Identification of a biomarker for sleep drivein flies and humansLaurent Seugnet*, Jaime Boero†, Laura Gottschalk*, Stephen P. Duntley†, and Paul J. Shaw*‡

*Anatomy and Neurobiology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8108, St. Louis, MO 63110;and †Department of Neurology, Washington University Sleep Medicine Center, 212 North Kingshighway, Suite 237, St. Louis, MO 63108

Communicated by Gene E. Robinson, University of Illinois at Urbana–Champaign, November 7, 2006 (received for review August 31, 2006)

It is a common experience to sacrifice sleep to meet the demandsof our 24-h society. Current estimates reveal that as a society, wesleep on average 2 h less than we did 40 years ago. This level ofsleep restriction results in negative health outcomes and is suffi-cient to produce cognitive deficits and reduced attention and isassociated with increased risk for traffic and occupational acci-dents. Unfortunately, there is no simple quantifiable marker thatcan detect an individual who is excessively sleepy before adverseoutcomes become evident. To address this issue, we have devel-oped a simple and effective strategy for identifying biomarkers ofsleepiness by using genetic and pharmacological tools that disso-ciate sleep drive from wake time in the model organism Drosophilamelanogaster. These studies have identified a biomarker, Amylase,that is highly correlated with sleep drive. More importantly, bothsalivary Amylase activity and mRNA levels are also responsive toextended waking in humans. These data indicate that the fly isrelevant for human sleep research and represents a first step indeveloping an effective method for detecting sleepiness in vul-nerable populations.

Drosophila � saliva � sleep deprivation

I t has been suggested that forced and self-inflicted sleep losshave reached epidemic proportions in Western industrialized

populations (1, 2), costing billions of dollars in lost productivityand creating hazardous conditions on our roadways (3), in ourskies (4), and in our hospitals (5). The National Highway TrafficSafety Administration estimates that 20% of motor vehiclecrashes are attributed to sleepiness and fully 37% of adult driversreport falling asleep at the wheel at some point in their lives.Moreover, both regional and long-haul pilots accumulate sleepdebt during trips, fall asleep in the cockpit, and experience levelsof sleepiness that are associated with performance decrements(6, 7). In the hospital setting, training demands frequentlydisrupt the sleep of medical residents, which is then associatedwith increased attentional failures and medical errors (8). In-deed, after a heavy call rotation, the driving performance ofmedical residents was similar to those with a blood alcohol levelof 0.05 g % (9) and is associated with increased risk of fallingasleep while driving (10).

Given the magnitude of this problem, it is not surprising thatthe sleep community, public health officials, and others havedevoted considerable attention toward minimizing the negativeimpact of sleep loss on public health and safety (11). In additionto more focused basic research and increased educational cam-paigns to create public awareness, both regulatory and legislativeinitiatives have been implemented to address this problem. Ageneral theme that has emerged from all of these efforts hasbeen the importance of identifying a simple and quantifiablebiomarker of sleepiness (11–13). A biomarker of sleepinessshould be responsive to increasing levels of sleep debt and shouldonly be activated by periods of waking that are followed bycompensatory increases in sleep time (sleep homeostasis). Un-fortunately, identifying such a marker in humans poses signifi-cant challenges.

In this work, we outline a strategy for identifying biomarkersof sleepiness using the model organism Drosophila melanogaster.This strategy is based on a unique set of genetic, behavioral, andpharmacological tools that have been identified in the fly thatallow for the disassociation of wake time with sleep drive(14–16). By using this approach, we have identified a molecule,Amylase, which is highly correlated with sleep drive in the fly andis also responsive to waking in humans.

ResultsSaliva is a suitable tissue for identifying biomarkers of sleepinessin humans, given that brainstem nuclei regulating salivary glandactivity receive inputs from neural structures in the forebrainthat are centers for homeostatic regulation (17, 18). In addition,saliva contains �3,000 mRNA species (19). We wonderedwhether any of these genes could be used as biomarkers ofsleepiness. To reduce the number of candidate genes that needto be tested in humans, we first looked for likely candidates byusing results obtained from several independent Drosophilamicroarray studies. Amylase was consistently modified by sleeploss using a variety of sleep deprivation (SD) protocols. Thisobservation coupled with the fact that amylase is abundant in areadily accessible fluid in humans prompted the current set ofexperiments.

