External and circadian inputs modulate synaptic protein expression in the visual system of Drosophila melanogaster

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ORIGINAL RESEARCH ARTICLE

published: 03 April 2014 doi: 10.3389/fphys.2014.00102

External and circadian inputs modulate synaptic protein expression in the visual system of D rosophila

m elanogaster

Wojciech Krzeptowskit, Jolanta Górska-Andrzejakt, Ewelina Kijakt, Alicja Görlich, Elżbieta Guzik, Gareth Moore and ElZbieta M. Pyza *

Department o f Cell Biology and Imaging, Institute o f Zoology, Jagiellonian University, Krakow, Poland

Edited by:

M atti Weckstrom, University o f Oulu, Finland

Reviewed by:

M ikko Juusola, University o f Sheffield, UK

Paivi Helena Torkkeli, Dalhousie University, Canada

*Correspondence:

Elżbieta M. Pyza, Department of Cell Biology and Imaging, Institute o f Zoology, Jagiellonian University, Gronostajowa 9, 30-387 Krakow, Poland

e-mail: elzbieta.pyza@uj.edu.pl f These authors have contributed equally to this work.

In th e visual s y s te m o f D rosophila m e la n o g a s te r th e retina p h o to re c e p to rs fo rm te tra d synapses w ith th e firs t o rd e r in te rn e u ro n s, am acrine cells and glial cells in th e firs t o p tic neuropil (lamina), in o rd e r to tra n s m it p h o tic and visual in fo rm a tio n to th e brain. Using th e s p e c ific a n tib o d ie s ag a inst syn a p tic p ro te in s; B ruchpilot (BRP), Synapsin (SYN), and D isc Large (DLG), th e synapses in th e distal lam ina w e re sp e c ific a lly labeled. Then th e ir abundance w a s m easured as im m u n o flu o re s c e n c e in te n s ity in flie s held in lig h t/d a rk (LD 12:12), c o n s ta n t darkness (DD), and a fte r lo c o m o to r and lig h t s tim u la tio n . M oreover, th e levels o f p ro te in s (SYN and DLG), and m R N A s o f th e brp, syn, and d lg genes, w e re m easured in th e fly 's head and brain, respectively. In th e head w e did n o t d e te c t SYN and DLG oscillations. W e found, however, th a t in th e lam ina, DLG o scilla te s in LD 12:12 and DD b u t SYN cycles only in DD. The abundance o f all syn a p tic p ro te in s w a s also changed in th e lamina a fte r lo c o m o to r and lig h t s tim u la tio n . O ne hour lo c o m o to r s tim u la tio n s a t d iffe re n t tim e p o in ts in LD 12:12 a ffe cte d th e pattern o f th e daily rh yth m o f syn a p tic p ro te in s. In turn, lig h t s tim u la tio n s in DD increased th e level o f all p ro te in s stu d ie d . In th e case o f SYN, however, th is e ffe c t w a s o b se rve d only a fte r a s h o rt lig h t pulse (15 min). In c o n tra st to p ro te in s s tu d ie d in th e lam ina, th e m R N A o f brp, syn, and dlg genes in th e brain w as n o t cycling in LD 12:12 and DD, e x c e p t th e m R N A o f dlg in LD 12:12. O ur earlier results and obta in e d in th e p re s e n t s tu d y s h o w e d th a t th e abundance o f BRP SYN and DLG in th e distal lamina, at th e te tra d synapses, is regulated by lig h t and a circadian clock w h ile lo c o m o to r s tim u la tio n a ffe c ts th e ir daily pattern o f expression. The o b se rve d changes in th e level o f syn a p tic m arkers re fle c t th e circadian p la s tic ity o f te tra d synapses regulated by th e circadian clock and external inputs, both s p e c ific and u n s p e c ific fo r th e visual syste m .

Keywords: synaptic plasticity, circadian rhythms, light stimulation, locomotor activity, the first optic neuropil

INTRODUCTION

In th e first o p tic n e u ro p il (lam ina) o f the fly’s visual system th e m a jo rity o f synaptic contacts is fo rm ed betw een th e te rm i

nals o f the c o m p o u n d eye p h o to recep to rs a n d th e ir p ostsynap- tic p a rtn e rs. T h ey are called te tra d synapses a n d use h istam in e (H a rd ie , 1987; M elzig et al., 1998; G avin et al., 2007) to tra n s m it p h o tic a n d visual in fo rm a tio n to fo u r lam in a po stsy n ap tic cells: L1 an d L2 m o n o p o la r cells, am ac rin e cell, a n d glial o r L3 m o n o p o la r cell (M ein ertz h ag en , 1989; M einertzhagen an d S o rra, 2001). C o u n tin g th e p resy n ap tic profiles o f these synapses (the so-called T -bars) a t th e electron m icroscope level revealed th a t th e ir n u m b e r oscillates d u rin g th e 24 h d ay /n ig h t cycle in b o th M usca dom estica (Pyza a n d M ein ertz h ag en , 1993) a n d Drosophila m elanogaster (W oznicka an d Pyza, u n p u b lish e d d ata). In th e p h o  to recep to rs o f D. m elanogaster th e n u m b e r o f te tra d ’s T -bars increases tw o tim es d u rin g the day: a t th e b eg in n in g o f th e day a n d at the b eg in n in g o f the n ig h t (W oznicka an d Pyza, u n p u b lished data). T his p a tte rn o f changes resem bles th e rh y th m o f lo c o m o to r activity o f the fru it fly. Recently, we also fo u n d th a t a sim ilar p a tte rn o f changes is characteristic fo r daily alteratio n s

in the level o f synaptic p ro te in B ru ch p ilo t (BRP) in th e lam in a a n d in th e w hole b ra in o f D. m elanogaster (G orska-A ndrzejak et al., 2013). BRP is a p resy n ap tic active zone p ro te in , h o m o lo gous to th e m a m m a lia n ELKS/CAST fam ily o f synaptic p ro tein s (W agh et al., 2006) th a t facilitates efficient vesicle release (K ittel et al., 2006). In D. melanogaster it has b een detected in dense b o d ies o f n e u ro m u sc u la r ju n c tio n s (N M J) (W agh et al., 2006;

F o u q u et et al., 2009) an d at the lam in a T -b ar synapses (G orska- A n d rzejak et al., 2009a, 2013; H a m a n a k a an d M ein ertz h ag en , 2010).

