31.05.

2013

What are microarrays teaching us about sleep?

What are microarrays teaching us about sleep?

Miroslaw Mackiewicz, John E. Zimmerman, [...], and Allan I. Pack

Abstract
Ma n y fu n da m en t a l qu est ion s a bou t sleep r em a in u n a n sw er ed. T h e pr esen ce of sleep a cr oss ph y la su g g est s t h a t it m u st ser v e a ba sic cellu la r a n d/or m olecu la r fu n ct ion . Micr oa r r a y st u dies, per for m ed in sev er a l m odel sy st em s, h a v e iden t ified cla sses of g en es t h a t a r e sleep-st a t e r eg u la t ed. T h is h a s led t o t h e follow in g con cept s: fir st , a fu n ct ion of sleep is t o m a in t a in sy n a pt ic h om eost a sis; secon d, sleep is a st a g e of m a cr om olecu le biosy n t h esis; t h ir d, ex t en din g w a k efu ln ess lea ds t o dow n r eg u la t ion of sev er a l im por t a n t m et a bolic pa t h w a y s; a n d, fou r t h , ex t en din g w a k efu ln ess lea ds t o en dopla sm ic r et icu lu m st r ess. In h u m a n st u dies, m icr oa r r a y s a r e bein g a pplied t o t h e iden t ifica t ion of biom a r k er s for sleepin ess a n d for t h e com m on debilit a t in g con dit ion of obst r u ct iv e sleep a pn ea .

High-throughput approaches to study gene expression

T h e sea r ch for g en es in v olv ed in r eg u la t ion by , a n d of, sleep a n d w a k efu ln ess a n d t h e qu est t o u n der st a n d t h e fu n ct ion s of sleep a t t h e m olecu la r lev el beg a n a deca de befor e t h e a dv en t of m icr oa r r a y s. Sev er a l ca n dida t e g en es w er e st u died t o elu cida t e t h eir r oles in sleep a n d w a k efu ln ess (for a r ev iew , see [1 ]). Su bt r a ct iv e h y br idiza t ion ca r r ied ou t on br a in m RNA fr om sleep-depr iv ed r a t s, per for m ed in t h e ea r ly 1 9 9 0 s, w a s t h e fir st h ig h -t h r ou g h pu t a t t em pt t o iden t ify su ch g en es [2 ]. La t er , t h e a pplica t ion of differ en t ia l displa y [3 ] br oa den ed t h e ca n dida t e g en e a n d su bt r a ct iv e h y br idiza t ion a ppr oa ch es t o scr een for g en es w h ose fu n ct ion s w er e poor ly ch a r a ct er ized or pr ev iou sly u n k n ow n . A s a r esu lt of sequ en cin g effor t s, t h er e h a s been a n ex pon en t ia l g r ow t h in t h e a m ou n t of in for m a t ion a v a ila ble a bou t t h e DNA sequ en ce of t h e h u m a n g en om e (e.g ., see [4 ]) a s w ell a s t h e g en om es of v a r iou s m odel or g a n ism s (e.g ., see [5 ,6 ]). Con sequ en t ly , t h ou sa n ds of g en es h a v e been discov er ed, in clu din g m a n y n ov el g en es for w h ich sequ en ces w er e u n a v a ila ble pr ev iou sly . T h e r ole of m a n y of t h ese g en es is u n k n ow n . T h is fa cilit a t ed t h e a dv en t of m icr oa r r a y a ppr oa ch es. A m icr oa r r a y con t a in s t en s of t h ou sa n ds of g en es a n d is a t ool for sim u lt a n eou s m ea su r em en t of t h eir ex pr ession . Sig n ifica n t a dv a n ces in g en e a n n ot a t ion s, pr og r ess in dev elopm en t a n d st a n da r diza t ion of m icr oa r r a y pla t for m s, a s w ell a s t h e ex pa n sion of da t a a n a ly sis t ools, in clu din g n ew a n d pow er fu l st a t ist ica l a ppr oa ch es t o a ssess ex pr ession da t a (r ev iew ed in [7 ,8 ]), m a k e a m icr oa r r a y a ppr oa ch t h e idea l w a y t o in it ia lly det er m in e t h e ch a n g es in t r a n scr ipt ion of t h e g en om e in r espon se t o, or a s a con sequ en ce of, sleep a n d/or w a k e st a t e. How ev er , m icr oa r r a y s descr ibe w h a t m ig h t h a ppen r a t h er t h a n w h a t does h a ppen in a cell or t issu e beca u se ch a n g es in a g iv en m RNA m ig h t n ot t r a n sla t e in t o ch a n g es in t h e r elev a n t pr ot ein . Micr oa r r a y s a r e a t ool of discov er y a n d, a s su ch , r esu lt s a r e h y pot h esis-g en er a t in g . T h er efor e, h y pot h eses a r isin g fr om m icr oa r r a y st u dies n eed t o be fu r t h er a ssessed. In pa r t icu la r , it is im por t a n t t o dist in g u ish w h et h er ch a n g es in g en e ex pr ession a r e bein g dr iv en by sleep v er su s dr iv in g sleep or w a k efu ln ess. Nov el pa t h w a y s a r e, h ow ev er , bein g iden t ified a n d, if con fir m ed, w ill pr ov ide n ew t a r g et s for t h er a peu t ic in t er v en t ion t o a ddr ess pr oblem s w it h sleep.

Microarrays in sleep research

Micr oa r r a y s offer a n ew w in dow on t h e differ en ce bet w een t h e sleepin g a n d a w a k e br a in a n d h old t h e pr om ise of fa cilit a t in g a n sw er s t o som e of t h e m a jor qu est ion s in sleep biolog y (Box 1 ; see a lso [9 1 1 ]).

