from the third cytoplasmic loop (Kim et al.,

2003). Antibodies were affinity purified. Antibody staining procedures and sources of other

immune reagents are described in Supplemental Methods.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

We thank Fred Wolf and Ulrike Heberlein for sharing data and early drafts of their manuscript as well as providing

the manifold for the puff-o-mat, Martin Heisenberg and members of the Anderson lab for helpful comments on the

manuscript, Eric Hoopfer for help with imaging, Michael Reiser for contributions to software development, Mary

Wahl for assistance with screening, Tim Tayler for sharing Gal4 stocks prior to publication, Shilpa Jeeda for

maintenance of stocks, Gaby Mosconi for lab management and Gina Mancuso for Administrative Support. Fly

stocks were obtained from the Bloomington and Harvard stock centers, and also generously provided by Doug

Armstrong. T.L. was a fellow of the Jane Coffin Childs Foundation and H.D. a fellow of the Alexander von

Humboldt Association. Supported in part by an NSF FIBR grant to D.J.A., P.P. and Michael Dickinson. D.J.A. is

an Investigator of the Howard Hughes Medical Institute. This paper is dedicated to the late Seymour Benzer.

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