2 Pigment-dispersing factor (PDF) - bioRxiv.org · 2 Pigment-dispersing factor (PDF) 3 4...

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Regulation of olfactory associative memory by the circadian clock output signal 1

Pigment-dispersing factor (PDF) 2

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Abbreviated Title: PDF regulates associative memory 4

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Johanna G. Flyer-Adams, Emmanuel J. Rivera-Rodriguez, Jacob D. Mardovin, Junwei Yu and 6

Leslie C. Griffith* 7

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Department of Biology, Volen National Center for Complex Systems and National Center for 9

Behavioral Genomics, Brandeis University, Waltham, MA 02454-9110, USA 10

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*Corresponding author: 12

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Leslie C. Griffith 14

Dept. of Biology MS008 15

Brandeis University 16

415 South St. 17

Waltham, MA 02454-9110 18

Tel: 781 736 3125 19

FAX: 781 736 3107 20

Email: [email protected] 21

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.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 18, 2020. . https://doi.org/10.1101/2020.04.17.046953doi: bioRxiv preprint

mailto:[email protected]

https://doi.org/10.1101/2020.04.17.046953

http://creativecommons.org/licenses/by-nc-nd/4.0/

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50 pages; 7 Figures; 1 Table; 2 Multimedia Figures 23

Number of words: Abstract, 217; Introduction, 642; Discussion, 1458 24

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The authors declare no competing financial interests. 26

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ACKNOWLEDGEMENTS 28

This work was supported by NIH R01 MH067284 to LCG, NIH F31 NS110273 to EJRR and NIH 29

T32 NS007292. We offer our thanks to Dr. Timothy D. Wiggin for his original versions of EPAC 30

data analysis scripts and his excellent feedback on the project overall. 31

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AUTHOR CONTRIBUTIONS 33

Designed research (JGFA, JY, LCG), Performed research (JGFA, EJRR, JDM, JY), Analyzed data 34

(JGFA), Wrote the paper (JGFA, LCG). 35

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KEY WORDS 37

Drosophila; Mushroom body; memory; clock; neuropeptide 38

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ABSTRACT 41

Dissociation between the output of the circadian clock and external environmental cues is a 42

major cause of human cognitive dysfunction. While the effects of ablation of the molecular 43

clock on memory have been studied in many systems, little has been done to test the role of 44

specific clock circuit output signals. To address this gap, we examined the effects of mutation 45

of Pigment-dispersing factor (Pdf) and its receptor, Pdfr on associative memory in male and 46

female Drosophila. Loss of PDF signaling significantly decreases the ability to form associative 47

memory. Appetitive short-term memory (STM), which in wildtype is time-of-day (TOD)-48

independent, is decreased across the day by mutation of Pdf or Pdfr, but more substantially in 49

the morning than in the evening. This defect is due to PDFR expression in adult neurons outside 50

the core clock circuit and the mushroom body Kenyon cells. The acquisition of a TOD difference 51

in mutants implies the existence of multiple oscillators that act to normalize memory formation 52

across the day for appetitive processes. Interestingly, aversive STM requires PDF but not PDFR, 53

suggesting that there are valence-specific pathways downstream of PDF that regulate memory 54

formation. These data argue that the circadian clock uses circuit-specific and molecularly 55

diverse output pathways to enhance the ability of animals to optimize responses to changing 56

conditions. 57

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SIGNIFICANCE STATEMENT 59

From humans to invertebrates, cognitive processes are influenced by organisms’ internal 60

circadian clocks, the pace of which is linked to the solar cycle. Disruption of this link is 61

increasingly common (e.g. jetlag, social jetlag disorders) and causes cognitive impairments that 62


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are costly and long-lasting. A detailed understanding of how the internal clock regulates 63

cognition is critical for the development of therapeutic methods. Here, we show for the first 64

time that olfactory associative memory in Drosophila requires signaling by Pigment-dispersing 65

factor (PDF), a neuromodulatory signaling peptide produced only by circadian clock circuit 66

neurons. We also find a novel role for the clock circuit in stabilizing appetitive sucrose/odor 67

memory across the day. 68

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INTRODUCTION 70

Cognition is influenced by the circadian clock. Within individual cells, time is kept by a 71

molecular clock; the coordination of time across tissues in the organism is performed by the 72

‘core clock’, a small population of individually cycling neurons bound together to form a 73

coherent circuit. The outputs of this circuit generate rhythms of physiology and behavior that 74

oscillate with a period aligned to the solar cycle. Circadian peaks and troughs in learning and 75

memory have been documented in humans as well as rodent and invertebrate models 76

(Gerstner & Yin, 2010; Wright et al., 2012) such that misalignments between an organism’s 77

cycling internal clock and external conditions (jetlag, social jetlag, and shift work disorders) 78

impair cognition/memory across phyla (Kott et al., 2012; Weingarten & Collop, 2013; Wittmann 79

et al., 2006; Wright et al., 2013). Irreversible neurodegenerative diseases that affect cognition 80

such as Parkinson’s and Alzheimer’s exhibit comorbid circadian dysfunction (Videnovic et al., 81

2014). Even natural aging degrades the fidelity of the body’s clock in a manner that has been 82

linked to cognitive decline (Reinhart & Nguyen, 2019). Thus a complete understanding of how 83

the body’s clock regulates cognition could generate new therapeutic approaches. 84

To date, the clock-memory link has largely been investigated by breaking the 85

transcriptional feedback loop providing intracellular timekeeping in cells of the clock circuit. 86

Eliminating cycling with altered light conditions affects novel object recognition in rodents 87

(Ruby et al., 2013) and time-of-day (TOD)-dependent associative memory in Drosophila 88

(Chouhan et al., 2015; Le Glou et al., 2012; Lyons & Roman, 2009). Clock gene mutations such 89

as Per1, Per2 and Bmal1 generate impairments in contextual fear and spatial memory tasks in 90

mice and are involved in all phases of memory processing (Rawashdeh et al., 2014; Snider & 91


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Obrietan, 2018; Wang et al., 2009; Wardlaw et al., 2014). In Drosophila, mutations of the clock 92

genes period and clock impair TOD memory and can disrupt memory generally (Chouhan et al., 93

2015; Fropf et al., 2014, 2018; Le Glou et al., 2012; Lyons & Roman, 2009; Sakai et al., 2004). 94

But studies using molecular clock mutants don’t fully replicate conditions that exist in common 95

human clock-related cognitive disorders; in most, a functional molecular clock is present but 96

outputs are misaligned with environmental cues. To fully understand the role of clock circuit 97

outputs on cognition, it is necessary to manipulate output pathways in the context of an intact 98

molecular clock. 99

Environmentally-cued circadian rhythms of behavior and physiology are generated by 100

the outputs of the core clock circuit which are organized by peptidergic signaling from a few 101

pacemaker neurons (Aton et al., 2005). In Drosophila, this peptide is Pigment-dispersing factor 102