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Box 1. Major questions in sleep biology

W h a t a r e t h e fu n ct ion s of sleep? W h ich g en es r eg u la t e sleep a n d w a k efu ln ess? W h a t m olecu la r ch a n g es set t h e du r a t ion of w a k efu ln ess t h a t ca n be su st a in ed w it h ou t im pa ir m en t ? W h a t a r e t h e m olecu la r con sequ en ces of sleep depr iv a t ion ? W h a t is t h e ba sis of in div idu a l differ en ces in r espon se t o sleep depr iv a t ion a n d ot h er sleep ch a r a ct er ist ics? A r e t h er e biom a r k er s for sleep dr iv e a n d for specific sleep disor der s? T h ese qu est ion s a r e r elev a n t t o t h e per v a siv e pr oblem of sleep depr iv a t ion in in du st r ia lized societ ies a n d t o t h e h ig h pr ev a len ce of sev er a l sleep disor der s [1 2 ]. In t h is r ev iew , w e descr ibe w h a t w e h a v e lea r n ed fr om m icr oa r r a y ex per im en t s desig n ed t o a ddr ess t h ese qu est ion s a n d discu ss fu t u r e oppor t u n it ies for t h e a pplica t ion of t h ese a n d r ela t ed a ppr oa ch es. Micr oa r r a y st u dies a ddr essin g sleep fu n ct ion h a v e focu sed on t h e br a in in v a r iou s species [1 3 1 9 ], a lt h ou g h a r ecen t st u dy ex a m in ed ot h er or g a n s [1 8 ]. A ssessm en t of t h e t r a n scr ipt om e in m odel or g a n ism s h a s dem on st r a t ed ch a n g es in t h e lev el of t r a n scr ipt s of m a n y g en es bet w een sleep a n d w a k efu ln ess; a s m a n y a s 1 0 % of g en es in m ou se br a in ch a n g e t h eir ex pr ession bet w een t h ese t w o beh a v ior a l st a t es [1 7 ]. Micr oa r r a y st u dies per for m ed t o da t e h a v e u sed differ en t pla t for m s a n d differ en t st u dy desig n s (Fig u r e 1 ). Som e st u dies h a v e ev a lu a t ed g en es ch a n g in g ex pr ession bet w een sleep a n d w a k efu ln ess u sin g fold ch a n g es (e.g ., see [1 3 ]), w h er ea s ot h er s h a v e u sed fa lse-discov er y r a t e st r a t eg ies (e.g . see [1 7 ]). Despit e t h ese differ en ces, t h er e is a sim ila r it y in t h e r esu lt s w it h r espect t o pa t h w a y s in t h e br a in t h a t a r e a ffect ed by sleep a n d w a k efu ln ess (T a ble 1 ). T h er e r em a in s deba t e a s t o t h e n u m ber of g en es t h a t a r e ex pr essed differ en t ia lly bet w een sleep a n d w a k efu ln ess (e.g ., see [1 3 ,1 4 ,1 7 ,1 9 ]). T h is pr oba bly r eflect s differ en t st u dy desig n s a n d, in pa r t icu la r , t h e sa m ple sizes (pow er ) u sed in differ en t st u dies [1 3 ,1 4 ,1 7 ,1 9 ].

Fi gur e 1 Ex ampl es of ex per i mental str ategi es i n mi c r oar r ay r esear c h to el uc i date the i denti ty of genes w hose ex pr essi on ex hi b i ts di f f er enc es b etw een b outs of sl eep and w ak ef ul ness. These c ompl ementar y str ategi es addr ess the c onf oundi ng ef f ec ts of sl eep depr i v ati on-i nduc ed ...

Tab l e 1 Mi c r oar r ay appr oac hes to study gene ex pr essi on dur i ng sl eep, w ak ef ul ness or sl eep depr i v ati on and the k ey f unc ti onal c ategor i es of genes ex pr essed di f f er enti al l y among b ehav i or al statesa

Model systems for the study of sleep

In r ecen t y ea r s, a n im por t a n t a dv a n ce in sleep r esea r ch h a s been t h e iden t ifica t ion of a sleep st a t e in n on -m a m m a lia n m odel sy st em s: n a m ely , t h e fr u it fly ( Dros ophila m elanogas ter) [2 0 2 2 ], t h e zebr a fish ( Danio rerio ) [2 3 2 5 ] a n d, m ost r ecen t ly , t h e n em a t ode ( Caenorhabditis elegans ) [2 6 ] (see a lso [2 7 2 9 ]). Cr u cia l t o t h e iden t ifica t ion of sleep in t h ese m odel sy st em s h a s been t h e u se of beh a v ior a l r a t h er t h a n elect r oph y siolog ica l cr it er ia t o defin e t h e sleep st a t e. T h e m a in beh a v ior a l cr it er ia , in a ddit ion t o qu iescen ce (la ck of m ov em en t ), a r e t h e follow in g : fir st , elev a t ed a r ou sa l t h r esh old (i.e. w h en a sleep, it t a k es a la r g er st im u lu s t o m a k e t h e a n im a l m ov e or a lon g er t im e t o r espon d t o a fix ed st im u lu s); secon d, h om eost a sis (i.e. follow in g sleep depr iv a t ion t h e a n im a l r et u r n s t o sleep fa st er , h a s in cr ea sed du r a t ion s of sleep bou t s, oft en con sider ed a m ea su r e of sleep
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dept h , a n d ca n sleep a t in a ppr opr ia t e cir ca dia n t im es); a n d, t h ir d, t h e t im in g of sleep pr open sit y cor r ela t es w it h t h e m olecu la r clock or r h y t h m ic ex pr ession of clock g en es. Not on ly a r e t h e beh a v ior a l a spect s of sleep con ser v ed a m on g t h e m odels bu t t h er e is con ser v a t ion of bot h n eu r ot r a n sm it t er sy st em s a n d t h e com pon en t s of t h e m olecu la r sig n a lin g pa t h w a y s t h a t r eg u la t e sleep [3 0 ]. For ex a m ple, sig n a lin g m ech a n ism s in v olv in g cy clic A MP, w h ich pr om ot es w a k efu ln ess [3 1 ,3 2 ], a n d epider m a l g r ow t h fa ct or , w h ich pr om ot es sleep [3 3 ,3 4 ], h a v e t h e sa m e r ole in differ en t m odel sy st em s (for r ev iew , see [3 0 ]). Model sy st em s pr ov ide a g r ea t oppor t u n it y for in v est ig a t ion by m icr oa r r a y s. Micr oa r r a y s h a v e been a pplied t o t h e st u dy of sleep in fr u it flies [1 4 ,1 9 ], r a t s [1 3 ,1 5 ] a n d m ice [1 7 ,1 8 ]. In a ddit ion , m icr oa r r a y st u dies h a v e been con du ct ed r ecen t ly in t h e w h it e-cr ow n ed spa r r ow ( Zonotrichia leuk ophrys ) [1 6 ], a lbeit w it h a lim it ed sa m ple size. T h is spa r r ow , lik e m a n y lon g -dist a n ce a v ia n m ig r a n t s, h a s t h e a bilit y t o g o w it h ou t sleep du r in g it s m ig r a t ion . A n a r ea r ipe for fu t u r e in v est ig a t ion is m icr oa r r a y st u dies of ch a n g es in g en e ex pr ession in t h e n em a t ode or zebr a fish . Micr oa r r a y st u dies t h a t h a v e been con du ct ed in flies, r oden t s a n d bir ds sh ow t h a t sim ila r m olecu la r pa t h w a y s a r e a lt er ed by sleep, w a k efu ln ess a n d sleep depr iv a t ion (T a ble 1 ).