To be effective, a biomarker should be responsive to increas-ing levels of sleep debt and should only be activated by periodsof waking that are followed by compensatory increases in sleeptime (sleep homeostasis). With this in mind, we have evaluatedthe reliability of Amylase as a biomarker of sleep drive by usinggenetic and pharmacological tools that differentially activatehomeostatic mechanisms in the model organism D. melanogaster.First, we evaluated the temporal dynamics of Amylase mRNA inflies mutant for the canonical clock gene cycle (cyc01) after 3, 6,9, and 12 h of SD. The detrimental effects of waking areaccelerated in cyc01 mutants and accrue over the course of ashort and well defined interval measured in hours (15, 20). Asseen in Fig. 1A, even small amounts of sleep loss (3 h) result inlarge compensatory increases in sleep. Importantly, as cyc01 f liesexperience greater amounts of sleep loss, they become increas-ingly longer sleepers. Amylase mRNA levels increased progres-sively with the duration of waking indicating that it is responsiveto increasing levels of sleep debt (Fig. 1B).

Next, we evaluated whether Amylase expression is associatedwith conditions where sleep drive is high or whether it isnonspecifically activated by waking. To accomplish this goal, weevaluated the temporal dynamics of Amylase expression in fliesmutant for timeless (tim01) after 3, 6, 9, and 12 h of SD (Fig. 2A).tim01 f lies are specifically resistant to short-term SD (3 and 6 h)but exhibit a normal homeostatic response after 9 and 12 h of SD

Author contributions: P.J.S. designed research; L.S., J.B., L.G., S.P.D., and P.J.S. performedresearch; L.S. and P.J.S. analyzed data; and L.S. and P.S. wrote the paper.

The authors declare no conflict of interest.

Abbreviations: SD, sleep deprivation; RT, reverse transcription.

‡To whom correspondence should be addressed. E-mail: shawp@pcg.wustl.edu.

© 2006 by The National Academy of Sciences of the USA

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(14, 15). As seen in Fig. 2B, Amylase mRNA levels were elevatedafter SD durations that activate sleep homeostatic mechanisms(9 and 12 h SD; filled bars). To determine whether these resultscould be generalized to other experimental conditions, weinduced periods of waking that differentially activated sleephomeostasis by using caffeine and methamphetamine. Bothcaffeine and methamphetamine each produce sustained periodsof waking and similar locomotor activity profiles. However,unlike caffeine, f lies do not compensate for the lost sleepaccrued during methamphetamine-induced waking (Fig. 2C)

(14, 16, 21). As seen in Fig. 2D, Amylase expression was stronglyactivated by caffeine (filled bars) but not by methamphetamine(open bars). To determine whether Amylase levels are associatedwith naturally occurring conditions where sleep drive is high, weevaluated its progression during the first few days of the flies’adult life. Flies, like humans, exhibit ontogenetic variations inbrain plasticity that are associated with increased sleep time (21,22). As seen in Fig. 2E, daytime sleep was high immediately aftereclosion and declined to adult levels by 3 days of age. Interest-ingly, the levels of Amylase followed these naturally occurring

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Fig. 2. Amylase is up-regulated during waking conditions associated with sleepiness. (A) tim01 flies show a minimal sleep rebound after 3 and 6 h of SD (openbars) but generate a sleep rebound typical of other clock mutants after 9 and 12 h of SD (filled bars). Percentage of sleep recovered is calculated as a ratio ofthe amount of sleep recovered divided by that lost. (B) Amylase mRNA levels remain low after deprivations that do not activate homeostatic mechanisms (3 and6 h SD; open bars) but are elevated after deprivations that activate homeostatic mechanisms (9 and 12 h SD; filled bars). (C) Waking induced by methamphetamine(meth; 1 mg/ml; open bars) did not induce a homeostatic response, whereas waking induced by caffeine (2.5 mg/ml; filled bars) exhibited a rebound of similarmagnitude as that seen after manual SD. (D) Amylase is elevated after caffeine administration but not after waking induced by methamphetamine. (E) Daytimesleep is highest in young flies and declines to stable adult values by 3 days of age. (F) Amylase levels decline with sleep time. Data are presented as percentageof 5-day-old flies.