(PDF), an 18 aa peptide produced in the adult brain solely by 16 ventrolateral clock neurons 103

(LNvs) (Park et al., 2000; Renn et al., 1999). PDF released from the LNvs signals within the core 104

clock circuit through its only known receptor PDFR (Hyun et al., 2005; Lear et al., 2005; Mertens 105

et al., 2005; Shafer et al., 2008) to coordinate the activity of the ~150 core clock neurons (Liang 106

et al., 2016; Lin et al., 2004; Peng et al., 2003; Stoleru et al., 2005; Yoshii et al., 2009). Core clock 107

circuit output directs rhythmic aspects of physiology such as locomotor activity and sleep, and 108

PDF also functions in these output pathways. In this way, the PDF/PDFR signaling pathway both 109

maintains free-running circadian activity and promotes output behaviors. 110

Here, we investigate the involvement of PDF signaling in Drosophila memory, examining 111

the role of clock output on associative olfactory memory for the first time in the context of a 112

functional molecular clock. We find a novel adult-specific requirement for PDF/PDFR signaling 113


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in memory that is distinct from its role in synchronizing the clock circuit. We also provide 114

evidence for the existence of a second PDF receptor that allows valence-specific regulation of 115

associative olfactory memory by PDF. 116

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MATERIALS AND METHODS 118

Fly Stocks and Husbandry. All experimental flies were collected directly after eclosion and 119

maintained in incubators at 25oC with a 12:12 LD cycle. Flies were reared and housed on 120

cornmeal dextrose food with yeast except those used for GeneSwitch experiments. For Figure 1 121

and 6 experiments, mixed males and females were used; for cell-specific PDFR expression 122

memory experiments and all activity experiments, males were used; for imaging experiments, 123

females were used. Fly strains used include: Canton-S wildtype (WT; Canton, Ohio), ;;pdf01 124

(BDSC#26654; backcrossed 6x into WT), han5304;; (BDSC#33068; backcrossed 6x into WT), 125

;;nsyb-GAL4/TM6B (Goodwin et al., 2018), han5304;;UAS-pdfr-myc13 (gift of Paul Taghert), ;;elav-126

GS-GAL4 (Osterwalder et al., 2001), VT030559-GAL4 (VDRC ID# v206077), pdfR-2A-LexA;; and 127

;;pdfattP (Deng et al., 2019), w-,UAS-mCD8-IVS-RFP,LexAop-mCD8-IVS-GFP;; (generated from 128

BDSC#61681), ;pdf-GAL4; (Park et al., 2000), UAS-P2X2 (Lima & Miesenböck, 2005), MB-LexA 129

(Pitman et al., 2011), ;LexAop-EPAC; (BDSC#76031), ;pdfR(10kb)-GAL4; (Parisky et al., 2016), 130

;clk856-GAL4; (gift from M. Rosbash). 131

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Temporal panneuronal expression of Pdfr (elav-GS-GAL4, adult only). GeneSwitch 133

experimental flies (han5304;;elav-GS-GAL4/UAS-Pdfr) were housed after eclosion on normal 134

yeast food containing 0.2g/L RU486 (Sigma, Cat#8046) diluted into 100% ethanol; control flies 135

were housed after eclosion on normal food containing equal volume 100% ethanol. Total 136

exposure time was 6-12d. Flies were starved for appetitive learning assays on nutrient-free 137

agarose ± RU486 or ethanol control. 138

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Learning assays. Appetitive and aversive associative olfactory memory assays were performed 140

in an environmental room in red light at 25oC with 65% ambient humidity. Flies were between 141

4-14 d old (STM) or 4-10 d old (LTM) and given at least 10 min acclimation to the environmental 142

room prior to training or testing. Data for each experiment was pooled from at least three 143

independent experimental days. 144

Appetitive learning assays were performed as previously described (Liu et al., 2012). 145

Briefly, flies were starved to 10% mortality. Filter papers were prepared blank or with 2 M 146

sucrose as null or unconditioned stimulus (US), and 10% MCH and OCT prepared as conditioned 147

stimulus (CS) odors. 50-100 starved flies were loaded into a vial and exposed to a one trial 148

training of sequential CSA-US and CSB-null pairings of 1 min (STM) or 2 min (LTM) duration. Flies 149

were then either tested for CS preference (2 min STM) or placed into fresh food vials for 4 h 150

and then starved for 20 h prior to CS preference testing (24 h LTM). Testing involved a 2 min 151

exposure to both CS odors, after which flies choosing either odor were counted. A preference 152

index (PI) was calculated for each trial as [(# of flies in CSA) - (# of flies in CSB)] / [(# of flies in 153

CSA) + (# of flies in CSB)]. This PI was averaged with the PI of a temporally paired CS reciprocal 154

trial to generate the final Learning Index (LI), calculated as a percentage. In this way, each 155

datapoint shown for these experiments represents 100-200 flies. 156

Aversive learning STM assays were performed similarly to appetitive STM assays with 157

the following modifications. Flies were not starved prior to training, and the US was provided by 158

supplying 12 1-sec 90mV shocks during the 1 min CS-US pairing. As for appetitive STM, flies 159

were immediately tested for CS preference and a final LI calculated from paired reciprocal tests. 160

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Activity Assays & Data Extraction. Male flies were collected immediately after eclosion and 162

individually loaded into sleep tubes and detectors as in (Haynes et al., 2015). Flies were 163

entrained for 3 d to 12:12 LD in 25oC incubators, after which 3 d of 12:12 LD baseline activity 164

was collected followed by release into free-running DD conditions for 9 d. During this time 165

locomotor activity data was collected using the Drosophila Activity Monitor (DAM; TriKinetics, 166

Waltham MA) as previously described (Agosto et al., 2008). Subsequently, beam break counts 167

were extracted using DAMFileScan (TriKinetics, Waltham MA) and analyzed for sleep and 168

activity using MATLAB SCAMP (v.2019, Chris Vecsey, Skidmore) as done previously (Haynes et 169

al., 2015). DD analysis was performed on days 2-8 DD data, with percent of population rhythmic 170

values calculated using cutoff criteria where a rhythmicity index below 0.2 was considered 171

arrhythmic. LD data was calculated from an average of days 1-3 in LD. Custom MATLAB scripts 172

were written to extract evening anticipation onset and evening peak values from 30 m binned 173

LD activity data of individual subjects and are available on GitHub (URL to be provided). Evening 174

anticipation onset was calculated as the max value of the 3 bin rolling average of the first 175

derivative for data between ZT6-11.5 (to prevent artifact from lights-off startle effects). Evening 176

peak was calculated as the max value between ZT6-12. 177

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Imaging 179

Functional imaging (Fig 3E) was performed on ex vivo whole brain dissections with imaging 180

hardware, solutions and acquisition software as described previously (Haynes et al., 2015). 181