What microarrays are teaching us about the molecular functions of sleep

A n a ly ses of t h e fu n ct ion a l ca t eg or ies of g en es ch a n g in g ex pr ession bet w een sleep a n d w a k efu ln ess, a s r ev ea led by m icr oa r r a y st u dies, h a v e led t o n ew h y pot h eses a bou t t h e fu n ct ion s of sleep a n d a r e n ow t h e focu s of h y pot h esis-dr iv en r esea r ch . T w o m a jor t h eor ies h a v e em er g ed t h a t a r e n ot m u t u a lly ex clu siv e: fir st , t h a t sleep a n d w a k efu ln ess r eg u la t e sy n a pt ic st r en g t h , w it h u p-sca lin g du r in g w a k efu ln ess a n d dow n -sca lin g du r in g sleep [3 5 ,3 6 ]; a n d, secon d, t h a t sleep is a st a g e of m a cr om olecu le biosy n t h esis [1 7 ]. In t h e follow in g sect ion s, w e ex plor e ea ch of t h ese t h eor ies.

Sleep and wakefulness and synaptic scaling

Micr oa r r a y st u dies con du ct ed in r a t s [1 3 ] iden t ified n u m er ou s g en es in v olv ed in t h e a cqu isit ion a n d pot en t ia t ion of sy n a pt ic pla st icit y (a t er m r ela t in g t o t h e a bilit y of sy n a pses t o ch a n g e in st r en g t h ); t h ese g en es ex pr essed in cr ea sed t r a n scr ipt lev els du r in g w a k efu ln ess. Con v er sely , t h e lev els of ex pr ession of g en es in v olv ed in sy n a pt ic con solida t ion or depr ession in cr ea se du r in g sleep [1 3 ]. T h ese obser v a t ion s h a v e been in cor por a t ed w it h in a h y pot h esis of sleep fu n ct ion r efer r ed t o a s t h e sy n a pt ic h om eost a sis t h eor y of sleepw a k e con t r ol [3 5 ] (for a r ev iew of t h is t h eor y , see [3 6 ]). It is post u la t ed t h a t w a k efu ln ess is a ccom pa n ied by sy n a pt ic pot en t ia t ion of cor t ica l n et w or k s t h r ou g h br a in -der iv ed n eu r ot r oph ic fa ct or (BDNF)-depen den t a n d ot h er sig n a lin g m ech a n ism s. Su ch sy n a pt ic pot en t ia t ion is a ch iev ed t h r ou g h a ct iv it ies a n d lea r n in g du r in g w a k efu ln ess (com m on ly t er m ed a s ex per ien ce) a n d occu r s in n eu r on a l cir cu it s t h a t a r e a ct iv a t ed by t h e r elev a n t ex per ien ce. In deed, st im u la t ion of w h isk er s in t h e r a t m odifies t h e loca l elect r oen ceph a log r a m (EEG) pa t t er n (t h e EEG is a m ea su r e of elect r ica l a ct iv it y pr odu ced by t h e br a in ) [3 7 ,3 8 ]. T h is obser v a t ion su ppor t s t h e n ot ion t h a t t h er e is a pot en t ia t ion of sy n a pt ic st r en g t h in t h e ba r r el cor t ex t h a t r eceiv es a n d pr ocesses t a ct ile in for m a t ion der iv ed fr om t h e con t r a la t er a l fa ce of t h e a n im a l. It is post u la t ed t h a t a n in cr ea se in ov er a ll sy n a pt ic pot en t ia t ion du r in g w a k efu ln ess w ill r equ ir e m or e r esou r ces (en er g y , spa ce) t o m a in t a in t h is lev el of pot en t ia t ion . It h a s been fu r t h er su g g est ed t h a t sy n a pt ic pot en t ia t ion is lin k ed ca u sa lly t o t h e in t en sit y of EEG slow -w a v e a ct iv it y du r in g su bsequ en t sleep a n d t h a t , du r in g slow -w a v e sleep, t h er e is sy n a pt ic dow n -sca lin g . Su ch dow n sca lin g of sy n a pt ic st r en g t h h a s a ben eficia l effect on n eu r on a l fu n ct ion by r et u r n in g t h e sy st em t o a n ov er a ll pr e-w a k efu ln ess ba la n ce, a lt h ou g h t h is lea v es t r a ces of ex per ien ces t h a t occu r r ed du r in g w a k efu ln ess a n d in cr ea ses sy n a pse sig n a l-t o-n oise r a t ios.