So far, BRP is th e on ly synaptic p ro te in w hose level o f expres

sion in the lam in a an d the w hole h ead has b een fo u n d to oscillate in a circadian m a n n e r (G orska-A ndrzejak et al., 2013). However, it is possible th a t th e a m o u n t o f o th e r key p ro tein s o f th e synap

tic m a c h in e ry m ay also be influenced b y the circadian system a n d /o r by vario u s in te rn a l a n d external factors. A n o th e r presy- n a p tic p ro te in (alth o u g h n o t th e stru c tu ra l on e like BRP) is Synapsin (SYN), an ev o lu tio n ary conserved p h o sp h o p ro te in asso

ciated w ith synaptic vesicles (H ilfiker et al., 1999; S u d h o f, 2004;

Evergren et al., 2007). In D. melanogaster, as w ell as in vertebrates,

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SYN has b een show n to m a in ta in vesicle release by recru itin g synaptic vesicles fro m the reserve p ool (Li et al., 1995; P ieribone et al., 1995; Sun et al., 2006; A kbergenova an d B ykhovskaia, 2007).

T he synaptic p ro te in re p o rte d to be im p o rta n t fo r the synap

tic assem bly a n d plasticity is Discs large (D LG), a m e m b e r o f th e M A GU K (M em brane-A ssociated G uanylate K inase) fam ily o f scaffolding p ro tein s. DLG has fo u r m a m m a lia n m e m bers; SA P97/hD lg, P S D -93/C hapsyn-110, PSD -95/SA P90, an d SA P102/N E-D lg, w h ich play im p o rta n t roles in th e d evelopm ent a n d reg u latio n o f th e nerv o u s system a n d epithelia (W ang et al., 2011). In D. melanogaster DLG is involved in th e d ev elo p m en t o f epithelia (W oods et al., 1996), asy m m etric cell division (O hshiro et al., 2000), th e d ev elo p m en t a n d plasticity o f the larval N M J (Lahey et al., 1994; B u d n ik et al., 1996; G uan et al., 1996; Tejedor et al., 1997; T h o m as et al., 1997), as w ell as in o th e r synaptic fu n ctio n s th a t are im p o rta n t fo r b eh av io r o f a d u lt flies, in c lu d ing th e ir circadian rh y th m s (M endoza-T opaz et al., 2008). DLG is th o u g h t to play a key role in clustering GluRIIB receptors at the N M J synapses an d Shaker K + channels th ro u g h o u t the CNS (C hen et al., 2008). In the b ra in o f D. melanogaster DLG is highly expressed in th e visual system (R ogero et al., 1997; Ruiz- C an ad a et al., 2002), w hile in th e la m in a it has b een localized in th e m e m b ra n e s o f p h o to re c e p to r term in als th a t su rro u n d the inv ag in atin g heads o f th e glial capitate p ro jectio n s (H am an ak a a n d M einertzhagen, 2010). A t N M Js, DLG is highly co n cen trated postsynaptically, in the subsynaptic re tic u lu m (SSR) a n d it is less p ro m in e n tly accu m u lated a t th e p resy n ap tic m e m b ra n e (G uan et al., 1996). H ow ever, DLG expression is im p o rta n t fo r the stru c tu re o f b o th p re- an d p o stsy n ap tic sites, since in dlg m u ta n ts, th a t survive only to th e p u p a l stage, th e n u m b e r o f N M J T -bars is increased a n d SSR is red u ced (Lahey et al., 1994).

In th e p re se n t study we exam ined circadian changes in th e level o f th ree crucial synaptic p ro tein s; BRP, SYN, a n d DLG, an d th eir gene expression in o rd er to learn a b o u t th e ir possible involve

m e n t in th e circadian synaptic plasticity o f te tra d synapses in the lam in a a n d in the b ra in . In a d d itio n we analyzed th e ir responses to external factors, such as d irect light exposure an d lo c o m o to r stim u latio n .

MATERIALS AND METHODS

A N IM A LS

All ex p erim en ts w ere co n d u cte d o n a w ild type C a n to n S strain o f Drosophila melanogaster. T he stock w as m a in ta in e d o n a sta n d a rd y east-co rn m eal-ag ar m e d iu m at 25 ± 1°C, u n d e r a d a y /n ig h t cycle (12 h o f light an d 12 h o f darkness; LD 12:12). Follow ing th e ir eclosion, the ex p erim en tal flies w ere k ep t in LD 12:12 fo r 1 w eek an d th e 7 days old m ales were d ecap itated at th e specific tim e p o in ts o f day a n d n ig h t. These tim e p o in ts were chosen to reflect significant features o f th e circadian lo c o m o to r activity p a tte rn . To exam ine th e expression o f stu d ied genes an d p ro tein s d u rin g the m o rn in g an d evening lo c o m o to r activity peaks, we chose ZT1 an d ZT12 o r ZT13 (ZT— Z eitgeber T im e; Z T0— th e b eg in n in g o f the day, Z T12— the b eg in n in g o f the n ig h t), respectively. T he after

n o o n siesta w as exam ined at ZT4 o r ZT6 a n d sleep at ZT16 or ZT18. To d eterm in e the role o f the circadian clock in regulating th e stu d ied rh y th m s, a n o th e r g ro u p o f th e exp erim en tal flies w as tra n sfe rre d to co n sta n t darkness (D D ) after 4 days in LD 12:12.

O n th e th ird day o f D D , flies w ere d ecap itated at CT1, CT4 o r CT6 a n d CT12 o r C T13, a n d CT16 o r CT18 (CT— C ircadian Tim e;

CT0— th e b eg in n in g o f th e subjective day, CT12— the b e g in n in g o f th e subjective n ig h t).

D ecap itatio n s o f flies in th e d a rk phase o f the LD 12:12, as w ell as all flies k ep t in D D w ere c o n d u cte d u n d e r dim red lighting (Pyza a n d M ein ertz h ag en , 1996).

LOCOMOTOR AND LIGHT STIMULATIONS

For lo c o m o to r stim u latio n experim ents, all flies w ere k ep t in em pty 250 m l E rlenm eyer flasks. O n e h o u r p rio r to d ecap itatio n th e ex p erim en tal flies w ere forced to fly by shaking th e flask. For light stim u latio n experim ents, flies w ere k ep t in LD 12:12 fo r 4 days before b ein g tra n sfe rre d to DD. O n the th ird day in c o n d i

tio n s o f co n sta n t darkness th e y w ere stim u lated w ith a w hite light pulse fo r eith er 15 o r 6 0 m in before th e ir decap itatio n at CT1.

C o n tro l g ro u p s w ere k ep t in co n sta n t darkness.