Planar image acquisition in MB calyx and surrounding soma was performed at 2Hz throughout a 182

3 min total experimental time for each brain, during which 30 s of baseline AHL perfusion was 183


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followed by 150s of ATP or AHL vehicle perfusion. Data was processed before analysis by 184

custom MATLAB scripts which normalized each trace to its baseline and then fit and subtracted 185

out any linear trends present in baseline due to bleaching (available on GitHub; URL to be 186

provided). Expression pattern imaging (Fig. 3B-D, Multimedia 1,2) was performed as follows: 187

Brains were fixed, stained, mounted and embedded according to Janelia FlyLight protocols 188

(‘Dissection and Fixation 2% PFA’, ‘IHC- Double Label’, ‘DPX Mounting’; 189

https://www.janelia.org/project-team/flylight/protocols). Primary antibodies included mouse 190

monoclonal anti-GFP (1:200; Roche Applied Science, Cat#11814460001), and rabbit polyclonal 191

anti-Ds-red (1:200; Clontech, Cat#632496). Secondary antibodies included Cy2 goat anti-mouse 192

(1:400; Jackson, Cat#115-225-166) and AlexaFluor594 donkey anti-rabbit (1:400; Jackson, 193

Cat#711-585-152) as recommended in Janelia protocols. Samples were imaged on a Zeiss LSM 194

880 microscope using a Plan-Apochromat 25x/0.8 Imm Corr DIC M27 objective. Data presented 195

in Fig.3B,C and Multimedia Fig.1 were imaged using 561/595 and 488/515 channels with a slice 196

depth of 0.65 m and subsequently processed with Airyscan. Data presented in Fig.3D and 197

Multimedia Fig.2 were acquired with a slice depth of 0.75 m using 458/550 and 633/698 198

channels. 3D rendering was performed on Zeiss Zen 2.3 software. 199

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Experimental design and statistical analysis. All statistical analyses used are detailed in Results 201

and Figure Legends and were performed using MATLAB R2019a (MathWorks, Inc.). As the 202

Kolmogorov-Smirnov test for normality is known to be overly sensitive with small sample sizes, 203

statistical approaches accommodating unequal variance or non-parametric data were used in 204

cases (1) where maximum standard deviation (SD) was equal or greater than three times’ the 205


https://www.janelia.org/project-team/flylight/protocols

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minimum SD, or (2) there was a known ceiling/floor imposing non-normality. Within a figure, 206

statistically similar groups (p>0.05) are identified by the same letter assignment; statistically 207

different groups (p<0.05) can be identified by having differing letter group assignments. For all 208

tests requiring posthoc comparisons (ANOVA, Kruskal-Wallis), the Bonferroni method was used. 209

For any significant (p<0.05) posthoc comparison p-values of an analysis, the largest significant 210

p-value informed the selection of the closest standard p-values ultimately reported in Figure 211

Legends (i.e. p<1E-4 in the case where the largest p-value was 0.000045). 212

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RESULTS 214

Wildtype appetitive olfactory STM is stable throughout the day 215

While it has previously been shown that there are time-of-day (TOD) effects on some 216

forms of olfactory memory in Drosophila (Chouhan et al., 2015; Fropf et al., 2014, 2018; Lyons 217

& Roman, 2009), there has been no investigation of the role of the clock in appetitive olfactory 218

STM. To address this, we first tested appetitive STM of Canton-Special wildtype (WT) flies 219

entrained to a 12:12 LD cycle at six timepoints evenly spaced throughout the 24 h day (Fig. 1A, 220

black). Grouped flies (100-200) were exposed to two neutral odors, one paired with sucrose 221

and the other unpaired. Memory of this experience was assessed directly after training by 222

allowing animals to choose between the two odors; WT flies prefer the odor previously paired 223

with sucrose (Tempel et al., 1983). While a double-plot of the mean learning index (LI) of each 224

timepoint was best fit with a nonlinear 1-term Fourier curve (R2=0.9889; linear fit R2=0.5400), 225

there were no statistically significant TOD changes (n=8 per timepoint, 2-way ANOVA, 226

pZT=0.0373; posthoc comparisons for WTxZT, all p=1.00). Thus, WT appetitive STM appears to 227

be relatively stable throughout the 24 h circadian cycle. 228

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The magnitude and TOD-independence of appetitive STM requires PDF/PDFR signaling 230

To determine if the circadian clock has a role in regulation of appetitive STM, we asked if 231

the major molecular output of the core clock, the neuropeptide Pigment-dispersing factor (PDF) 232

(Renn et al., 1999; Shafer & Yao, 2014) and its receptor PDFR (Choi et al., 2009; Hyun et al., 233

2005; Lear et al., 2005; Mertens et al., 2005), affected this behavior. Mutants lacking PDF (pdf01) 234

or PDFR (han5304) were tested for appetitive STM alongside WT flies (Fig. 1A). Both pdf01 and 235


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han5304 showed a clear STM deficit compared to WT at all time points (2-way ANOVA: pgenotype = 236

6.43E-23; Bonferroni posthoc comparisons: pWT,pdf = 3.62E-23, pWT,han = 4.31E-12, ppdf,han = 237

3.44E-05). These animals had normal odor and sugar responses (data not shown). Given the 238

requirement found for both PDF and its receptor PDFR, we conclude that the PDF signaling 239

pathway supports STM throughout the day. 240

These pdf01 and han5304 data were also well fit by non-linear Fourier curves (R2 = 0.7812 241

and 0.8472 respectively; linear fit R2=0.0052 and 0.0987 respectively), but their apparent 242

amplitudes of oscillation were larger than those of WT. This is made more obvious if the data 243

are normalized to their means to allow direct comparison between genotypes (Fig. 1B). While 244

this normalization damps the amplitude of the WT oscillation, the pdf01 and han5304 curves 245

show even larger excursions, implying that the PDF signaling pathway may in fact stabilize STM 246

formation over the course of the day. 247

To test this, we assayed WT STM compared to pdf01 or han5304 at the times of day where 248

we had previously observed largest deviations from the mean: dawn (ZT1) and dusk (ZT13) (Fig. 249

1C,D). These experiments replicated the prior overall deficits seen in pdf01 and han5304 250

appetitive STM and confirmed that WT appetitive STM does not change with TOD (2-way 251

ANOVA, posthoc comparisons: Fig. 1C pWT1,13=1.00; Fig. 1D pWT1,13 = 1.00). However, as 252

predicted by our previous findings, both pdf01 and han5304 had significantly lower learning 253

scores at ZT1 relative to ZT13 (2-way ANOVA, posthoc comparisons: Fig. 1C ppdf1,13 = 0.008; Fig. 254

1D phan1,13 = 0.003). The loss of PDF signaling through PDFR therefore uncovers strong TOD 255

effects on the ability to learn. We conclude that PDF and its receptor PDFR are generally 256