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changes in sleep time (Fig. 2F). Together, these data indicatethat Amylase is not simply a marker for waking but also forconditions where sleep drive is elevated.

To determine whether Amylase could provide an assessmentof sleep drive in real time, we evaluated a Drosophila line inwhich the promoter for Amylase was linked to the fireflyluciferase (Amy/Luc) (23). As shown in Fig. 3, bioluminescencewas high after 16 h of SD and declined during 12 h of recovery(squares). In contrast, bioluminescence was low in siblings thathad a full nights’ sleep (circles) but increased as these flies spenttime on caffeine consistent with the effects of caffeine on sleephomeostasis described above (14, 21). Thus, changes in biolu-minescence are correlated with rising and falling levels of sleepdrive in real time.

Our data indicate that Amylase is highly correlated with sleepdrive, but we wondered whether it plays a more direct role insleep regulation. In Drosophila, �-Amylase enzymes are encodedby a pair of genes located on the right arm of the secondchromosome, amy-p and amy-d (24). As shown in Fig. 3B, f liescarrying a null mutation for both amylase genes (amy-pn amy-dn)display normal sleep time and architecture. Moreover, amy-pn

amy-dn f lies respond to SD with a homeostatic response similar

to wild-type flies, indicating that Amylase is not mechanisticallyinvolved in sleep regulation (Fig. 3C).

We wondered whether Amylase activity could be used as abiomarker for sleep drive in humans. Fig. 4 A and B demonstratethat Amylase activity was increased in both humans and flies after28 h of sustained waking compared with untreated circadian-matched controls; total salivary protein and volume were notsignificantly altered (P � 0.10; data not shown). In flies, paraquatdid not alter Amylase activity, suggesting that these changes werenot due to stress (Fig. 4A). Similarly, salivary cortisol was notaltered by SD (0.254 � 0.06 �g/dl) compared with untreatedcircadian-matched controls (0.251 � 0.06 �g/dl), indicating thatstress is also an unlikely explanation for the changes in Amylase inhumans (n � 9). Recent reports indicate that mRNA from cell-freesaliva extracts can be used to identify biomarkers of disease (25).Therefore, we evaluated Amylase mRNA levels in our sleep-deprived subjects. To ensure that the integrity of mRNA wasconsistent between subjects, only samples with intact �-actin wereevaluated (25). As shown in Fig. 4 C and D, Amylase mRNA wasincreased �2-fold in both humans and flies after SD. Together,these data indicate that, as a group, Amylase activity and mRNAlevels are elevated by sustained waking in humans. Because indi-viduals vary greatly in their response to sleep loss (12), it isimportant to present data from individual subjects. As shown in Fig.5A, Amylase activity was numerically elevated in five of ninesubjects. In contrast, all subjects with intact salivary mRNA showednumerically increased Amylase mRNA levels, including four sub-jects whose Amylase activity was not altered (Fig. 5B). Interestingly,data from both the fly and human suggest that Amylase mRNA isa more sensitive measurement of sleepiness than Amylase activity.

DiscussionWe have outlined a simple and effective strategy for identifyingbiomarkers of sleepiness by using unique genetic and pharma-cological tools in Drosophila. With this approach, it is possible toevaluate the behavior of a candidate molecule under a variety ofexperimental conditions and determine whether it is consistentlyassociated with high sleep drive or whether it is nonspecificallyactivated by a particular experimental protocol. In addition, wehave shown that it is possible to evaluate biomarkers of sleep-iness in human subjects from samples derived from a readilyaccessible biological f luid by using noninvasive procedures.More importantly, we have identified a molecule, Amylase, thatis highly correlated with sleep drive in the fly and is alsoresponsive to waking in humans. It is important to note thatrecent studies have emphasized that the utility of saliva as adiagnostic tool will likely require a panel of biomarkers to beeffective (26). As such, our data demonstrating that 28 h ofwaking increased salivary Amylase activity and mRNA representthe first step in developing an effective method for detectingsleepiness in vulnerable populations.