IM M U NO HISTOCHEM ISTRY

T he heads o f th e flies collected at the ex p erim en tal tim e p o in ts (ZTs o r CTs) w ere processed in parallel, u n d e r the sam e c o n ditions. Flies w ere im m o b ilized w ith C O2 an d decapitated in a d ro p o f fixative— 4% form aldehyde (PFA) in a 0.1 M p h o s

ph ate b u ffer (PB). Follow ing tissue fixation an d overn ig h t infil

tra tio n in a 25% so lu tio n o f sucrose, the cryostat sections o f heads w ere cu t an d in cu b ated w ith one o f the th ree m o u se m o n o c lo n a l antibodies: nc82, 3C11, o r 4F3 [D evelopm ental Studies H y b rid o m a B ank (D SHB)] detecting th e synaptic p ro teins BRP, SYN, a n d D isc Large (DLG), respectively (K ittel et al., 2006; W agh et al., 2006; H o fb au er et al., 2009; H am a n a k a and M einertzhagen, 2010). T he 4F3 an tib o d y w as p ro d u c e d against th e P S D -95/D L G /zona occludens-1 2 (PD Z -2) (Parnas et al., 2001). A fter several w ashes in a 0.01 M so d iu m p h o sp h ate b uffer (PBS) c o n tain in g 0.2% T riton-X (Sigm a), th e tissue was in c u b a te d w ith th e g o at an ti-m o u se secondary an tib o d y conjugated w ith Cy3 (Jackson Im m u n o R esearch L aboratories Inc.). Sections w ere exam ined using a Zeiss LSM 510 M eta confocal m ic ro scope follow ing w ashing a n d m o u n tin g in a V ectashield m e d iu m (Vector).

QUANTIFICATION OF IMM UNOLABELING

In each ex p erim en t, the im ages o f th e lam in a o f flies decapitated at different tim e p o in ts w ere collected at n o n -s a tu ra te d settings, using id en tical im age acquisition p aram eters. T he changes in the level o f fluorescence o f BRP, SYN, a n d D LG -specific im m u n o la - beling (b rig h tn ess), c o rresp o n d in g to th e changes in the a m o u n t o f th e given p ro te in (BRP, SYN, o r D LG ), w ere qu an tified using Im ageJ softw are (N IH ). In each ex p erim en t th e in ten sity o f flu orescence w as m easu red in the cartridges o f th e distal p a rt o f the cross-sectioned lam in a (the second a n d th e th ird row o f cartridges fro m th e lam in a cortex) (Figure 1B) , as rep resen ted b y the M ean Gray Value, defined by the su m o f th e gray values o f all pixels in th e selected area, divided by th e n u m b e r o f pixels w ith in the selection. In th e Im ageJ softw are, th e range o f gray values in 8-bit im ages is divided in to 256 bins. For each tim e p o in t (ZTs o r CTs) we collected d a ta fro m 7 to 12 individuals. T he b a c k g ro u n d signal w as su b tracted .

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Krzeptowski et al. Synaptic proteins and plasticity

FIGURE 1 | (A) The synaptic neuropils of the lamina (L), medulla (M) and the central brain of Drosophila melanogaster visualized by anti-BRP immunostaining using Mab nc82. There is a strong fluorescent signal in the mushroom body pedunculi in the central brain as well (arrow heads). R, retina. Scale bar: 50 pm. (B) The array of cartridges (arrows) in the cross sectioned neuropil of lamina as revealed by immunostaining using Mab 4F3 against DLG. Scale bar: 20 pm. Insert: the magnification of a single cartridge. Scale bar: 1 pm. (C-E) Cross sections of cartridges of GMR-Gal4 x UAS-S65T-GFP transgenic flies showing targeted expression of a green fluorescent protein (GFP) in photoreceptors (green) and immunolabeling (magenta) with Mab nc82 against BRP (C), 3C11 against SYN (D) and 4F3 against DLG (E). R—the terminals of six photoreceptors, L1, L2—interneurons receiving photic and visual information from photoreceptors, the so-called large monopolar cells (LMCs), Gl—the epithelial glial cells that surround lamina cartridges. Scale bar: 1 pm.

DETERMINATION OF mRNA

For b ra in dissection we u sed th e m e th o d d escribed by F ujita et al.

(1987) . To su m m arize, ex p erim en tal flies, som e 15-20 in d iv id u als p e r tim e p o in t, w ere frozen in liq u id n itro g e n . Subsequently, flies w ere tra n sfe rre d in to glass tu b es filled w ith 2 m l o f cold acetone ( - 8 0 ° C) for d eh y d ratio n , an d stored at - 8 0 ° C fo r 1 week. D u rin g th a t tim e, th e acetone was replaced twice. T hen, flies b ra in s w ere dissected a n d to ta l RN A was isolated using a N ucleoSpin RNA XS k it (M acherey-N agel) according to the m a n u fa c tu re r’s pro to co l. To avoid c o n ta m in a tio n by genom ic D NA, sam ples w ere treated w ith rD N ase. RNA was eluted in 11 p l o f RN ase-free w ater. Reverse tra n sc rip tio n was p e rfo rm e d according to th e m a n u fa c tu re r’s p ro to co l using a S uperS cript

III F irst-S tran d Synthesis System for RT-PCR k it (Invitrogen), fo r dlg analysis o r a H ig h C apacity cD N A Reverse T ran scrip tio n Kit (A pplied Biosystem s) for analysis o f brp a n d syn. cDNA w as p rep ared in 20 p l volum es using an o lig o (d T )20 p rim e r or ra n d o m p rim ers. G ene q u an tificatio n was p e rfo rm e d using th e Step O n e Plus R eal-Tim e PC R System (A pplied Biosystem s) an d TaqM an G ene Expression Assays (A pplied B iosystem s). dlg (Assay ID: D m 01799278_g1), brp (Assay ID: D m 0194336_g1), an d syn (Assay ID: D m 02148572_m 1) genes w ere exam ined. Q u an titativ e PC R w as p e rfo rm e d w ith 1 p l o f cD N A as a tem p late in a final reactio n v o lu m e o f 20 p l. T h e rm a l cycling c o n d itio n s w ere as fol

lows: 2 m in at 50°C, 1 0 m in at 9 5 °C follow ed b y 40 repeats o f 15 s at 95°C, a n d 1 m in at 60°C. D ata collection was p e rfo rm e d d u rin g each annealing phase. Raw Ct values w ere collected a n d analysis was p e rfo rm e d according to th e 2-AACT m e th o d , using rpl32 (Assay ID: D m 02151827_g1) as a reference gene.

G ene q u an tificatio n s w ere co m p leted using th e softw are su pplied by, a n d according to th e in s tru c tio n s o f th e m an u factu rer. All ex p erim en ts w ere p e rfo rm e d in d e p e n d e n tly at least th re e tim es, a n d each e x p erim en t co m p rised th ree parallel sam ples. In each r u n a negative co n tro l (distilled w ater in stead o f cDNA) was included.