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necessary for STM but also have a critical role in ensuring that animals can learn equally well at 257

all times of day. 258

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Appetitive STM requires PDF signaling in adult neurons 260

In the mature adult fly, PDF is uniquely produced in the LNv neurons of the clock, but 261

PDFR is present in both neurons and glia (Im & Taghert, 2010). To distinguish between a 262

neuronal and a glial PDF target involved in STM, we asked if neuron-specific expression of a Pdfr 263

cDNA would rescue the han5304 appetitive learning deficit. Panneuronal expression of Pdfr on a 264

han5304 background using the nsyb-GAL4 driver significantly increased STM compared to 265

parental controls (Fig. 2A, 1-way ANOVA, p=4.61E-06; posthoc comparisons pgal4,UAS = 0.808, 266

pgal4,UAS+gal4 = 6.10E-06, pUAS,UAS+gal4 = 2.07E-04). We excluded the possibility of neomorphic 267

effects from the broad nsyb-GAL4 expression pattern by investigating the same panneuronal 268

PDFR overexpression on a WT background, which failed to generate significant changes in STM 269

compared to parental controls (data not shown). Therefore we concluded that PDF/PDFR 270

signaling in neurons is sufficient for normal appetitive STM. 271

To determine if adult-specific PDF signaling was sufficient for normal STM, we used the 272

drug-inducible driver elav-GS-GAL4 to panneuronally express Pdfr on the han5304 mutant 273

background solely during adulthood. Flies were placed onto food containing RU486 or EtOH 274

vehicle directly after eclosion to fully induce transgene expression (Depetris-Chauvin et al., 275

2011; Osterwalder et al., 2001). Under these conditions, han5304;;elav-GS>Pdfr flies fed with 276

RU486 (Fig. 2B) showed significantly increased appetitive STM compared to their vehicle-fed 277

sibling controls and had learning scores equivalent to the concurrently run WT control (1-way 278


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ANOVA, p = 8.0235e-04; posthoc comparisons pEtOH,RU486 = 0.0144, pEtOH,WT = 5.872e-04, 279

pRU486,WT = 0.3288). Taken together, we conclude that PDFR in a population of adult neurons 280

promotes appetitive STM. 281

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Mushroom body Kenyon cells are not direct targets of PDF 283

We began our search for the relevant PDFR+ neurons in the mushroom body (MB), a site 284

of integration essential for olfactory learning and memory in the fly. Olfactory information is 285

delivered to the MB neuropil by sparse random activation of subpopulations of roughly 2000 286

Kenyon cells (KCs) whose soma surround the calyx of the MB. Within the compartmentalized 287

MB neuropil lobes, KC axonal projections receive stimulus-specific information from 288

dopaminergic inputs, allowing for time-space coincidence of odor-stimulus pairings (for review, 289

see Busto et al., 2010). Dorsomedial projections from PDF+ sLNv clock cells skew posteriorly in 290

close proximity to the MB KCs and calyx (Fig. 3A, and (Helfrich-Förster, 1995); while these 291

projections contain small clear core vesicles (Yasuyama & Meinertzhagen, 2010) the recently 292

published Drosophila adult brain connectome shows no synaptic routes between sLNv and MC 293

KC neurons (Xu et al., 2020) permitting us to exclusively focus on sLNv extrasynaptic dense core 294

vesicle signaling as a communication mechanism between sLNv and MBs. In fact, sLNv 295

projections show TOD-dependent changes in PDF immunoreactivity (Park et al., 2000) and low 296

levels of PDFR mRNA have been detected in some KC subpopulations (Crocker et al., 2016). 297

These prior findings led us to ask if KCs could be a direct target of PDF. 298

We labeled KCs with membrane-bound RFP using the VT030559-GAL4 driver and looked 299

for colocalization with membrane-bound GFP expressed with Pdfr-2A-LexA (Deng et al., 2019), a 300


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gene-fusion in the Pdfr locus (Fig. 3B-D) which should recapitulate the endogenous expression 301

pattern of Pdfr. Given the dense packing of the KC soma, we took particular care to maximize 302

our confocal Z-resolution and visualize independent soma by utilizing AiryScan deconvolution. A 303

Z-axis maximum image projection (MIP) of the KC soma and MB calyx (Fig. 3B) shows potential 304

overlap of reporter signal; however a flythrough of the stacked dataset (Multimedia Fig. 1) 305

shows that the entirety of this ‘overlap’ can be attributed to the extremely close proximity of 306

the PDFR+ DN1 clock cell soma and PDFR+ DN1/LNv projections to the calyx and KC soma. In an 307

examination of ~6000 KCs from three independent brains we saw only a single cell that 308

expressed both GFP and RFP (Fig. 3C, inset). We also examined the MB lobe region to detect 309

any innervation of the neuropil by PDFR+ projections; again, though the Z-axis MIP implies 310

potential overlap (Fig. 3D), examination of 3D rotational data (Multimedia Fig. 2) revealed no 311

detectable PDFR+ signal within the MB lobe neuropil. GFP signal closest in proximity belonged 312

to EB-projecting PDFR+ cells. 313

We also attempted to visualize a functional connection between the PDF+ LNvs and the 314

MB. PDFR is a G-protein coupled receptor which activates Gs to increase intracellular cAMP 315

(Duvall & Taghert, 2012, 2013). To allow us to see PDF-dependent changes in cAMP we 316

expressed the FRET-based reporter EPAC1cAMPs (Shafer et al., 2008) in the entirety of the MB 317

with MB247-LexA. In these flies, exclusive activation of LNvs was accomplished by expressing 318

ATP-gated P2X2 channels under the control of pdf-GAL4 and perfusing dissected brains with ATP 319

(Lima & Miesenböck, 2005; Yao et al., 2012). Though we performed experiments at five 320

different timepoints spanning the 24 h circadian day, we failed to detect any significant 321

response to ATP within the MB calyx or KC soma (pooled data, Fig. 3E; paired T-test, p = 0.054; 322


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unpaired T-test, p = 0.153) relative to vehicle or genetic controls. The PDFR+ neuron(s) involved 323

in the regulation of appetitive STM must therefore be extrinsic to the MB, acting as 324

interneurons downstream of the PDF+ LNvs to regulate the memory center. 325

326

Intra-clock PDF signaling is not sufficient for appetitive STM 327

The two most extensively characterized functions of PDF are regulation of daily 328

locomotor activity timing and maintenance of clock circuit intracellular cycling (manifest by 329

rhythmic locomotor activity in constant conditions). Both of these functions are carried out by 330

PDF activation of PDFR in neurons of the core clock. Because we found that PDF is required for 331

STM but does not directly signal to memory-relevant MB KCs, we next considered PDF signaling 332

within the clock itself as an indirect regulator of memory formation. If cycling of the core clock 333

was required to produce an STM-promoting non-PDF output or if circadian-regulated 334

locomotion itself was a factor in memory formation, intra-clock signaling might be memory-335

relevant. We therefore asked if PDFR expression sufficient for the rescue of canonical han5304 336