A noninvasive peripheral marker of sleepiness may be usefulas an independent evaluation of sleep phenotypes both in thelaboratory and in the field where electrophysiological measure-ments are not feasible. For example, genetic studies have begunto identify mutations in humans, mice, and flies that alter sleeptime and sleep homeostasis (27–30). Similarly, ethological stud-ies have reported instances where animals can sustain waking forextended periods of time without exhibiting a homeostaticresponse [e.g., cetacean (31) and white-crown sparrow (32)].Frequently, it is not clear whether a mutation or an adaptationhas altered sleep need or whether it has disrupted sleep-regulatory mechanisms and thus simply degraded the ability ofthe animal to respond appropriately to sleep loss. To distinguishbetween these possibilities, it is necessary to evaluate outcomemeasurements that should be adversely affected by inadequatesleep (e.g., lifespan, learning, and memory) but are not them-

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Fig. 3. Amylase can provide an assessment of sleep drive in real-time. (A)Bioluminescence (counts/min � SEM) in Amy/Luc flies is high immediatelyafter 12 h of SD (n � 9) and declines during recovery (squares). In contrast,Amy/Luc flies that have slept all night and are placed onto caffeine (2.5 mg/ml)in the morning (n � 9) show a progressive increase in bioluminescence as sleepdebt accrues (circles). Flies were maintained on 0.5% sucrose supplementedwith 1 mM beetle luciferin. (B) Flies null for amylase display normal sleep timeand sleep consolidation (average sleep bout duration (n � 32/group). (C)amy-null flies exhibit wild-type homeostatic response after SD; data indicatecumulative sleep lost then gained after 6 h of SD. A negative slope indicatessleep lost, and a positive slope indicates sleep gained; when the slope is zerorecovery is complete.

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selves dependent on sleep regulatory networks. These experi-ments are not only time-consuming and costly, they are alsoconfounded by the possibility that the mutation/adaptationcould adversely alter sleep and the outcome measurementindependently. Although not definitive, a biomarker that isindependent of sleep regulatory mechanisms may provide ad-ditional mechanistic insight into the nature of the mutationand/or adaptation. Interestingly, f lies mutant for Amylase re-spond to SD with a homeostatic response similar to wild-typeflies, indicating that Amylase is not mechanistically involved insleep regulation. Thus, Amylase is suitable to evaluate whetheranimals that experience sustained waking accrue sleep debt orwhether they have a reduced need for sleep.

A limitation of our human study is the lack of subjectivemeasures of sleepiness and objective measurements of perfor-mance decrements. However, it should be noted that a singlenight of sleep loss is known to reliably produce subjectivefeelings of sleepiness, negative mood, cognitive impairment,reduced attention, and driving impairments equivalent to bloodalcohol level of 0.1 g % (33–35). Moreover, increased Amylaseactivity and mRNA levels were observed after 28 h of waking, 4more waking hours than what would be considered criminal fordrivers who cause accidents in New Jersey under ‘‘Maggie’s Law’’(11). Thus, the 28 h of waking in this experiment is more than

sufficient to increase sleepiness and impair performance. Anadditional concern is whether the changes we see in Amylase aredue to increased sleepiness or to stress. In flies, paraquat did notalter Amylase activity or mRNA levels, suggesting that thesechanges were not due to stress. A previous study in humansfound that shorter amounts of SD (�8 h) do not activate salivaryAmylase activity (36), and cortisol levels were unchanged after28 h in our experiment. Thus, it is unlikely that stress plays a rolein modifying Amylase during sustained periods of waking ineither humans or flies.

Finally, one can ask whether Amylase will be effective indetecting sleepiness in individuals or whether it will only beeffective using population measurements. This question is com-plicated by the growing awareness that individuals vary in theirsensitivity to SD both in the extent to which they report beingsleepy and in subsequent neurobehavioral deficits (12). Thesedeficits are both task-specific and highly reproducible for a givensubject, suggesting that they reflect trait characteristics. Al-though our sample size was not sufficient to evaluate individualdifferences directly, our data indicate that Amylase mRNA willbe effective in measuring sleepiness in individuals. As seen inFig. 5B, all subjects with intact salivary mRNA displayed nu-merical increases in Amylase after 28 h of waking. Whether theobserved variability in Amylase mRNA levels will ultimatelyreflect individual trait characteristics in sensitivity to sleep lossawaits future investigations.