W ESTERN BLOT ANALYSIS

Flies collected at different tim e p o in ts (ZTs a n d CTs) w ere im m e diately frozen in liq u id n itro g en . T h eir heads (2 0 -3 0 heads per tim e p o in t) w ere cu t off, frozen a n d h o m o g en ized by sonification in a RIPA b uffer (1.5 p l/1 head) co n tain in g a com plete protease in h ib ito r cocktail (B oehringer). A fter being gently shaken fo r 1 h o n ice, th e o b ta in e d h o m o g en ates w ere sto red at — 20° C u n til cen trifu g atio n . Sam ples w ere centrifuged at 13,200 rp m for 1 h at 4°C an d su p p lem en ted w ith a 2 x L aem m li buffer (1:1) before d e n a tu ra tio n at 100° C for 5 m in . T he p ro te in level was m easu red w ith a Q u a n t-iT P ro tein Assay Kit using a Q u b it flu o ro m eter (Invitrogen). P ro tein extracts w ere subjected to electrophoresis using th e NuPAGE SDS-PAGE Gel System. T he 4 -1 2 % Bis-Tris gel w as lo ad ed w ith 20 o r 10 p g (SYN a n d DLG, respectively) o f th e to ta l p ro te in p er lane. T he p ro tein s w ere b lo tte d b y ele c tro tra n s

fer o n to a PV DF m e m b ra n e th a t was blocked w ith 5% n o n fa t d ry m ilk in PBS w ith 0.1% Tween-20. Follow ing blocking, th e m e m b ra n e w as im m u n o p ro b e d eith er w ith M ab 3C11 (1:10,000) or M ab 4F3 (1:20,000). T he effectiveness o f p ro te in loading (loading co n tro l) was defined by p ro b in g th e b lots w ith th e m o u se m o n o clonal a n ti-a tu b u lin an tib o d y (AA4.3), d ilu ted 1:20,000 (DSHB).

T he ECL d etectio n system (Perkin Elm er Inc.) was applied for im m u n o d e te c tio n , w hereas th e A lphaEaseFC S tan d A lone im age analysis p ro g ra m (A lpha In n o tech , Cell Bioscience,) was used fo r th e d en sito m etric analysis o f th e o b ta in e d W estern blots.

T h e collected d a ta derived fro m th ree in d e p e n d e n t ex p erim en ts p ro v id in g th re e d istin ct p ro te in p rep aratio n s.

STATISTICS

All statistical tests w ere p e rfo rm e d using th e Statistica v.10 (StatSoft, Inc.). T he significance o f differences observed betw een tw o in d e p e n d e n t gro u p s (e.g., day an d n ig h t) w ere statistically analyzed using th e T -test o r M a n n -W h itn e y Test, d ep en d in g o n th e resu lt o f th e S h a p iro -W ilk W -Test. Statistical significance

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across groups, ZT o r C T tim e p o in ts, was analyzed using O n e W ay ANOVA follow ed by a Tukey post-hoc Test o r th e n o n p a ra - m e tric c o u n te rp a rt o f ANOVA— K ruskal-W allis Test (one-w ay test), follow ed by th e M ultiple C o m p ariso n Test. To assess d if

ferences b etw een LD 12:12 an d D D w e p e rfo rm e d th e Two-W ay ANOVA regarding th e light co n d itio n s a n d th e tim e p o in ts (ZTs a n d CTs) as tw o key factors.

Im m u n o lab elin g q u an tificatio n d ata are show n as a p e rc e n t

age o f th e hig h est value (100% ) th a t was o b ta in e d in a given e x p erim en t (m eans ± SD).

For visualization o f qPC R data, gene expression was n o rm a l

ized o n an a rb itra ry scale, w here th e ZT1 tim e p o in t was set to 1.00 (m eans ± SE).

RESULTS

T he p a tte rn s o f im m u n o lab elin g o f BRP, SYN, an d DLG in the w hole b ra in o f D. melanogaster was sim ilar, if n o t identical. In each case, th e im m u n o flu o rescen ce was confined to n eu ro p ils o f th e visual system a n d th e cen tral b ra in , as displayed by an exem plary im age o f BRP-specific im m u n o lab elin g in Figure 1A.

Analysis o f these labelings in th e cross sectioned la m in a show ed th a t each o f th e stu d ied p ro tein s is c o n cen trated w ith in th e sy n ap tic u n its o r cartridges, revealing th e array o f cartridges th a t co n sti

tu te th is n e u ro p il (Figure 1B) . However, th e p a tte rn o f expression w ith in th e cartrid g e (Figures 1C -E ) is different for each o f these p ro tein s, fo rm in g well defined spots in case o f BRP (w hich local

izes to T -b ar rib b o n s) a n d being m o re dispersed in th e case o f SYN o r DLG (Figures 1C -E ) .

FIGURE 2 | The relative level of brp mRNA in the brain of flies raised in LD 12:12 (A) and in constant darkness (B). The level of brp mRNA is constant during the day and night in LD and DD.

BRUCHPILOT

T he brp m R N A d id n o t cycle in th e b ra in o f flies raised either in LD 12:12 (Figure 2A ) [K ruskal-W allis Test: H (3, n = 12) = 2.63, p = 0.453] o r in D D (F ig u re2 B ) [K ruskal-W allis Test:

H (³, n = 12) = 3.04, p = 0.38].

A fter 1 h lo c o m o to r stim u latio n , th e level o f BRP flu c tu ated (ANOVA, p < 0.0001), being h ig h er at ZT1 a n d ZT13 in c o m p ariso n to Z T4 a n d ZT16 (Figure 3A ) (Tukey’s H SD Test, differences betw een ZT1 a n d Z T4 at p < 0.05; betw een ZT1 an d ZT16 at p < 0.01; betw een Z T4 a n d ZT13 at p < 0.001, an d betw een ZT13 a n d ZT16 at p < 0.001). However, th e a m p litu d e o f th e observed changes was larger th a n in n o n -s tim u la te d flies (G orska-A ndrzejak et al., 2013).

Light stim u la tio n p ro fo u n d ly increased th e level o f BRP in th e lam in a o f D. melanogaster [K ruskal-W allis Test:

H (2, n = 57) = 11.08, p = 0.004] (Figure 3B ) . W h en co m p ared to th e n o n -s tim u la te d controls, th e in ten sity o f BRP-specific im m u n o flu o rescen ce in th e la m in a o f flies exposed to th e lig h t pulse at CT1 for eith er 15, o r 60 m in , increased by 58 a n d 63% , respectively (M ultiple C o m p a riso n Test, p = 0.02 a n d p = 0.005, respectively).