LD and DD locomotor phenotypes is also sufficient for STM rescue. To do this we first sought to 337

identify clock-related GAL4 lines that were capable of rescuing these phenotypes, with later 338

intent to examine their efficacy in the STM assay. 339

The han5304 LD locomotor activity phenotype has an advance in the timing of evening 340

anticipation onset and an early evening peak activity (Hyun et al., 2005). We observed han5304 341

activity for 3 consecutive days in 12:12 LD using the Drosophila Activity Monitor (DAM) system 342

and, consistent with prior reports, han5304 flies showed accelerated evening onset and evening 343

peak activity compared to WT (Fig. 4A-C; Student’s unpaired T-test: Fig. 4B, p=8.45E-14; Fig. 4C, 344


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p=4.38E-08). We then rescued expression of Pdfr in han5304 using two different clock-related 345

drivers: PdfR(10kb)-GAL4 and clk856-GAL4, and recorded locomotor activity under similar 346

conditions. The PdfR(10kb)-GAL4 expression pattern (Fig. 4H) includes the ellipsoid body and 1-347

2 cells of each clock subset (Parisky et al., 2016). We found that Pdfr expression under the 348

PdfR(10kb)-GAL4 promotor was sufficient to delay the han5304 evening onset and return evening 349

peak timing back to WT (Fig.4E-G) (Fig. 4F,G Kruskal-Wallis with Bonferroni posthoc 350

comparisons, (F) p=5.56E-10, posthoc comparisons: pgal4,UAS =1.00, pgal4,UAS+gal4 =5.44E-05, 351

pUAS,UAS+gal4 =2.67E-06, pgal4,WT =3.86E-05, pUAS,WT =1.89E-06, pUAS+gal4,WT =1.00; (G) p=9.60E-09, 352

posthoc comparisons: pgal4,UAS =1.00, pgal4,UAS+gal4 =2.86E-05, pUAS,UAS+gal4 =9.71E-08, pgal4,WT 353

=2.67E-02, pUAS,WT =5.61E-04, pUAS+gal4,WT =0.568). Similarly, clock-limited PDFR expression with 354

clk856-GAL4 (Fig. 4L) (Gummadova et al., 2009) significantly delayed the accelerated han5304 355

evening onset and evening peak timing (Fig. 4I-K) (Fig. 4J,K Kruskal Wallis with Bonferroni 356

posthoc comparisons, (J) p=1.58E-10, posthoc comparisons: pgal4,UAS =1.00, pgal4,UAS+gal4 =1.24E-357

07, pUAS,UAS+gal4 =3.40E-09; (K) 1.94E-13, posthoc comparisons: pgal4,UAS =0.772, pgal4,UAS+gal4 358

=8.39E-09, pUAS,UAS+gal4 =2.96E-12). 359

The other phenotype of the han5304 mutant is an inability to maintain circadian 360

rhythmicity of locomotor activity after transfer to total darkness (Hyun et al., 2005). Although 361

our STM assays utilize flies entrained to a 12:12 LD cycle, it was still possible that a PDFR+ 362

component of the clock involved in maintenance of circadian rhythmicity was required for STM. 363

We therefore examined the free-running activity of those flies in DD conditions, analyzing 364

activity from DD days 2-8 (Table 1). Compared to WT, han5304 had significantly decreased 365

rhythmicity index (RI) (Student’s unpaired T-test, p=2.84E-08) and periodicity (Welch’s T-test, 366


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p=0.0363) and the overall population was much more arrhythmic (70.4 % vs 95.6 %). PDFR 367

expression with the pdfR(10kb) promotor failed to increase han5304 RI (1-way ANOVA, p=4.75E-368

14, posthoc comparisons pgal4,UAS =0.19, pgal4,UAS+gal4 =0.0280, pUAS,UAS+gal4 =1, pgal4,WT =4.38E-14, 369

pUAS,WT =1.05E-08, pUAS+gal4,WT =5.575E-07), periodicity (Kruskal-Wallis, p=2.31E-05, posthoc 370

comparisons pgal4,UAS =1, pgal4,UAS+gal4 =1, pUAS,UAS+gal4 =1, pgal4,WT =1.79E-04, pUAS,WT =9.60E-05, 371

pUAS+gal4,WT =6.23E-03) or percent rhythmic values to WT levels. han5304;clk856>Pdfr flies had 372

significantly increased RI (1-way ANOVA, p=1.61E-08, posthoc comparisons pgal4,UAS =0.013, 373

pgal4,UAS+gal4 =8.37E-04, pUAS,UAS+gal4=8.16E-09) and percent rhythmic population values compared 374

to their parental controls, while periodicity showed decreased variance but no rescue in mean 375

values (Kruskal-Wallis, p=1.49E-03, posthoc comparisons pgal4,UAS =0.263, pgal4,UAS+gal4 =9.27E-04, 376

pUAS,UAS+gal4=0.219). 377

Taken together, we find that pdfR(10kb)-GAL4 driven expression of PDFR rescues only 378

LD activity phenotypes of han5304 mutants, while clk856-GAL4 PDFR expression is sufficient to 379

rescue both LD and DD phenotypes. These drivers therefore allowed us to ask whether PDF’s 380

role in appetitive STM is mediated through core-clock-driven LD locomotor patterns or 381

maintenance of circadian rhythmicity. Both of these hypotheses were disproven as we found 382

that neither pdfR(10kb)-GAL4 nor clk856-GAL4 expression of PDFR was able to rescue the 383

han5304 appetitive STM deficit (Fig. 5A,B; 1-way ANOVA, (A) p = 0.239, (B) p = 3.21E-05, posthoc 384

comparisons: pgal4,UAS =4.19E-05, pgal4,UAS+gal4 =0.879, pUAS,UAS+gal4 =0.001). Furthermore, as the 385

clk856-GAL4 expression pattern includes nearly all core clock neurons, it’s reasonable to 386

entirely infer that clock-based PDFR expression alone is insufficient to support normal 387

appetitive STM. However to fully exclude any possible synergistic interaction of PDF with the 388


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clock and MB in STM behavior, we co-expressed PDFR in the clock and MB simultaneously using 389

clk856-GAL4 and the strong KC-specific driver VT030559-GAL4 on a han5304 background (Fig. 390

5C). This manipulation not only failed to rescue appetitive STM compared to parental controls, 391

but in fact further impaired learning, likely due to either genetic background effects or to a 392

neomorphic effect of strong overexpression of PDFR in the MB (1-way ANOVA, p = 6.15E-03, 393

posthoc comparisons: pVTgal4,UAS =1.00, pVTgal4,clkgal4UAS+VTgal4 =8.17E-03, pclkgal4UAS,clkgal4UAS+VTgal4 394