Sleep and wakefulness and macromolecule biosynthesis

Micr oa r r a y st u dies h a v e a lso iden t ified ot h er cla sses of g en es w h ose pa t t er n s of ex pr ession ch a n g e
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du r in g sleep [1 7 ]. In m ou se cer ebr a l cor t ex a n d, t o a lesser ex t en t , h y pot h a la m u s t h er e is u pr eg u la t ion du r in g sleep of g en es en codin g pr ot ein s in v a r iou s, pr edom in a n t ly in t er m edia r y , biosy n t h et ic pa t h w a y s for h em e, pr ot ein a n d lipid [1 7 ]. A sig n ifica n t n u m ber of g en es en codin g t h e st r u ct u r a l con st it u en t s of t h e r ibosom es, t r a n sla t ion -r eg u la t ion a ct iv it y a n d for m a t ion of t r a n sfer RNA (t RNA ; a sm a ll RNA t h a t t r a n sfer s a specific a m in o a cid t o a g r ow in g poly pept ide du r in g pr ot ein sy n t h esis) a n d r ibosom e biog en esis a r e a lso u pr eg u la t ed du r in g sleep. T h u s, sy n t h esis of pr ot ein s a n d ot h er m a cr om olecu les seem s lik ely t o occu r du r in g sleep, m or e so t h a n du r in g w a k efu ln ess. Gen es w h ose ex pr ession in cr ea ses pr og r essiv ely du r in g sleep in clu de g en es en codin g m u lt iple en zy m es of t h e ch olest er ol-sy n t h esis pa t h w a y , pr ot ein s in v olv ed in ch olest er ol u pt a k e a n d t r a n spor t a n d r elev a n t t r a n scr ipt ion fa ct or s a n d ch a per on es r espon sible for t r a n scr ipt ion a l r eg u la t ion of ch olest er ol-r ela t ed g en es [1 7 ]. T h u s, m em br a n e ch olest er ol is ex pect ed t o in cr ea se du r in g sleep. Ch olest er ol h a s a n im por t a n t r ole in m em br a n e st a bilit y a n d is t h e k ey st r u ct u r a l com pon en t of m em br a n e m icr odom a in s ca lled lipid r a ft s [3 9 ]. T h e t r a n scr ipt lev els of sev er a l g en es en codin g lipid-r a ft -r esiden t pr ot ein s, su ch a s flot illin , a lso in cr ea se du r in g sleep [1 7 ]. Ra ft s br in g t og et h er n eu r ot r a n sm it t er r ecept or s w it h ot h er sig n a lin g m olecu les a n d, by so doin g , a lt er t h e st r en g t h of sig n a lin g [3 9 ]. T h e r ea ssem blin g of lipid r a ft s du r in g sleep w ou ld a lt er sig n a lin g of sev er a l n eu r ot r a n sm it t er s [4 0 4 3 ]. T h is m ig h t be in pr epa r a t ion for su bsequ en t w a k efu ln ess, w h en t h er e is en h a n ced r elea se of v a r iou s n eu r ot r a n sm it t er s on a r ou sa l fr om sleep. Rea ssem bly of lipid r a ft s du r in g sleep w ou ld en a ble m a x im a l sig n a lin g on a w a k en in g . It is u n clea r h ow a n in cr ea se in ch olest er ol sy n t h esis du r in g sleep w ou ld im pa ct on sy n a pt ic dow n -sca lin g du r in g sleep t h a t is pr oposed in t h e sy n a pt ic h om eost a sis t h eor y of sleepw a k e con t r ol [3 5 ]. In a ddit ion , du r in g sleep t h er e is a n u pr eg u la t ion of g en es en codin g pr ot ein s in v olv ed in t h e fu n ct ion in g of v esicle pools, a s w ell a s in t h e fu n ct ion in g of a ll a n t iox ida n t en zy m es a n d com pon en t s of in t r a cellu la r t r a n spor t . T h ese obser v a t ion s su g g est t h a t sleep is a st a g e of r ebu ildin g a n d r epa ir in pr epa r a t ion for su bsequ en t w a k efu ln ess (Fig u r e 2 ).
Fi gur e 2 Dur i ng w ak ef ul ness, upr egul ati on of genes i nv ol v ed i n sy napti c pl asti c i ty oc c ur s and, w i th ex tended w ak ef ul ness, upr egul ati on of mol ec ul ar c haper ones f ol l ow s. If w ak ef ul ness i s ex tended, a major mec hani sm pr omoti ng sl eep mi ght b e the pr ogr essi v e dec l i ne ...

What microarrays are teaching us about mechanisms limiting the duration of wakefulness
In h u m a n s, w a k efu ln ess ca n be su st a in ed for 1 6 h w it h ou t im pa ir m en t of per for m a n ce [4 4 ]. In m ice, t h e du r a t ion of t h e lon g est episodes of su st a in ed w a k efu ln ess is m u ch sh or t er , bein g in t h e or der of 3 h [4 5 ,4 6 ]. A ccor din g ly , t h er e m u st be a cost t o ex t en din g w a k efu ln ess a n d t h er e is lik ely t o be m olecu la r m ech a n ism s t h a t lim it t h e du r a t ion of w a k efu ln ess. If so, ex t en din g w a k efu ln ess bey on d t h ese lim it s, a s in a cu t e sleep depr iv a t ion , sh ou ld be delet er iou s. Micr oa r r a y st u dies h a v e r ev ea led t w o k ey con cept s: fir st t h a t ex t en din g w a k efu ln ess lea ds t o en dopla sm ic r et icu lu m (ER) st r ess; a n d, secon d, t h a t m u lt iple g en es in k ey cellu la r pa t h w a y s a r e dow n r eg u la t ed w it h pr olon g ed w a k efu ln ess.

Extended wakefulness leads to cellular stress

St u dies of g en e ex pr ession h a v e r ev ea led t h a t ex pr ession of t h e HSPA5 ( heat s hock 70 k DA protein 5) g en e, w h ich en codes t h e m olecu la r ch a per on e com m on ly k n ow n a s BiP, in cr ea ses w it h ex t en ded w a k efu ln ess in differ en t species a n d differ en t br a in r eg ion s [1 3 ,1 6 ,1 7 ,4 7 ] (see a lso [4 8 ,4 9 ]). T h ese fin din g s su g g est t h a t , w it h ex t en ded w a k efu ln ess, t h er e is st r ess in t h e ER. In r oden t s, ot h er g en es
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en codin g h ea t -sh ock pr ot ein s a n d m olecu la r ch a per on es a r e a lso u pr eg u la t ed du r in g sleep depr iv a t ion [1 7 ,4 9 ]. In cr ea sed ex pr ession of t h e Hs pa5 g en e occu r s a s pa r t of t h e sig n a lin g pa t h w a y ca lled t h e u n folded-pr ot ein r espon se (UPR). T h is is a n a da pt iv e r espon se t h a t en a bles cells t o su r v iv e st r ess in t h e ER, r esu lt in g fr om per t u r ba t ion s in ca lciu m h om eost a sis, r edox st a t u s, elev a t ed secr et or y pr ot ein sy n t h esis, m isfolded pr ot ein s, g lu cose depr iv a t ion or a lt er ed g ly cosy la t ion [5 0 ]. T h e UPR h elps t o r est or e n or m a l ER fu n ct ion by r edu cin g pr ot ein t r a n sla t ion a n d by u pr eg u la t in g t h e ex pr ession of ch a per on es t o in cr ea se t h e ER ca pa cit y for foldin g or t o pr om ot e deg r a da t ion of m isfolded pr ot ein s (for r ev iew s, see [5 1 ,5 2 ]). A ll com pon en t s t h a t a r e a ct iv a t ed a s pa r t of t h e UPR a r e elev a t ed in m ou se cer ebr a l cor t ex a ft er 6 h of sleep depr iv a t ion [4 8 ]. In t h is st u dy , sleep depr iv a t ion w a s per for m ed a t t h e beg in n in g of t h e lig h t s-on per iod, t h u s pr olon g in g w a k efu ln ess t h a t n or m a lly occu r s in t h e lig h t s-off per iod. A r ecen t m icr oa r r a y st u dy in dica t es t h a t ex pr ession of t h e Hs pa5 g en e in cr ea ses w it h sleep depr iv a t ion n ot on ly in br a in bu t a lso in liv er [1 8 ]. T h u s, ER st r ess m ig h t be a g en er a l r espon se t o sleep depr iv a t ion t h a t is n ot specific t o br a in . Hen ce, t h e pr ev a ilin g et h os t h a t sleep is of t h e br a in , by t h e br a in a n d for t h e br a in [5 3 ] is ch a llen g ed by r ecen t m icr oa r r a y da t a [1 8 ]. T h er e is, h ow ev er , lim it ed in for m a t ion cu r r en t ly on h ow sleep a n d pr olon g ed w a k efu ln ess im pa ct on g en e ex pr ession in per iph er a l t issu es a n a r ea r ipe for fu t u r e st u dy .