SYNAPSIN

T he level o f syn m R N A d id n o t cycle in th e b ra in o f flies held e ith er in LD 12:12 [K ruskal-W allis Test: H (3, N = 12) = 1.37, p = 0.7], o r in D D [K ruskal-W allis Test: H (3, N = 12) = 3.82, p = 0.3]

(Figures 4A ,B ). Also th e to ta l a m o u n t o f SYN in w hole head h o m o g en ates stu d ied using th e W estern b lo ttin g m e th o d , did

n o t change significantly d u rin g th e 24 h p erio d , eith er in LD 12:12 (ANOVA, F = 0.1 8, p = 0.9) o r in D D [K ruskal-W allis Test H (3, n = 12) = 5.1, p = 0.16] (d ata n o t show n).

Sim ilar results w ere o b tain ed for SYN p ro te in in th e la m in a o f flies h eld in LD 12:12. T he analysis o f th e fluorescence in te n sity o f im m u n o la b e le d SYN show ed th a t th e a b u n d a n c e o f this p ro te in d id n o t change significantly in th e course o f day an d n ig h t in LD 12:12 (ANOVA, F = 0.54, p = 0.7) (Figure 5A ) . T he average level o f SYN-specific fluorescence d u rin g th e day an d th e n ig h t was th e sam e (t-test, t = 0.02, d f = 26, p < 0.98). In DD, o n th e o th e r h a n d , th ere w ere significant changes in th e level o f S Y N -im m unospecific fluorescence in th e la m in a over th e 2 4 h p e rio d [K ruskal-W allis Test (3, N = 31) = 14.7, p = 0.002] (Figure 4 B ). T he fluorescence in ten sity was th e highest in th e m id d le o f th e subjective day, at CT4, a n d th e low est in th e m id d le o f th e subjective n ig h t, at CT16 (Figure 5B ). T he differ

ence b etw een these tw o tim e p o in ts was statistically significant (M ultiple C o m p a riso n Test, p = 0.002). T he average level o f flu  orescence d u rin g th e subjective day w as th e sam e as d u rin g th e day in LD 12:12, b u t was 31% low er d u rin g th e subjective nig h t, th a n d u rin g th e n ig h t in LD 12:12. T he difference betw een th e subjective day a n d th e subjective n ig h t was statistically signifi

c an t (M a n n -W h itn e y Test, U = 32, p = 0.0005) a n d so w as th e difference betw een th e daily p a tte rn s o f SYN-specific im m u n o flu orescence in D D a n d LD 12:12 (MANOVA, F = 3.2, p = 0.03).

M oreover, th e average level o f fluorescence in D D was 22% low er th a n in LD 12:12 (M a n n -W h itn e y Test, U = 254.5, p = 0.007).

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Krzeptowski et al. Synaptic proteins and plasticity

FIGURE 3 | The level of BRP-specific immunofluorescence intensity (brightness) in the fly's lamina after locomotor (A) and light (B) stimulations. Both stimulations increase BRP level (*p < 0.05, p < 0.01,

p < 0.001).

FIGURE 4 | The relative level of synmRNA in the brain of flies raised in LD 12:12 (A) and in constant darkness (B). The mRNA of syn does not cycle in LD 12:12 or in DD.

A fter 1 h lo c o m o to r activity stim u la tio n at different tim es o f th e day, th e in ten sity o f SYN-specific im m u n o flu o rescen ce in the la m in a displayed significant changes in th e course o f th e day [K ruskal-W allis Test (4, N = 53) = 18, p = 0.001] (Figure 5C ).

It was th e low est in th e la m in a o f flies stim u lated at th e b eg in n in g o f th e day (ZT1— 47% ). A t th a t tim e th e fluorescence in ten sity was considerably low er (M ultiple C o m p a riso n Test, p = 0.02) th a n at th e b eg in n in g o f th e n ig h t (Z T13), w h en it reached the hig h est value m easu red fo r th is set o f d ata (65% ). Also th e average level o f fluorescence d u rin g th e day (ZT1 a n d ZT4) was signif

ican tly low er (11% ) th a n d u rin g th e n ig h t (ZT13 a n d ZT16) (M a n n -W h itn e y Test, U = 88, p = 0.001).

A fter light stim u latio n at CT1 th e level o f SYN in th e lam in a increased b u t only after th e s h o rt 1 5 m in light pulse [K ru sk al- W allis Test (2, N = 35) = 11.05, p = 0.004] (Figure 3 D ). T he sh o rt pulse increased SYN-specific im m u n o flu o rescen ce in ten sity by 21% w h en co m p ared w ith th e control. H owever, after a longer, 60 m in light pulse, th e level o f SYN was sim ilar to th e control. T he differences b etw een th e 15 m in light pulse a n d th e co n tro l (67 vs.

47% ), as well as betw een th e 60 m in a n d 15 m in pulses (67 vs.

45% ), w ere significant (M ultiple C o m p ariso n Test, p = 0.02 an d p = 0.006, respectively).

DISC LARGE

R eal-tim e PC R q u an tificatio n revealed significant changes in the expression o f dlg in th e b ra in o f D. melanogaster collected at

different tim e p o in ts o f th e LD 12:12 cycle [K ruskal-W allis Test:

H (3, n = 23) = 17.7, p = 0.0005] (Figure 6A ). T he hig h est level o f dlg m R N A was detected in th e m id d le o f th e day (ZT6) an d at th e en d o f th e day (ZT12), w hereas th e low est was ty p i

cal fo r th e en d o f th e n ig h t (ZT18). T he differences betw een ZT1 a n d ZT6 (63% ) o r ZT1 a n d ZT18 (103% ) w ere statistically significant (M ultiple C o m p a riso n Test, p = 0.01 a n d p = 0.005 respectively), as was th e difference betw een ZT12 a n d ZT18 (86% , M ultiple C o m p a riso n Test, p = 0.05). T he expression o f dlg in th e b ra in o f flies raised in c o n sta n t co n d itio n s o f darkness did n o t change d u rin g th e subjective day a n d n ig h t [K ruskal-W allis Test: H (3, n = 28) = 4.83, p = 0.18] (Figure 6B ). In tu r n , th e to tal a m o u n t o f DLG in th e w hole h e a d h o m o g en ates o b ta in e d fro m flies kept in LD 12:12 o r DD, was th e sam e d u rin g th e 24 h cycle [K ruskal-W allis Test (3, N = 12) = 1.17, p = 0.76; K ru sk al- W allis Test (3, N = 8) = 1.13, p = 0.72, respectively] (d ata n o t show n).

T he a b u n d a n c e o f DLG in th e distal la m in a o f flies held in LD 12:12, show ed significant changes d u rin g th e lig h t/d a rk cycle [K ruskal-W allis Test: H (3, N = 174) = 34.2, p < 0.0001]

(Figure 7A ) . T he p a tte rn o f changes in th e DLG level in th e la m in a was b im o d a l w ith tw o peaks, at ZT1 a n d ZT12, as in th e case o f BRP (G orska-A ndrzejak et al., 2013). T he average in ten sity o f D LG -specific fluorescence at these tw o tim e p o in ts o f th e cycle was th e sam e (M ultiple C o m p ariso n Test: p < 0.1), w hereas it was low er in th e m id d le o f th e day (ZT6) a n d in

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FIGURE 5 | Synapsin-specific immunofluorescence (brightness) measured from confocal images of the fly's lamina. The level of this protein is the same during the day and night of LD 12:12 (A), oscillates in constant darkness (B), changes after 1 h of locomotor stimulation (C) and increases after 15 min of light stimulation (D) (*p < 0.05, *'*p < 0.01).