A major question about Drosophila sleep research is whetherit has relevance for human sleep studies. We demonstrate herethat 28 h of waking in human subjects significantly increasedAmylase activity and mRNA levels compared with untreated,circadian-matched controls. Thus, a marker originally identifiedin flies is also modified by extended episodes of waking inhumans. To our knowledge, results obtained in the fly anddirectly applied to human sleep research have not been reportedpreviously. Thus, this work supports findings from other fieldssuch as circadian rhythms, memory, and development that the flycan generate data that is immediately relevant for human studies.

MethodsFlies. Flies were cultured at 25°C in 50–60% humidity for a12 h:12 h light:dark cycle on yeast, dark corn syrup, and agar food

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Fig. 4. Amylase can be used as a marker in flies and humans. (A and B) Amylase activity, but not protein levels, is increased in homogenates from Drosophilaheads after 28 h of enforced waking (n � 20) (A) and in saliva samples taken from normal healthy humans (n � 9; P � 0.05) (B). In flies, 20 �M paraquat did notalter Amylase activity. Human saliva samples were collected at the same circadian time over consecutive weekends where each subject served as his or her ownuntreated control. (C and D) Amylase mRNA is increased in homogenates from Drosophila heads after 28 h of enforced waking (n � 20) (C) and in saliva samplestaken from normal healthy humans (n � 6) after 28 h of waking (D) (P � 0.05).

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Fig. 5. Individual differences. (A) Amylase activity was increased in five ofnine subjects after 28 h of waking. (B) Amylase mRNA levels were increased inall six saliva samples with intact �-actin. Arrows designate subjects withAmylase activity levels that were not elevated but that displayed increases inAmylase mRNA.

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as described (15). Lights came on at 8:00 a.m. cyc01;ry andyw;tim01f lies were obtained from J. C. Hall (Brandeis University,Waltham, MA); Canton-S flies were obtained from the Bloom-ington Stock Center (Bloomington, IN), and Amy/Luc flies wereobtained from Donnel Hickey (University of Ottawa, Ottawa,ON, Canada). Newly eclosed adult f lies were collected fromculture vials daily under CO2 anesthesia.

Procedure. Three-day-old female flies were placed into 65-mm glasstubes, and sleep parameters were continuously evaluated through-out all experiments by using the Trikinetics activity monitoringsystem as described (refs. 15 and 21; see also www.Trikinetics.com).Flies were subjected to SD by using an automated SD apparatusthat has been found to produce waking without nonspecificallyactivating stress responses (15, 21). Three independent replicates of32 flies were conducted for each time point. After SD, two-thirdsof the flies from each group were frozen, and RNA was extractedfrom whole heads. The remaining flies (one-third) were monitoredfor an additional 24 h to assay the size of the homeostatic response.By using this protocol, both gene expression and behavior can beevaluated in siblings that have been exposed to identical environ-ments and experimental manipulations.

Pharmacological studies were conducted on female Canton-sf lies. After 2 days of baseline recordings, f lies were placed ontocaffeine (2.5 mg/ml), methamphetamine (0.5 mg/ml), or vehicle2 h before the beginning of their primary sleep period. The nextmorning, after 14 h of drug treatment, two-thirds of the fliesfrom each group were frozen, and RNA was extracted fromwhole heads. The remaining flies were monitored for an addi-tional 24 h to assay the size of the homeostatic response.

Amylase activity was evaluated after 28 h of SD in Cs f lies tomatch the duration of waking used in the human experiments.Female Cs f lies were sleep deprived for 28 h beginning at lightsout (8:00 p.m.). Whole-body homogenates were extracted fromfour groups of five flies and compared with untreated circadian-matched controls; experiments were conducted in duplicate.Amylase activity was determined by using the Infinity Amylasereagent (TR25421; Thermo Electron Corp., Louisville, CO)according to the manufacturer’s instructions. All samples wereassayed in quadruplicate. To evaluate the effects of stress onAmylase activity, Cs females were placed onto 20 �M paraquatdissolved in 1% agar/5% sucrose for 16 h and killed at the samecircadian time as the 28-h SD flies and their controls. We limitedthe duration on paraquat to 16 h because flies begin to die duringlonger exposure times (15).