=0.0379). These data imply that PDF must be acting on a PDFR+ neuronal target exclusive from 395

both the core clock and the MB KCs to regulate appetitive STM. Our results also argue that the 396

multiple behavioral roles of PDF signaling (on locomotor activity, DD rhthyms, and memory) are 397

independently controlled by the expression of PDFR in distinct PDF target neurons. 398

399

Aversive olfactory STM requires PDF but not PDFR 400

Differential distribution of PDFR, the only known receptor for PDF, within and external 401

to the clock allow it to have many different and independent behavioral roles. Other signaling 402

pathways, however, often use an additional strategy for diversification: multiple receptors. We 403

were struck by the difference in STM scores between pdf01 and han5304 mutants (Fig. 1A)—the 404

pdf01 STM phenotype is significantly and consistently more severe than han5304. Since both of 405

these alleles are protein null (Hyun et al., 2005; Renn et al., 1999) in theory they should have 406

the same magnitude of deficit if PDFR is the sole receptor for PDF. If, however, there were a 407

second receptor for PDF, it could provide some PDF signaling in the han5304 mutant and result in 408

a milder phenotype. 409


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To further explore the learning and memory roles of PDF and PDFR and determine if the 410

disparity in STM deficits was common to other types of memory, we assayed appetitive long-411

term memory (LTM) and aversive STM. We first investigated a possible difference between 412

pdf01 and han5304 24 h appetitive LTM (Fig. 6A). While pdf01 flies were slightly more impaired 413

than han5304 flies were, there was no statistically significant disparity between the mutants (1-414

way ANOVA, p = 8.00E-04, posthoc comparisons: pWT,pdf01= 7.79E-04, pWT,han5304 = 0.0215, 415

ppdf01,han5304 = 0.770). However we found strong evidence for the existence of a second PDF 416

receptor when we tested pdf01 and han5304 mutants for aversive STM (Fig. 6B). In this assay, 417

flies are tested for memory to an odor that was previously paired with shock; instead of 418

approaching the paired odor (as in the appetitive assay), flies demonstrate memory by avoiding 419

the paired odor. As before, pdf01 mutants had impaired aversive STM compared to WT, but 420

quite surprisingly, han5304 mutants showed no aversive STM deficit at all (1-way ANOVA, p = 421

5.08E-06, posthoc comparisons: pWT,pdf01= 4.90E-05, pWT,han5304 = 1.00, ppdf01,han5304 = 1.97E-05). 422

To definitively confirm a requirement for PDF in aversive STM and rule out the influence of 423

genetic background in pdf01, we crossed pdf01 flies to WT, pdf01, and the recently created pdfattP 424

mutant (Deng et al., 2019) and tested progeny for aversive STM (Fig. 6C). pdf01/+ heterozygotes 425

were indistinguishable from WT for STM, confirming the recessive nature of the defect. pdf01 426

homozygotes and pdfattP/pdf01 transheterozygotes had significant STM defects (Kruskal-Wallis, p 427

=4.78E-04, posthoc comparisons: pCS/pdf01,pdfattP/pdf01 = 0.0269, pCS/pdf01,pdf01/pdf01 = 3.91E-04, 428

ppdfattP/pdf01,pdf01/pdf01 = 0.676). Collectively, these data suggest that PDF normally promotes 429

aversive STM by acting not on PDFR, but on a novel, and still unidentified, receptor. 430

431


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DISCUSSION 432

A role for the circadian clock in cognition and memory has been demonstrated in many 433

ways and in many organisms, including humans (for review, see Gerstner & Yin, 2010; Krishnan 434

& Lyons, 2015; Smarr et al., 2014). To date, efforts to understand these phenomena have 435

largely involved manipulations of intracellular molecular oscillator components such as the 436

period (per) gene to abolish intracellular clock cycling. However this type of manipulation is not 437

fully representative of the nature of human circadian misalignment disorders, as a role for 438

intercellular signaling has also been shown (Yamaguchi et al., 2013). In Drosophila, where 439

investigators have tools to interrogate highly conserved circadian processes in great 440

mechanistic detail, manipulation of the signaling peptide PDF allowed us to specifically examine 441

the role of a key neuromodulatory output of the core clock circuit in cognition. Our work 442

demonstrates a requirement for PDF in the formation of associative olfactory memory 443

(schematized in Figure 7). Putting our data in the context of what is known about the molecular 444

clock’s influence on cognition, we explore below the importance of TOD on WT and PDF mutant 445

STM and provide evidence which supports the existence of a second PDF receptor. 446

447

PDF acts on multiple targets to regulate different behaviors 448

In the adult fly, we and others have shown that PDF released from the LNv neurons of 449

the core clock circuit acts, via PDFR, both on the clock circuit itself and outside of the clock to 450

drive key circadian features and rhythmicity of locomotor activity (Fig. 4, Table 1). Our data 451

show that PDF and PDFR are also required for robust formation of appetitive olfactory memory 452

and for equalizing the ability to form memory across the day (Figs. 1,2). Surprisingly, the intra-453


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clock circuit PDF signaling pathway, which is sufficient for normal locomotor activity, plays no 454

role in PDF’s regulation of memory, demonstrating that PDF independently directs multiple 455

behaviors depending on the location of its target receptor PDFR. Regulation of associative 456

olfactory memory requires PDF signaling outside both the clock and the MB KCs, a key memory 457

formation center (Figs. 3-5). An additional layer of valence-specific control over memory 458

formation is provided by the fact that PDF appears to act in aversive olfactory memory 459

formation independently of PDFR, implying the existence of a second receptor system (Fig. 6). 460

461

The role of core-clock driven circadian rhythms themselves in olfactory STM formation is 462

limited 463

In Drosophila, a requirement for molecular clock components like per in cognitive tasks 464

has varied, dependent on the type of learning and stage of memory studied (Chouhan et al., 465

2015; Fropf et al., 2014, 2018; Gailey et al., 1991; Inami et al., 2020; Le Glou et al., 2012; Lyons 466

& Roman, 2009; Sakai et al., 2004). Interestingly, in cases where the locus of action for per has 467

been mapped, PER acts outside the core clock circuit to regulate memory but these studies do 468

not explore TOD effects. In studies which examine TOD effects on cognition (Fropf et al., 2014, 469

2018; Lyons & Roman, 2009), per mutants or animals rendered arrhythmic by altered light 470

conditions lose TOD differences. Thus the role of the cycling core clock itself in learning and 471

memory has been mostly restricted to TOD modulation, and PER, like PDF and PDFR, appears to 472

have a role in memory that is independent of its function in the core clock circuit. 473

474

PDF signaling opposes a latent TOD rhythm in appetitive STM 475


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Our data suggest that the requirement for PDF in supporting normal levels of appetitive 476

STM is TOD-independent i.e. PDF is required at all times of day to form robust appetitive STM. 477