FIGURE 6 | The relative level of dlg mRNA in the brain of flies held in LD 12:12 or DD. In LD 12:12 the mRNA of dlg changes during 24 h cycle w ith a peak in the middle of the day (ZT6) and a trough in the middle of the night (A). No rhythm was observed in DD (B); *p < 0.05, * * p < 0.01.

th e m id d le o f th e n ig h t (ZT18). T he differences betw een ZT1 a n d ZT6 (16% ) o r ZT1 a n d ZT18 (15% ) w ere statistically sig

n ifican t (M ultiple C o m p a riso n Test: p < 0.0001 a n d p < 0.0001, respectively), as w ell as th e differences detected betw een ZT6 a n d ZT12 (13% , M ultiple C o m p a riso n Test: p = 0.002) o r ZT12 a n d ZT18 (12% , M ultiple C o m p a riso n Test: p = 0.006). Sim ilar oscillations in th e level o f D LG -specific im m u n o flu o rescen ce w ere also observed in th e distal la m in a o f flies k ep t in c o n stan t darkness [K ruskal-W allis Test: H (3, N = 198) = 27.3, p < 0.0001]

(Figure 7B ). Interestingly, th e circadian p a tte rn o f changes was th e sam e as th e o n e observed in LD 12:12, since th e flu o res

cence in ten sity was significantly h ig h er at CT1 a n d C T12 th a n at C T4 a n d CT18. T he differences betw een CT1 an d CT6 (12% ) o r CT18 (8% ), a lth o u g h slightly sm aller th a n betw een ZT1 an d ZT6 o r ZT16, w ere statistically significant (M ultiple C o m p ariso n Test: p = 0.0008 a n d p = 0.04, respectively). Also th e differences betw een th e second p eak o f DLG a b u n d an ce at C T12 a n d CT6 (15% ) o r CT18 (12% ) w ere significant (M ultiple C o m p ariso n Test: p < 0.0001 a n d p = 0.004, respectively).

T he above d escribed p a tte rn o f DLG rh y th m in th e la m in a o f flies k ep t in LD 12:12 w as changed by lo c o m o to r s tim u latio n (Figure 7C ). A fter a 1 h long lo c o m o to r stim u latio n o f flies, ad m in istered p rio r to th e ir d ecap itatio n , th e in ten sity o f D LG -specific fluorescence was still flu ctu atin g d u rin g th e

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Krzeptowski et al Synaptic proteins and plasticity

FIGURE 7 | DLG-specific immunofluorescence (brightness) measured from confocal images of the fly's lamina. In both LD 12:12 (A) and DD (B) the level of DLG oscillates and its pattern is similar in both conditions with tw o peaks at ZT1 or CT1 and ZT12 or CT12. One hour of locomotor stimulation changes the rhythm of DLG (C). Light stimulation increases DLG abundance in the lamina of wild type flies (D) (*p < 0.05, **p < 0.01,

***p < 0.001).

2 4 h c y c le [K ruskal-W allisT est: H (3, n = 149) = 24.8, p < 0.0001].

H ow ever, th e daily p a tte rn w as altered w ith a p eak at the end o f th e n ig h t (ZT18) w h en the fluorescence level w as 20, 22, an d 17% m o re intensive th a n at Z T1, Z T6, an d Z T12, respectively (M ultiple C o m p ariso n Tests: p = 0.002, p = 0.0001, p = 0.02, respectively).

To exam ine the d irect exposure o f light on th e DLG level in th e lam in a, we exam ined D LG -specific im m u n o flu o rescen ce in flies after 15 o r 60 m in light pulses in D D (Figure 7 D ). T he results revealed th e significant influence o f such stim u latio n o n th e DLG level in the lam in a [K ruskal-W allis Test: H (2, n = 254) = 51.4, p <

0.0001]. A significant increase in the in ten sity o f fluorescence (in co m p ariso n to th e n o n -s tim u la te d co n tro l g ro u p ), w as observed n o t on ly after th e 60 m in light pulse (21% ), b u t also after the 15 m in pulse (11% ) (M ultiple C o m p ariso n Test: p < 0.0001 for b o th co m p ariso n s). A significant difference (10% ) in th e level o f fluorescence w as also revealed betw een th e tw o stim u lated g ro u p s (M ultiple C o m p ariso n Test: p = 0.004).

DISCUSSION

All th ree synaptic m ark ers exam ined in o u r p revious (G orska- A n d rzejak et al., 2013) a n d p re se n t stu d y show ed b o th cyclic an d stim u li-d e p e n d e d changes in th e ir ab u n d an ce in the lam in a o f D. melanogaster. T his is th e first re p o rt th a t the ab u n d an ce o f th e synaptic stru c tu ra l p ro tein s (BRP a n d DLG) a n d the p ro teins involved in synaptic vesicle release (SYN) is co n tro lled by th e circadian clock n o t only in n e u ro n s o f the circadian clocks (Shapiro-R eznik et al., 2012) b u t also in o th e r b ra in regions, such as th e first o p tic n e u ro p il (lam ina). M oreover, the observed changes in the a b u n d an ce o f BRP, DLG, an d SYN w ere specific for each p ro tein . In co n trast, n o oscillations in these p ro tein s, except BRP (G orska-A ndrzejak et al., 2013), w ere detected in the w hole head. Also th e ir gene expression, except dlg, w as n o t cycling w hen m easu red as th e m R N A levels in th e b ra in o f flies sacrificed at different tim e p o in ts in the 24 h cycle. These results indicate th a t th e expression o f th e synaptic p ro tein s is specifically regulated in different regions o f the b rain .

O f th e th ree synaptic p ro tein s, BRP has already b een stu d ied in the b ra in a n d la m in a o f D. melanogaster an d its a b u n d an ce dis

played b o th daily an d circadian rh y th m s (G orska-A ndrzejak et al., 2013). In th e lam in a the level o f BRP increases tw ice d u rin g the 24 h cycle o f LD 12:12; at the b eg in n in g o f th e day (ZT1) a n d at th e o n set o f the n ig h t (ZT13). T he sam e changes w ere observed in w hole h ead h o m o g en ates analyzed using W estern b lo ttin g . In D D th e level o f BRP increases on ly once d u rin g th e 24 h cycle— at th e b eg in n in g o f th e subjective n ig h t (C T 13) (G orska-A ndrzejak et al., 2013). T he circadian rh y th m o f BRP is correlated w ith the circadian rh y th m in th e lo c o m o to r activity o f D. m elanogaster an d th e circadian plasticity o f m o n o p o la r cells in th e lam in a (Pyza and M einertzhagen, 1999; W eber et al., 2009).