Downregulation of genes for multiple cellular processes as wakefulness is prolonged

In bot h fr u it -fly br a in s a n d m ou se cer ebr a l cor t ex a n d h y pot h a la m u s, t h e la r g est cla ss of g en es t h a t a r e ex pr essed differ en t ia lly bet w een sleep-depr iv ed a n im a ls a n d sleepin g con t r ols a r e t h ose t h a t sh ow declin e in ex pr ession w it h pr olon g ed w a k efu ln ess [1 7 ,1 9 ]. A s t h e du r a t ion of w a k efu ln ess pr og r esses, t h er e is a r edu ct ion in t h e ex pr ession of g en es in v olv ed in m u lt iple ph y siolog ica l pr ocesses; r edu ct ion in t h ese pr ocesses m ig h t a ct t og et h er t o lim it w a k efu ln ess. In t h e br a in of t h e fr u it fly , m u lt iple g en es in v olv ed in differ en t st eps in t h e pr ot ein -pr odu ct ion pa t h w a y a r e dow n r eg u la t ed w it h ex t en ded w a k efu ln ess [1 9 ]. In m ou se cer ebr a l cor t ex a n d h y pot h a la m u s, t h er e is a r edu ct ion du r in g ex t en ded w a k efu ln ess in t h e ex pr ession of g en es en codin g pr ot ein s in v olv ed in t h e m a in pa t h w a y s of ca r boh y dr a t e, en er g y , t r ica r box y lic a cid (T CA ) a n a bolism a n d v a r iou s m et a bolic pa t h w a y s, su ch a s lipid, a ldeh y de a n d a m in e sy n t h esis [1 7 ]. T h u s, a m ech a n ism pr om ot in g sleep m ig h t be t h e declin e in pr ocesses t h a t h elp su st a in w a k efu ln ess, t h er eby en a blin g sleep t o occu r (Fig u r e 2 ).

Genetic perturbations can test the functional roles of genes identified using microarrays
On e disa dv a n t a g e of m icr oa r r a y st r a t eg ies is t h a t on e does n ot k n ow w h et h er t h e g en es iden t ified a s ch a n g in g ex pr ession w it h beh a v ior a l st a t es a r e doin g so a s a con sequ en ce of t h e st a t e (t h ey cou ld be pa r t of t h e fu n ct ion of sleep) or w h et h er t h ey cou ld be in v olv ed in r eg u la t in g t h e sleep st a t e. T h ese possibilit ies a r e n ot m u t u a lly ex clu siv e. T h is disa dv a n t a g e h a s led r esea r ch er s t o u se t h e for w a r d-g en et ic st r a t eg y of scr een in g m u t a n t s for a n a lt er ed sleep ph en ot y pe. If a m u t a n t a n im a l w it h a n a lt er ed ph en ot y pe is iden t ified, t h en it ca n be a ssu m ed t h a t t h e g en e t h a t is m u t a t ed is in v olv ed in r eg u la t in g t h e pr ocess. T h is st r a t eg y , a pplied in D. m elanogas ter [5 4 5 6 ], h a s iden t ified t w o g en es in v olv ed in r eg u la t in g sleep Sh , en codin g t h e sh a k er K + ch a n n el [5 4 ] a n d t h e g en e en codin g ex t r a cellu la r GPI-a n ch or ed pr ot ein t er m ed Sleepless [5 5 ] a n d, w h en a pplied in C. elegans , h a s iden t ified a cy clic-GMP-depen den t pr ot ein k in a se [2 6 ,5 7 ]. Usin g cu r r en t ly a v a ila ble r esou r ces, h ow ev er , pa r t icu la r ly in t h e fr u it fly , a n in v est ig a t or ca n det er m in e qu ick ly w h et h er t h e a lt er ed ex pr ession of g en es iden t ified in m icr oa r r a y st u dies w ill a ffect sleep. In t h e fr u it fly , t h er e a r e lin es a v a ila ble w it h t r a n sposa ble elem en t s t h a t disr u pt g en e fu n ct ion by in ser t ion in t o iden t ified g en es [5 8 6 0 ]. A lso a v a ila ble is a libr a r y of RNA in t er fer en ce (RNA i) lin es t h a t t a r g et 8 8 % of pr ot ein -codin g g en es [6 1 ]. T h ese t r a n sposon -in ser t ion lin es a n d t h e
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RNA i lin es ca n lea d t o r edu ced g en e fu n ct ion . T h e pow er of a v a ila ble D. m elanogas ter g en et ic t ools is t h a t spa t ia l a n d t em por a l con t r ol of g en e ex pr ession a r e a lso possible u sin g t w o- a n d t h r eecom pon en t t r a n sg en ic sy st em s (for a r ev iew , see [6 2 ]). Gen e ex pr ession ca n be a lt er ed in bot h t h e posit iv e a n d n eg a t iv e dir ect ion a n d ca n disr eg u la t e t h e g en e of in t er est iden t ified in m icr oa r r a y st u dies. It is, t h er efor e, fea sible t o t est w h et h er a g en e in den t ified in a m icr oa r r a y ex per im en t a lso r eg u la t es sleep. T h is t ech n iqu e w a s u sed in t h e st u dy of t h e m olecu la r ch a per on e g en e Hs pa5 in t h e fr u it fly . T h e ex pr ession of Hs pa5 in cr ea ses w it h sleep depr iv a t ion in fr u it flies [4 7 ], m ice [1 7 ], r a t s [1 3 ] a n d bir ds [1 6 ]. A lt h ou g h n u m er ou s st u dies in dica t e t h a t ex t en ded w a k efu ln ess lea ds t o in cr ea sed ex pr ession of t h e m olecu la r ch a per on e Hs pa5, it h a s been sh ow n r ecen t ly t h r ou g h g en et ic m a n ipu la t ion of Dros ophila t h a t a lt er a t ion of Hs pa5 lev els does a ffect t h e a m ou n t of r ecov er y sleep follow in g sleep depr iv a t ion . T h er e is n o a lt er a t ion in t h e a m ou n t of ba selin e sleep or w a k efu ln ess. In cr ea sed ex pr ession of Hs pa5 in cr ea ses t h e a m ou n t of sleep r ecov er y follow in g sleep depr iv a t ion [6 3 ]. By con t r a st , in cr ea sed ex pr ession of a dom in a n t -n eg a t iv e for m of Hs pa5 decr ea ses t h e a m ou n t of sleep r ecov er y a ft er sleep depr iv a t ion [6 3 ]. T h ese obser v a t ion s a r e com pa t ible w it h t w o possible ex pla n a t ion s. Hs pa5 it self cou ld be a sleep-pr om ot in g m olecu le w it h h ig h er lev els of Hs pa5 lea din g t o m or e r ecov er y sleep. A lt er n a t iv ely , a n d m or e lik ely , is t h a t h ig h er lev els of Hs pa5 dela y a ct iv a t ion of t h e UPR, a s r ev ea led by in vitro st u dies [6 4 ,6 5 ]. Hen ce, t h er e is a n eed for m or e r ecov er y sleep, t h e fin a l m ech a n ism of defen se. T h u s, pr ocesses con t r ollin g t h e ba selin e a m ou n t s of sleep v er su s w a k e a n d t h ose con t r ollin g r ecov er y sleep follow in g sleep depr iv a t ion ca n be sepa r a t ed a t t h e m olecu la r lev el. T h e Hspa 5 pr ot ein is in v olv ed in r eg u la t in g r ecov er y sleep. T h e r ole of Hspa 5 pr ot ein , w h ich w a s iden t ified by m icr oa r r a y s a s im por t a n t t o sleepw a k e fu n ct ion , w a s n ot u n der st ood fu lly u n t il g en e ex pr ession w a s a lt er ed ex per im en t a lly .