PDF has been implicated in a wide variety of behaviors that are influenced by circadian time 478

(Chen et al., 1999; Chen et al., 2016; Chung et al., 2009; Keene et al., 2011; Kim et al., 2013; 479

Krupp et al., 2013; Mertens et al., 2005; Nagy et al., 2019) but its only previously known roles in 480

learning and memory are courtship-related (Inami et al., 2020). The PDF peptide is anatomically 481

well placed to be an agent of general learning enhancement since it can act on long range 482

targets by virtue of extrasynaptic release and diffusion, allowing it to provide neuromodulation 483

over the entire circadian cycle. The fact that PDF has been characterized as an arousal-484

promoting signal (Chung et al., 2009; Parisky et al., 2008) may be germane to this general role 485

since arousal state is critical to attention state in working memory (Ricker et al., 2018). 486

An intriguing additional feature of the pdf and Pdfr mutant phenotypes is the 487

emergence of a latent TOD oscillation in STM (Fig. 1). Daily changes in sLNv structure and 488

cycling accumulation of PDF peptide at sLNv terminals (Park et al., 2000) have minima and 489

maxima at ZT1 and ZT13 (Fernández et al., 2008; Gorostiza et al., 2014) which align with our 490

observed peak and trough of appetitive STM in PDF signaling mutants. The role of PDF signaling 491

in morning arousal (Parisky et al., 2008) may explain the early day appetitive STM trough of pdf 492

and Pdfr animals: since a heightened arousal state facilitates learning, an absence of PDF 493

signaling reduces morning arousal and cripples the ability of animals to make associations with 494

food-predictive odors. Furthermore, the latent nature of TOD effects in PDF signaling mutants 495

relative to the stable nature of WT STM throughout the day allows us to infer that this stability 496

is the sum of oscillating contributions from PDF signaling and one or more other cycling factors. 497


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This unknown oscillator may be complex, dependent on interactions between sleep drive, the 498

sleep homeostat and arousal systems. In humans, multiple oscillators for working memory have 499

been proposed (Folkard et al., 1983). 500

501

TOD-independent appetitive memory has survival value 502

The diversity and abundance of roles for PDF in behavior illustrates how essential the 503

output of the core clock is to an organism’s optimal function and survival. It’s important 504

nevertheless to explore why, in the context of appetitive STM (which is ultimately TOD-505

independent), an organism’s capacity for appetitive learning should be so critically linked to its 506

timekeeping capabilities. Though prior work is conflicting regarding TOD influence on 507

associative STM (Fropf et al., 2018; Lyons & Roman, 2009), TOD effects are consistently found 508

in associative LTM (Chouhan et al., 2015, 2017; Fropf et al., 2014, 2018; Lyons & Roman, 2009). 509

In this context we ask: why is it important for appetitive learning to remain stable throughout 510

the day? Given the nutrient-based nature of our assay, we posit that at the time of training, an 511

organism lacks information necessary to evaluate the benefit of devoting metabolic resources 512

to consolidation of that learning—i.e. future food availability is unknown. Though the widely-513

used appetitive STM paradigm requires starvation for expression of memory immediately after 514

training, significant expression of appetitive LTM requires starvation only at the time of 515

retrieval but not at the time of acquisition (Chouhan et al., 2017; Krashes et al., 2009). 516

Furthermore, feeding after appetitive training prompts the decay of appetitive STM within 10-517

30 min, though LTM can still be observed with subsequent starvation (Krashes et al., 2009). In 518

light of a STM requirement for PDF throughout the day, we hypothesize that PDF may help 519


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maximize information acquisition at the front end of the process, to allow memory to be 520

dispensed with or consolidated in a manner dependent on future internal metabolic state 521

information. 522

523

PDF signaling in aversive memory utilizes an unknown receptor 524

We also find that PDF is required for aversive olfactory STM. Surprisingly, however we 525

did not uncover a requirement for PDFR, the only characterized receptor for this peptide. 526

Aversive associative STM is therefore the only behavior known for which PDF is required but 527

PDFR is dispensable, providing the first evidence in Drosophila for a second PDF receptor. In 528

other organisms, circadian output peptides have multiple receptors. Three GPCRs for the C. 529

elegans homolog of PDF have been identified, each of which is highly similar to Drosophila PDFR 530

and related to VIPR1 and VIPR2, the two known mammalian receptors for VIP (a mammalian 531

peptide with similar clock roles) (Janssen et al., 2008, 2009). We suggest that receptor diversity 532

allows the valence-specific regulation of associative STM by regionally segregated and distinct 533

receptors. Since the circuitry involved in acquisition of appetitive versus aversive memory 534

involves different sets of neurons (Liu et al., 2012; Riemensperger et al., 2005; Yamazaki et al., 535

2018), this implies that that PDF will act at circuit nodes in each pathway that are valence-536

specific and is consistent with our finding that PDF does not act on Kenyon cells, the final 537

common substrate of memory. Ultimately, identification and characterization of the second 538

PDF receptor will be required to fully understand this peptide’s multiple roles in behavior. 539

540


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Table 1. Free-running circadian locomotor activity properties of han5304 mutants and rescue 805

genotypes. 806

Genotype Rhythmicity Index (RI) % Rhythmic Periodicity

han5304 0.267±0.026 70.4 23.5±0.281

WT 0.501±0.021 95.6 24.1±0.047

han5304;pdfR(10kb)-GAL4/+ 0.218±0.017 50.0 23.5±0.233

han5304;;UAS-Pdfr/+ 0.275±0.016 72.9 23.3±0.137

han5304;pdfR(10kb)-GAL4/+;UAS-Pdfr/+ 0.295±0.021 73.9 23.6±0.155

WT 0.445±0.020 93.5 24.0±0.071

han5304;clk856-GAL4/+ 0.308±0.025 71.0 23.1±0.154

han5304;;UAS-Pdfr/+ 0.216±0.019 60.7 23.8±0.293

han5304;clk856-GAL4/+;UAS-Pdfr/+ 0.422±0.019 93.8 23.7±0.055

Table 1. Free-running circadian locomotor activity properties of han5304 mutants and rescue 807

genotypes. Flies used in Figure 4 were released into free-running DD conditions for 8 days 808

following Figure 4 LD recordings; circadian properties are calculated from DD days 2-8. Shaded 809

groupings indicate independent experimental comparison groups. 810

811


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FIGURE LEGENDS 812

813

Figure 1. The core clock supports appetitive short-term memory throughout the circadian 814

cycle via a PDF signaling pathway. Appetitive olfactory 2 min short-term memory (STM) of WT 815

and PDF pathway mutants. (A,B) Learning index (LI) scores for STM tested every 4 h through the 816

24 h cycle and double-plotted, with white and grey background indicating lights on/off of 817

entrainment phase. Each genotype was fit with 1-term Fourier curve, R2 values shown in (A). (A) 818