In th e p re se n t study we also fo u n d th a t th e daily rh y th m o f BRP is n o t altered by lo c o m o to r stim u latio n . T he stim u latio n , how ever, increases th e level o f BRP, especially at ZT1 an d ZT13 w h en BRP n o rm ally peaks d u rin g the 24 h lig h t/d a rk cycle. The level o f BRP is also affected by light, since it increases after light pulses, a n d the circadian rh y th m o f BRP is e n tra in e d by light (G orska-A ndrzejak et al., 2013). M oreover, a m o n g tw o peaks

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observed in BRP a b u n d an ce d u rin g th e day an d n ig h t o f LD 12:12, th e m o rn in g p eak is co n tro lled by light b u t th e evening one by the circadian clock, alth o u g h b o th v an ish in m u ta n ts w ith th e n u ll m u ta tio n o f th e clock gene period (per) (G orska-A ndrzejak et al., 2013).

O u r p re se n t resu lts, as w ell as o u r p revious findings (G orska- A n d rzejak et al., 2013) in d icate th a t BRP a b u n d an ce a t te tra d synapses is co n tro lled by th e circadian clock a n d m o d u la te d by light a n d o th e r stim uli. T herefore, BRP organizes the te tra d presy- n a p tic elem ents according to th e activity o f a fly a n d flow o f visual a n d p h o tic in fo rm a tio n fro m th e eye p h o to recep to rs.

T he BRP level is also affected b y sleep d ep riv atio n (G ilestro et al., 2009) a n d b y activity stim u latio n , how ever, a sh o rt, 1 h lo co m o to r stim u latio n does n o t change th e daily p a tte rn o f BRP b u t m agnifies the am p litu d e o f th e rh y th m .

In the case o f SYN th e results are different th a n those o b tain ed fo r BRP in LD 12:12 (G orska-A ndrzejak et al., 2013). T he level o f SYN in th e lam in a, m easu red as SYN-specific im m u n o flu orescence intensity, d u rin g the 24 h cycle o f LD 12:12 d id n o t change. T here are also n o significant changes in SYN level in the h ead analyzed using th e W estern b lo t tech n iq u e. These results are su rp risin g , since SYN, like BRP, co n stitu tes p resy n ap tic m a c h in ery. H ow ever, SYN is involved in the reg u latio n o f the reserve p o o l o f vesicles (A kbergenova a n d B ykhovskaia, 2007), w h ich is k n o w n to be released exclusively u p o n p ro lo n g ed stim u latio n an d h ig h synaptic activity (D en k er et al., 2011). D u rin g the n o rm a l activity o f flies, th e m ajo rity o f these vesicles do n o t u n d erg o eith er exocytosis, o r recycling. It has b een show n th a t d u rin g such circum stances only a surprisingly sm all percentage o f vesi

cles (approxim ately 1 -5 % ) m a in ta in s synaptic activity a n d is used repeatedly. Taking in to acco u n t th a t n o rm ally active synapses ap p ear n o t to req u ire n u m e ro u s reserve vesicles to sustain n e u ro tra n s m itte r release (D en k er et al., 2011), it seem s plausible th a t th e level o f SYN stays th e sam e d u rin g the day a n d nig h t. In D D on th e o th e r h a n d , th e circadian rh y th m w as detected, w ith a h ig h er level o f SYN d u rin g th e subjective day th a n d u rin g th e subjective n ig h t. This reveals th e influence o f b o th th e circadian clock an d light o n th e expression o f SYN. It also indicates th a t th e circadian in p u t increases th e level o f SYN synthesis d u rin g th e subjective day. In LD 12:12 th e circadian reg u latio n seem s to be m asked by light, suggesting th a t th e visual system is ready to re c ru it synap

tic vesicles at any tim e d u rin g th e day an d n ig h t. In D D the SYN level w as increased on ly b y the sh o rt (15 m in ) lig h t pulse an d n o t b y th e lo n g (60 m in ) one, w h ich indicates th e fast a d a p ta tio n o f SYN to lig h t exposure.

SYN has b een used, as a synaptic m arker, in m any studies in v erteb rates an d in D. melanogaster. T he level o f SYN increases d u rin g w akefulness a n d after sleep d ep riv atio n in th e b ra in o f Drosophila (G ilestro et al., 2009) a n d after exercise in the h ip  p o c a m p u s an d d en tate gyrus o f rats (V aynm an et al., 2004;

B echara et al., 2013). A fter lo c o m o to r stim u latio n , th e increase in th e SYN level in th e lam in a w as observed only d u rin g the n ig h t. At th e b eg in n in g o f th e day, th e forced activity decreased th e expres

sion o f SYN. T his decrease m ay p ro te c t th e visual system against o v erstim u latio n by light an d the visual stim u li d u rin g h ig h lo co m o to r activity at th e b eg in n in g o f th e day. This is also consistent w ith k n o w n d ata on SYN responses to intense stim u latio n . The

S Y N -dependent m ain ten an ce o f synaptic release occurs u n d e r h ig h -freq u en cy nerve stim u latio n . T he C a2+ triggered increase in the p h o sp h o ry la tio n o f SYN, p ro m o te s re c ru itm e n t o f vesicles fro m the reserve p o o l to th e active zone o f th e presy n ap tic elem ent (P ierib o n e et al., 1995; B loom et al., 2003; M enegon et al., 2006;

Sun et al., 2006; G affield an d Betz, 2007). In synapsin-null m u ta n t o f D. melanogaster, th e p ro p o rtio n o f vesicle recycling in vivo is increased b y 30% (D en k er et al., 2011).

T herefore, th e tw o p ro tein s, BRP an d SYN, alth o u g h b o th p resy n ap tic a n d engaged in synaptic vesicle release d u rin g n e u ro tra n sm issio n , ap p ear to fu n c tio n in d istin ct m o d es th a t are activated u p o n low — o r hig h — freq u en cy stim u latio n s (K napek e ta l., 2011).

T he th ird stu d ied p ro tein , DLG, belongs to the M A GU K fam ily o f p ro tein s, w h ich play a cen tral role in m o lecu lar m echanism s involved in th e reg u latio n o f spine m o rp h o g en esis a n d co n tact actin regulating p ro tein s th a t are crucial fo r co n tro llin g the size a n d shape o f d e n d ritic spines (Z h en g et al., 2011).