Before luminescence recordings, sleep was evaluated for 2days in female Amy/Luc flies as described above with theexception that they were maintained on 0.5% sucrose supple-mented with 1 mM beetle luciferin (Promega, Madison Wiscon-sin). On day 3, f lies were sleep deprived for 12 h (n � 9) or servedas untreated controls (n � 9). Flies that were sleep deprived wereremoved from their 65-mm glass tubes and placed into Petridishes with 200 �l of fresh 0.5% sucrose/1 mM beetle luciferin.Siblings that had not been sleep deprived were placed into Petridishes with 2.5 mg/ml caffeine added to the 0.5% sucrose/1 mMbeetle luciferin. Bioluminescence was subsequently recorded in1-min bins for 12 h under photomultiplier tubes (HC135–11MOD; Hamamatsu, Shizouka, Japan) as described (37). Amy/Luc flies that had never been exposed to luciferin were recordedfor 12 h and served as a blank. Luminescence for SD andcaffeine-treated f lies was subtracted from the blank andsmoothed by using a 3-h running average. Two independentreplicates were conducted.

Quantitative PCR. Total RNA was isolated from fly heads by usingTRIzol (Invitrogen, Carlsbad, CA) following the manufacturer’sprotocol. Reverse-transcription (RT) reactions were carried outin parallel on DNase I-digested total RNA as described (15). RTproducts were stored at �80°C until use. PCRs to measure levelsof artificial transcript were performed to confirm uniformity ofRT within sample groups and between samples. ComparableRTs within a sample group were pooled. All reverses wereperformed in quadruplicate. At least two quantitative PCRreplications were performed for each condition. Values wereexpressed as a percentage of untreated animals and wereevaluated by using one-way ANOVA.

Human Subjects. Nine healthy human adult volunteers (seven menand two women) were enrolled in the study after providing theirconsent. The study was approved by the Institutional ReviewBoard at Washington University School of Medicine.

Procedure. The subjects were randomly separated in two groups,which where scheduled to alternate 2 weekends of either normalsleep or 28 h of continuous waking. The sleep protocol wascarried out at the Sleep Medicine Center, Department ofNeurology, Washington University School of Medicine. On thenormal-sleep weekend, the volunteers were allowed to fall asleepat 10:00 p.m. Normal sleep architecture and absence of signif-icant respiratory abnormalities during sleep, periodic limb move-ment disorder, parasomnias, and nocturnal seizures were con-firmed by standard polysomnography. The polysomnogramswere evaluated and scored following standard criteria (38). TheSD group remained awake and was allowed free access to waterduring the night. However, meal times were restricted to 8:00a.m., 12:00 noon, and 6:00 p.m. No efforts were taken to limitcaffeine consumption during the day, and logs were kept for eachparticipant. The participants were constantly monitored by twoexperienced, certified sleep technicians. Saliva was collectedfrom plain (noncitric acid) cotton Salivettes (Sarstedt, Newton,NC) that had been chewed for �1 min. The samples were rapidlyfrozen over dry ice and kept at �80°C until assayed. Totalprotein was evaluated in saliva by using the Pyrogallol Redmethod (TP0400–1KT; Sigma, St. Louis, MO). Amylase activitywas evaluated by Salimetrics Analytical Laboratory Services byusing the Salivary �-Amylase Assay Kit (1-1902; Salimetrics,State College, PA). Saliva samples were assayed for cortisol byusing a commercially available immunoassay protocol (Salimet-rics). RNA was isolated from cell-free supernatant as described(19). RNA was treated with RNase-free DNase I (TURBODNA-free; Ambion, Austin, TX) according to the manufactur-er’s instructions. Isolated RNA was reverse-transcribed by usingSuperScript III (Invitrogen Life Technologies, Carlsbad, CA)according to the manufacturer’s instructions. Quantitative PCRwas performed by using the 7000 Real-Time PCR System(Applied Biosystems, Foster City, CA). Predesigned TaqManGene Expression Assays (Applied Biosystems) were used foranalyzing the mRNA levels of �-actin and Amylase. A 9-�laliquot of the cDNA was used in each reaction, and all reactionswere performed in duplicate.

We thank Mathew Thimgan and David Van Essen for helpful commentsand input; Michael Morrisey, Janine Kempleman, Min Quan, Lucy Vine,and Jordan Weitzner for assisting with the study; and David T. Wong fortechnical assistance. This work was supported by National Institutes ofHealth Grant R01-NS051305-01A1.

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