Microarrays and quantitative trait loci and the genetic basis of inter-individual differences
A spect s of h u m a n sleep, su ch a s t im in g [6 6 ] a n d sleep du r a t ion [6 7 ], a r e h er it a ble. Sim ila r ly , m a n y sleep-r ela t ed t r a it s a r e h er it a ble in m ice [4 5 ,4 6 ,6 8 ] (for r ev iew , see [6 9 ,7 0 ]). Usin g m ou se r ecom bin a n t -in br ed st r a in s a n d a qu a n t it a t iv e t r a it locu s (QT L) a ppr oa ch , a r eg ion on ch r om osom e 1 3 in v olv ed in t h e r espon se t o sleep depr iv a t ion w a s iden t ified [4 5 ]. QT L da t a w er e fu r t h er ch a r a ct er ized by con sider in g g en es in t h e r eg ion t h a t w er e ex pr essed differ en t ia lly du r in g sleep a n d w a k efu ln ess in m icr oa r r a y st u dies [1 7 ,1 8 ]. By com bin in g h a plot y pe a n a ly sis w it h da t a fr om g en eex pr ession pr ofilin g by m icr oa r r a y s, h u n dr eds of g en es loca t ed w it h in t h e QT L in t er v a l w er e n a r r ow ed dow n t o a few g en es [7 1 ]. How ev er , on ly on e of t h ese g en es, a s det er m in ed by ex pr ession pr ofilin g [i.e. Hom er1a (a splice v a r ia n t of t h e Hom er1 g en e)], h a s a differ en t ia l in cr ea se in ex pr ession w it h sleep depr iv a t ion a m on g t h e r ecom bin a n t in br ed st r a in s u sed for QT L m a ppin g [1 8 ,7 1 ]. Mor eov er , t h er e is a poly m or ph ism in t h e r eg u la t or y r eg ion of t h e Hom er1 g en e t h a t pr oba bly im pa ct s on t h e t r a n scr ipt lev el obser v ed in m icr oa r r a y pr ofilin g [7 1 ]. T h e pr om ot er r eg ion of t h e Hom er1 g en e con t a in s m u lt iple CRE sit es t h a t w ill bin d t h e cy clic-A MP r espon se elem en t -bin din g pr ot ein (CREB) [7 2 ], a n d t h e poly m or ph ism pr esen t in t h is pr om ot er m ig h t im pa ct CREB bin din g . Mice w it h low lev els of CREB ow in g t o a delet ion of t h e a n d isofor m s of CREB h a v e r edu ced w a k efu ln ess du r in g t h e ea r ly pa r t of t h eir n ig h t -t im e a ct iv e per iod [3 1 ]. W h et h er t h is r edu ct ion in w a k efu ln ess is m edia t ed, a t lea st in pa r t , by r edu ced in cr ea ses in Hom er1a w it h w a k efu ln ess in CREB h y pom or ph m ice is cu r r en t ly u n k n ow n . T h u s, m icr oa r r a y st u dies su ppor t t h e h y pot h esis t h a t a v a r ia t ion in Hom er1a ex pr ession ex pla in s differ en ces in r espon se t o sleep depr iv a t ion [1 8 ,7 1 ].