In D .m elanogaster several tra n sc rip ts are coded b y the dlg gene, w hilst tw o p ro tein s, DLGA (PSD -95-like p ro te in ) a n d DLGS97 (SAP97-like p ro tein ) are m ain isoform s (M endoza et al., 2003).

T he DLGA is expressed in th e epithelia, so m atic m uscles an d n e r

vous system a n d th e dlga m u ta tio n is lethal in th e d evelopm ent (W oods an d B ry an t, 1991).

T he dlgs97 m u ta n ts survive u n til th e a d u lt stage b u t th eir behavior, in c lu d in g circadian rh y th m s, are d isru p te d (M endoza- Topaz et al., 2008). In th e p resen t stu d y we used the an tib o d y w h ich labels b o th DLGA a n d DLGS97 p ro tein s, how ever, the observed changes are related to DLGS97 since th is isoform p re d o m in ates at th e synapses in th e b ra in o f the a d u lt flies (M endoza- Topaz et al., 2008). In the lam in a, we detected th e circadian rh y th m o f DLG. M oreover, th e p a tte rn o f th e rh y th m in D D was th e sam e as in LD 12:12, w ith tw o m o rn in g /su b jectiv e m o rn in g a n d evening/subjective evening peaks. T his p a tte rn is ch aracter

istic fo r the daily rh y th m s in lo c o m o to r activity, plasticity o f the lam in a m o n o p o la r cells (Pyza an d M ein ertz h ag en , 1999), activ

ity o f the so d iu m p u m p in th e lam in a (G orska-A ndrzejak et al., 2009b; D am ulew icz et al., 2013) an d fo r th e rh y th m o f BRP (G orska-A ndrzejak et al., 2013).

We also fo u n d th a t dlg show s daily expression in th e b rain , w h ich suggests th a t th e cyclic expression o f dlg m ig h t be c ru  cial fo r rh y th m s in b ra in fu n ctio n s a n d in behavior. In dlgS97 m u ta n ts , defects in p h o to tax is a n d c o u rtsh ip behavior, as w ell as d isru p tio n o f th e circadian rh y th m in lo c o m o to r activity have b een observed (M endoza-T opaz et al., 2008). However, th e rh y th m in dlg expression w as n o t m a in ta in e d in co n stan t darkness, so th e circadian clock does n o t co n tro l its expression directly. Surprisingly th e level o f DLG, m easu red using W estern b lo ttin g , does n o t oscillate in w hole h ead ho m o g en ates. It m ay resu lt fro m different rh y th m s o f DLG iso fo rm s o r different p a t

te rn s o f DLG rh y th m in v ario u s b ra in regions. In the lam in a we observed D LG — im m u n o flu o rescen ce in p re- an d p ostsynap- tic cells b u t we w ere n o t able to exam ine p re- a n d po stsy n ap tic changes in th e a b u n d an ce o f DLG separately.

H aving analyzed the daily rh y th m s o f DLG a n d BRP, we can conclude th a t b o th scaffolding p ro tein s organize p ro p e r synaptic contacts d u rin g h ig h activity o f th e visual system a n d lo c o m o to r

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Krzeptowski et al Synaptic proteins and plasticity

activity offlies. T he co rrelatio n betw een activity an d expression o f DLG has also b een fo u n d after sleep d ep riv atio n an d lo n g perio d s o f w akefulness (G ilestro et al., 2009). In th e first n e u ro p il o f the visual system we observed th a t the DLG level increases after light pulses in D D , activating th e visual system b u t n o t after lo c o m o to r stim u latio n . T he 1 h forced m o to r activity at fo u r tim e p o in ts o f LD 12:12 increased th e a b u n d an ce o f DLG in th e m id d le o f the n ig h t. These results indicate th a t DLG, like SYN, m ay p ro te c t the visual system against o v erstim u latio n d u rin g th e day by tw o stim uli, light, an d lo c o m o to r activity, so DLG expression is suppressed d u rin g the day w h en active flies are additionally forced to fly.

E ach o f th e th ree synaptic p ro tein s th a t w ere stu d ied appears to be endogenously regulated a t synapses an d additionally m o d u lated by external stim uli. In case o f th e visual system , light is a stro n g stim u lu s th a t can synchronize, m o d u late, a n d /o r m ask th e circadian expression o f synaptic p ro tein s. H ow ever, these p ro tein s can also be m o d u la te d by n o n -v isu a l stim uli, such as m o to r stim u latio n , w h ich n o n eth eless have a stro n g er im p a c t on th e visual system th a n th e visual stim u latio n (Kula an d Pyza, 2007). In the lam in a o f D. melanogaster, light a n d m o to r stim  u latio n s n o t only affect synaptic p ro te in expression b u t also the m o rp h o lo g y o f p o stsy n ap tic cells, w h ich are rem o d eled in th eir stru c tu re d u rin g th e day an d n ig h t (Pyza an d M ein ertz h ag en , 1995, 1999; W eber et al., 2009). T he m ech an ism s o f BRP, SYN, a n d DLG circadian reg u latio n , an d th e effects o f light an d m o to r stim u latio n s on th e ir a b u n d an ce are u n k n o w n . In o u r prev i

ous study on BRP we fo u n d th a t the circadian rh y th m o f this p ro te in is abolished in th e clock m u ta n t per0 a n d its p a tte rn is changed in a n o th e r clock m u ta n t tim 0, in th e circadian p h o  to re c e p to r m u ta n t cry0, an d in th e p h o to tra n s d u c tio n m u ta n t norpA (G orska-A ndrzejak et al., 2013). These cyclic a n d stim uli- d e p e n d e n t changes o f synaptic p ro tein s m ay be responsible for several daily an d circadian rh y th m s in th e m o rp h o lo g y o fn e u ro n s (Pyza an d M ein ertz h ag en , 1995, 1999; W eber et al., 2009), glial cells (Pyza a n d G orska-A ndrzejak, 2004), activity o f io n p u m p s (Pyza et al., 2004; G orska-A ndrzej ak et al., 2009b; D am ulew icz et al., 2013) a n d in o th e r rh y th m s detected in the fly’s lam in a (Pyza a n d G orska-A ndrzejak, 2008; Pyza, 2010).

ACKNOWLEDGMENTS

T he study w as su p p o rte d by N N 301 038240 g ra n t fro m the Polish M inistry o f Science a n d H ig h E d u catio n a n d by the E u ro p ean FP7-P eople-2012-IT N IN secTIM E p ro ject n r 316790 to Elzbieta M . Pyza. W ojciech K rzeptow ski w as su p p o rte d fro m the Jagiellonian U niversity g ra n t w ith in th e SET p ro ject co -fo u n d ed by th e E u ro p ean U n io n w ith in the E uro p ean Social Fund.

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