Mean LI (circles; n=8 for each timepoint) and (B) normalized mean LI (triangles; mean 819

subtracted, then divided by standard deviation). (C,D) STM of pdf01 and han5304 mutants 820

compared to WT at ZT1 and ZT13. Mean LI scores are shown + SEM, with individual datapoints 821

(circles). Letter categorization indicates groups of statistical equivalence (p>0.05) or difference 822

found by 2-way ANOVA with interactions, Bonferroni posthoc comparisons (all significant 823

comparisons, panel C: p<0.01, panel D: p<0.005). 824

825

Figure 2. Adult-specific neuronal PDF-PDFR signaling is sufficient for appetitive short-term 826

memory formation. Appetitive olfactory STM tested between ZT0-4 in han5304 mutants with 827

panneuronal PDFR expressed (A) constitutively, compared to genetic controls and (B) adult-828

specifically using the RU486-inducible GeneSwitch system, compared to EtOH vehicle and WT 829

controls. Mean LI scores are shown + SEM, with individual datapoints (circles). Letter 830

categorization indicates groups of statistical similarity (p>0.05) or difference found by 1-way 831

ANOVA with Bonferroni posthoc comparisons (all significant comparisons, panel A: p<0.0005, 832

panel B: p<0.05). 833


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834

Figure 3. The MB is not a direct target of the PDF signaling pathway. (A) Cartoon 835

representation of LNv/MB anatomy. Purple PDF+ LNv soma (light:small, dark:large) are shown 836

with other core clock cells in light grey. sLNvs send projections anterodorsomedially in close 837

proximity to the MB (magenta) calyx, as indicated. (Inset) Area shown in main panel indicated 838

by dotted outline on whole brain. (B-D) Confocal images of MB (magenta) and putative Pdfr 839

(green) expression patterns. (B) Maximum intensity projection (MIP) of calyx, with single planar 840

slice shown in (C) where dotted outline shows the only cell with colocalization of markers seen 841

in n=3 brains (magnified,inset). (D) MIP of MB lobe region. (E) LNv neurons were activated by 842

pdf-GAL4 expression of the ATP-stimulated P2X2 channel and compared to AHL vehicle and 843

empty driver controls. Data shows MB calyx intracellular cAMP as reported by EPAC1camps 844

FRET signal imaged at 2 Hz; line mean, shadow + SEM. N of each genotype (shown in 845

parentheses) pooled from five timepoints throughout the 24 h cycle. T-test comparisons 846

(paired, unpaired) of mean terminal 10 s are non-significant. 847

848

Figure 4. Clock-based PDFR supports normal locomotor activity. 849

LD activity and appetitive STM for han5304 and cell-specific Pdfr rescue mutants. (A,E,I) 850

Population LD activity (mean + SEM) shown in 30 min bins averaged from 3 consecutive days. In 851

(A), mean population values of evening anticipation onset (arrows) and evening peak 852

(arrowheads) are shown. Evening anticipation onset (B,F,J) and the evening activity peak 853

(C,G,K) were extracted for individual subjects (circle datapoints; overlaid box-and-whisker). (D) 854

In addition to other cells, core clock neurons (categorized by subtype DN, LNd, LPN, and LNv) 855


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and EB soma and ring neuropil in han5304 lack PDFR (indicated by empty outlined ROIs). (H,L) 856

Compared to (D), filled ROIs demonstrate (H) PdfR(10kb)-GAL4 and (L) clk856-GAL4 mediated 857

PDFR rescue expression patterns on the han5304 background. Letter categorization indicates 858

groups of statistical similarity (p > 0.05) or difference found by (B,C) Student’s unpaired t-test (p 859

< 1E-7); (F,G,J,K) Kruskal-Wallis with Bonferroni posthoc comparisons (all significant 860

comparisons panel F: p < 0.0001; panel G: p < 0.05; panel J: p < 1E-7; panel K p < 1E-9). 861

862

Figure 5. PDF signaling in the clock and MB does not support STM. Appetitive STM of han5304 863

mutants with cell-specific constitutive Pdfr expression. Mean LI scores are shown + SEM, with 864

individual datapoints (circles). Letter categorization within each panel indicates groups of 865

statistical similarity (p > 0.05) or difference found by 1-way ANOVA with Bonferroni posthoc 866

comparisons (all significant comparisons, panel B: p<0.005; C: p < 0.05). 867

868

Figure 6. Multiple receptor targets permit valence-specific support of STM by a single clock 869

output. Mean LI scores are shown + SEM, with individual datapoints (circles). (A) 24 h 870

appetitive LTM of pdf01 and han5304 mutants. (B,C) Aversive STM of WT and PDF pathway 871

mutants. Letter categorizations indicate groups of statistical similarity (p>0.05) or difference 872

found by 1-way ANOVA with Bonferroni posthoc comparisons (all significant comparisons, 873

panel A: p<0.05; panel B: p<1E-4; panel C: p<0.05). 874

875

Figure 7. The core clock regulates distinct behaviors via discrete PDF targets: a proposed 876

model. Localization of PDFR to the clock permits control of locomotor activity independently 877


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from control of memory. Signaling through PDFR in a population of interneurons extrinsic to 878

both the clock and MB (here shown as IN1) permits regulation of appetitive memory. We 879

propose that, in place of PDF-PDFR signaling, PDF activation of a novel unidentified receptor 880

(here called PDFR2) in a separate population of interneurons (IN2) is required for aversive 881

memory. 882

883

Multimedia Figure 1. Putative PDFR expression is excluded from the MB Kenyon cells. 884

Confocal image of MB calyx and Kenyon cell soma, acquired with a slice depth of 0.65 m, 885

processed with Airyscan, and rendered as a fly through 5 fps video (posterior anterior). MB 886

calyx and Kenyon cell soma are shown in magenta (fixed with dsRed antibody staining of 887

VT030559-GAL4>UAS-mCD8-IVS-RFP); putative PDFR expression pattern is shown in green 888

(fixed with GFP antibody staining of pdfR-2A-LexA>LexAop-mCD8-IVS-GFP). 889

890

Multimedia Figure 2. Putative PDFR expression is excluded from the MB lobe neuropil. 891

Confocal image of MB lobe neuropil, acquired with a slice depth of 0.75 m and rendered as a 892

3D rotation around the X axis. MB lobes are shown in magenta (fixed with dsRed antibody 893

staining of VT030559-GAL4>UAS-mCD8-IVS-RFP); putative PDFR expression is shown in green 894

(fixed with GFP antibody staining of pdfR-2A-LexA>LexAop-mCD8-IVS-GFP). The MB ’ lobe 895

tips hug the PDFR+ EB projection rings without overlap. 896

897


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FIGURE 1 898

899

900

901

902

FIGURE 2 903

904

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FIGURE 3 907

908

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FIGURE 4 911

912

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FIGURE 5 915

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917

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921

FIGURE 6 922

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FIGURE 7 925

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