Microarrays and molecular signatures of sleep deprivation and sleep disorders

Micr oa r r a y s a r e a lso pow er fu l t ools t o iden t ify t h e m olecu la r sig n a t u r es of disea ses (for r ev iew s, see [7 3 ,7 4 ]). How ev er , t h ey a r e on ly ju st beg in n in g t o be a pplied t o t h e st u dy of sleep depr iv a t ion a n d
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sleep disor der s [7 5 7 8 ]. In D. m elanogas ter, a m icr oa r r a y st u dy iden t ified t h a t t h e ex pr ession of t h e g en e en codin g a m y la se in cr ea ses w it h pr olon g ed w a k efu ln ess [7 5 ]. In h u m a n s, a m y la se in cr ea ses in sa liv a w it h sleep depr iv a t ion [7 5 ]. T h u s, a ssessm en t of a m y la se pr ov ides a pot en t ia l biom a r k er of sleep loss. T h e im por t a n ce of ev a lu a t in g w h et h er t h er e a r e biom a r k er s for sleep h om eost a sis (sleepin ess) h a s been em ph a sized pr ev iou sly [7 9 8 1 ]. Su ch biom a r k er s w ill pr ov ide a ssessm en t s of sleepin ess t h a t cou ld be u sed in v a r iou s set t in g s a n d pr ov ide a m ea n s for dist in g u ish in g in div idu a ls w h o a r e pa r t icu la r ly sen sit iv e t o t h e effect s of sleep depr iv a t ion . Molecu la r sig n a t u r es m ig h t a lso be dev eloped for sleep disor der s, in pa r t icu la r , t h e com m on con dit ion k n ow n a s obst r u ct iv e sleep a pn ea (for a r ev iew , see [8 2 ]). In t h is con dit ion , br ea t h in g st ops r epet it iv ely du r in g sleep, r esu lt in g in r epea t ed in t er r u pt ion of sleep a n d in cy clica l r epea t ed h y pox ic episodes [8 3 ]. T h ese h y pox ic episodes r esu lt in fr ee-r a dica l pr odu ct ion a n d a ct iv a t ion of t h e t r a n scr ipt ion fa ct or NF B a n d in fla m m a t or y pa t h w a y s (for r ev iew , see [8 3 ]). Micr oa r r a y s h a v e sh ow n t h a t , in pa t ien t s w it h obst r u ct iv e sleep a pn ea , t h er e a r e ch a n g es in t r a n scr ipt lev el in sev er a l g en es in v olv ed in m odu la t ion of r ea ct iv e-ox y g en species (ROS), in clu din g h em e ox y g en a se, su per ox ide dism u t a se a n d ca t a la se [7 7 ]. T h ese ch a n g es in obst r u ct iv e sleep a pn ea pa t ien t s a r e su g g est iv e of t h e a ct iv a t ion of m ech a n ism s t o m odu la t e, a n d a da pt t o, in cr ea sed ROS dev elopin g in r espon se t o t h e fr equ en t episodes of in t er m it t en t h y pox ia [7 7 ]. T h u s, t em por a l ch a n g es in m olecu la r -pa t h w a y com pon en t s a cr oss t h e sleep per iod m ig h t pr ov ide a sig n a t u r e of t h e pr esen ce of t h is a n d per h a ps ot h er sleep disor der s [8 4 ].

Concluding remarks
Her e, w e h a v e a r g u ed t h a t m icr oa r r a y s a r e a discov er y st r a t eg y t h a t h a s h a d a m a jor im pa ct in g en er a t in g h y pot h eses a bou t fu n da m en t a l qu est ion s in sleep biolog y a n d is beg in n in g t o iden t ify biom a r k er s for bot h t h e effect s of sleep depr iv a t ion a n d for specific sleep disor der s. Recen t da t a su g g est t h a t ex t en din g w a k efu ln ess lea ds t o ER st r ess a n d sev er a l differ en t st r a t eg ies, in clu din g t h e u se of specific ph a r m a colog ica l a g en t s t h a t m odify t h e pr ocess [8 5 ], su ppor t t h is con cept . W h et h er sleep depr iv a t ion lea ds t o ER st r ess in t h e br a in in h u m a n s is u n k n ow n cu r r en t ly , a lt h ou g h it seem s lik ely t h a t t h is w ill be t h e ca se beca u se t h is h a s been dem on st r a t ed in a ll species st u died t o da t e. Giv en t h a t ER st r ess in du ced by sleep depr iv a t ion is a lso fou n d in t h e liv er , it is con ceiv a ble t h a t ER st r ess m ig h t be dem on st r a t ed in per iph er a l leu k ocy t es in h u m a n s a n a r ea for fu r t h er st u dy . T h e con cept of m odu la t ion of sy n a pt ic pla st icit y w it h sleep or w a k e st a t e, a r isin g fr om m icr oa r r a y st u dies, is a lso su ppor t ed by in v est ig a t ion of ph osph or y la t ion of t h e g lu t a m a t e r ecept or s ca lciu m /ca lm odu lin -depen den t pr ot ein k in a se II (Ca MKII) a n d g ly cog en sy n t h a se k in a se 3 (GSK3 ) [8 6 ]. How ev er , ot h er a ppr oa ch es, a t lea st in dev elopin g a n im a ls [8 7 ], su g g est t h a t st r en g t h en in g of sy n a pt ic con n ect ion s occu r s du r in g sleep, n ot w a k efu ln ess, w h ich is com pa t ible w it h t h e im pr ov em en t s in per for m a n ce of specific t a sk s t h a t occu r in h u m a n s fr om befor e t o a ft er sleep (for r ev iew , see [8 8 ]). T h u s, fu r t h er st u dy of t h is con cept is r equ ir ed beca u se t h e sit u a t ion m ig h t be m or e com plex . It is con ceiv a ble t h a t differ en t n eu r on a l g r ou ps r espon d differ en t ly w it h r espect t o sleep or w a k e ch a n g es in sy n a pt ic pla st icit y . T h e con cept t h a t sleep is a st a g e of m a cr om olecu la r sy n t h esis is r ela t iv ely r ecen t , a lt h ou g h com pa t ible w it h ea r lier da t a t h a t pr ot ein sy n t h esis occu r s du r in g sleep [8 9 ,9 0 ]. Fu r t h er st u dies t o v a lida t e t h is a r e r equ ir ed. In pa r t icu la r , it w ill be im por t a n t t o det er m in e t h e m olecu la r ba sis for t h e sw it ch fr om en er g y r esou r ces bein g u sed du r in g w a k efu ln ess t o su ppor t n eu r on a l fir in g t o t h ose bein g u sed du r in g sleep for sy n t h esis of k ey m olecu les. A lt h ou g h m u ch h a s been a ccom plish ed, m u ch m or e r em a in s t o be don e (Box 2 ). Micr oa r r a y st u dies of ch a n g es in g en e ex pr ession du r in g t h e cy cle of sleep a n d w a k efu ln ess in t h e n em a t ode a n d zebr a fish sh ou ld fu r t h er cla r ify w h a t fu n ct ion s of sleep a r e con ser v ed a cr oss ph y log en y . Sleep a n d
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w a k efu ln ess, u n lik e t h e cir ca dia n clock , w h ich ca n fu n ct ion a s a cell-a u t on om ou s m olecu la r n eg a t iv e-feedba ck loop, a r e con t r olled by in t er a ct in g n eu r on a l cir cu it s (for r ev iew , see [5 3 ,9 1 9 3 ]). T h u s, u n der st a n din g sleep a n d w a k efu ln ess a t t h e lev el of t r a n scr ipt ion r equ ir es t h e st u dy of ch a n g es in g en e ex pr ession w it h in iden t ified popu la t ion s of n eu r on a l cells. Iden t ifica t ion a n d isola t ion of pa r t icu la r n eu r on a l cell popu la t ion s w ou ld t h en be t h e ba sis of a m icr oa r r a y -ba sed ex a m in a t ion of g en e ex pr ession in m odel or g a n ism s. T h is is possible w it h la ser m icr odissect ion , h ig h -t h r ou g h pu t in s itu h y br idiza t ion or t h e u se of flow cy t om et r y for cell sepa r a t ion (for a r ev iew of t h ese m et h ods, see [9 4 ]). T h e fir st a t t em pt t o a ssess ch a n g es in t h e t r a n scr ipt lev el in a specific n eu r on a l popu la t ion w a s m a de by Ma r et et al. , w h o u sed m icr oa r r a y s t o st u dy RNA isola t ed fr om n eu r on s ex pr essin g Hom er1a g en e [1 8 ]. In t h is ex per im en t , a poly -A bin din g pr ot ein (PA PB) w a s ex pr essed in t r a n sg en ic m ice u n der t h e con t r ol of t h e pr om ot er of t h e Hom er1 g en e, follow ed by t h e pu r ifica t ion of t h e PA PB a n d t h e bou n d m RNA . T h is st u dy iden t ified sev er a l t r a n scr ipt s w it h ch a n g in g ex pr ession in Hom er1a -ex pr essin g n eu r on s a ft er sleep loss, in clu din g , a s pr edict ed, t h e t r a n scr ipt of t h e Hom er1a g en e [1 8 ].

Box 2. Future applications of microarrays in sleep research

T o iden t ify con ser v ed g en e ex pr ession ch a n g es in sleepw a k e per iods in a ddit ion a l m odel or g a n ism s t o iden t ify k ey con ser v ed sleep fu n ct ion s. T o det er m in e ch a n g es in g en e ex pr ession du r in g sleepw a k e per iods a n d on sleep depr iv a t ion in iden t ified n eu r on a l popu la t ion s. T o det er m in e ch a n g es in g en e ex pr ession du r in g sleepw a k e a n d sleep depr iv a t ion in per iph er a l or g a n s; sepa r a t in g effect s of sleepw a k e a n d sleep depr iv a t ion fr om clock in flu en ces. T o dev elop m olecu la r sig n a t u r es for dia g n ost ic a pplica t ion s a n d for iden t ify in g in div idu a ls pa r t icu la r ly a t r isk of a dv er se ou t com es st em m in g fr om sleep depr iv a t ion a n d sleep disor der s. Recen t a dv a n ces in sequ en cin g t ech n olog ies, su ch a s sh or t -r ea d h ig h -t h r ou g h pu t sequ en cin g (RNA Seq), m a k e it clea r t h a t cu r r en t m et h ods, in clu din g m icr oa r r a y s, a r e n ot iden t ify in g a ll of t h e t r a n scr ipt ion a l la n dsca pe of m a m m a lia n cells [9 5 ]. RNA -Seq det ect s a s m a n y a s 2 5 % m or e g en e t r a n scr ipt s t h a n m icr oa r r a y s. T h u s, h ig h -t h r ou g h pu t sequ en cin g of t h e t r a n scr ipt om e du r in g sleep a n d w a k efu ln ess w ill pr ov ide a ddit ion a l in for m a t ion con cer n in g ch a n g es in g en e ex pr ession bet w een t h ese beh a v ior a l st a t es. A lt h ou g h t h e con t r ol of sleep a n d w a k efu ln ess or ig in a t es in t h e br a in a n d m a n y fu n ct ion s of sleep a r e cen t er ed on t h e br a in , la ck of sleep a ffect s per iph er a l or g a n s, a s seen by t h e effect s of sleep depr iv a t ion on m et a bolism [9 6 ] a n d ca r dia c h ea lt h [9 7 ]. T h er efor e, m icr oa r r a y st u dies a lso n eed t o ex t en d t o per iph er a l or g a n s so t h a t t h e effect of sleep a n d w a k efu ln ess, a s w ell a s sleep depr iv a t ion on g en e ex pr ession in h ea r t , lu n g a n d k idn ey , a m on g ot h er s, ca n be a ssessed. In fu r t h er in v est ig a t ion s in differ en t species, differ en t n eu r on a l g r ou ps a n d differ en t or g a n s, st u dy desig n s n eed t o be u sed t o sepa r a t e t h e effect s of sleep or w a k e fr om cir ca dia n clock in flu en ces, a s h a s been don e in r ecen t st u dies [1 8 ]. On e of t h e g oa ls of clin ica l r esea r ch is t o iden t ify t h e ea r liest possible st a g e of pr og r essiv e disea ses, so t h a t t r ea t m en t ca n be in it ia t ed. T h u s, a lt h ou g h t h e sea r ch es for pr edict iv e biom a r k er s of sleep disor der s a n d t h e delet er iou s effect s of sleep depr iv a t ion h a v e on ly ju st beg u n , m icr oa r r a y -ba sed st r a t eg ies a r e a lr ea dy beg in n in g t o iden t ify pa t h w a y s t o pr ov ide bot h dia g n ost ic in for m a t ion a bou t t h e pr esen ce of a specific sleep disor der a n d pr og n ost ic in for m a t ion .

Acknowledgements
W e a r e g r a t efu l t o Da n iel Ba r r et t a n d Jen n ifer Mon t oy a for t h eir h elp in pr epa r a t ion of t h is
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What are microarrays teaching us about sleep?

m a n u scr ipt . T h e or ig in a l r esea r ch w a s su ppor t ed by NIH g r a n t s HL6 0 2 8 7 , A G1 7 6 2 8 a n d HL6 6 6 1 1 , a n d by t h e NIH/NHGRI Ru t h L. Kir ch st ein Post doct or a l Fellow sh ip HG0 0 3 9 6 8 t o K.R.S.

Footnotes
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Article information
Trends Mol Med. Author manuscript; available in PMC 2010 September 20. Published in final edited form as: Trends Mol Med. 2009 February; 15(2): 7987. Published online 2009 January 21. doi: 10.1016/j.molmed.2008.12.002 PMCID: PMC2942088 NIHMSID: NIHMS230888 Miroslaw Mackiewicz,1,2 John E. Zimmerman,1 Keith R. Shockley,3 Gary A. Churchill,3 and Allan I. Pack1,2
1 Center for Sleep 2 Division 3 The

and Respiratory Neurobiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA

of Sleep Medicine/Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA

Jackson Laboratory, Bar Harbor, ME 04609, USA

Corresponding author: Pack, A.I. (Email: pack/at/mail.med.upenn.edu) Copyright notice and Disclaimer Publisher's Disclaimer The publisher's final edited version of this article is available at Trends Mol Med See other articles in PMC that cite the published article.

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