Stress inhibition of melatonin synthesis in the pineal organ of rainbow trout (Oncorhynchus mykiss) is mediated by cortisol

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1 214. Pulished y The Compny of iologists Ltd The Journl of Experimentl iology (214) 217, doi:1.1242/je RESEARCH ARTICLE Stress inhiition of meltonin synthesis in the pinel orgn of rinow trout (Onorhynhus mykiss) is medited y ortisol Mros A. López-Ptiño*, Mnuel Gesto, Mrt Conde-Sieir, José L. Soengs nd Jesús M. Míguez ASTRACT Cortisol hs een suggested to medite the effet of stress on pinel meltonin synthesis in fish. Therefore, we imed to determine how pinel meltonin synthesis is ffeted y exposing rinow trout to different stressors, suh s hypoxi, hsing nd high stoking density. In ddition, to test the hypothesis tht ortisol is meditor of suh stress-indued effets, set of nimls were intrperitonelly implnted with oonut oil lone or ontining ortisol (5 mg kg 1 ody mss) nd smpled 5 or 48 h post-injetion t middy nd midnight. The speifiity of suh effet ws lso ssessed in ultured pinel orgns exposed to ortisol lone or with the generl gluoortioid reeptor ntgonist, mifepristone (RU486). Stress (in prtiulr hsing nd high stoking density) ffeted the ptterns of plsm nd pinel orgn meltonin ontent during oth dy nd night, with the gretest redution ourring t night. The derese in noturnl meltonin levels in the pinel orgn of stressed fish ws ompnied y inresed serotonin ontent nd deresed AANAT2 enzymti tivity nd mrna undne. Similr effets on pinel meltonin synthesis to those eliited y stress were oserved in trout implnted with ortisol for either 5 or 48 h. These dt indite tht stress negtively influenes the synthesis of meltonin in the pinel orgn, thus ttenuting the dy night vritions of irulting meltonin. The effet might e medited y inresed ortisol, whih inds to trout pinel orgn-speifi gluoortioid reeptors to modulte meltonin rhythms. Our results in ultured pinel orgns support this. Considering the role of meltonin in the synhroniztion of dily nd nnul rhythms, the results suggest tht stress-indued ltertions in meltonin synthesis ould ffet the vilility of fish to integrte rhythmi environmentl informtion. KEY WORDS: Meltonin, Pinel orgn, AANAT2, Cortisol, Rinow trout, Stress INTRODUCTION In teleost fish, the pinel orgn pereives nd trnsdues the light drk signl (romge et l., 21) into neurl nd humorl signls, one of whih is the hormone meltonin. Meltonin is rhythmilly synthesized minly from the pinel orgn nd relesed into the lood. Highest plsm levels our t night nd sl meltonin vlues our during the dy. The penultimte step of meltonin synthesis in the pinel orgn is rried out y the enzyme ryllkylmine N-etyltrnsferse (AANAT), whih is onsidered s the rte-limiting enzyme sed on its dily vritions of tivity tht prllel those of meltonin (Klein, 27). One meltonin is relesed into the lood, its rhythmi profile onveys photi Lortorio de Fisioloxí Animl, Deprtmento de ioloxí Funionl e Cienis d Súde, Fultde de ioloxí, Universidde de Vigo, 3631 Vigo, Spin. *Author for orrespondene (mlopezpt@uvigo.es) Reeived 7 Mrh 213; Aepted 22 Deemer 213 informtion to the orgnism (reviewed y Flón et l., 21) nd ts s synhronizer of vriety of proesses inluding lrvl development, loomotor tivity, sedtion, skin pigmenttion, oxygen onsumption, thermoregultion nd food intke ehvior (Ekström nd Meissl, 1997; Rees, 22; Flón et l., 21; Zhdnov nd Rees, 26). In ddition, nnul rhythms of reprodution, growth, immune response nd migrtion, re lso timed y meltonin in different fish speies (romge et l., 21; Oliveir nd Sánhez-Vázquez, 21). The dily meltonin profile persists even fter exposing fish to onstnt drkness s desried for most teleost speies (Chill, 22; Migud et l., 27). This is euse the pinel orgn hosts true irdin light sensitive pemker whih drives meltonin rhythms. Only slmonids, inluding rinow trout, re n exeption to this rule. In ll slmonid speies investigted to dte it hs een demonstrted tht pinel meltonin synthesis does not involve n endogenous lok, so tht lk of meltonin osilltion hs een desried under onstnt onditions (Thiult et l., 1993; Gern nd Greenhouse, 1988; Mizusw et l., 2; Migud et l., 27). However even under onstnt drkness severl ore irdin genes ontinue to yle in other trout neurl regions (retin nd hypothlmus) (López-Ptiño et l., 211) tht re involved in the regultion of dily rhythms of severl prmeters suh s feeding ehvior nd loomotor tivity (Cuen nd de l Higuer, 1994; Sánhez-Vázquez nd Tt, 1998). In ddition to externl ftors nd the irdin influene, severl internl ftors modulte meltonin synthesis in fish (Ekström nd Meissl, 1997). Roles hve een suggested for proltin (De Vlming nd Olese, 1981), estrogens (égy et l., 1994; Forlno et l., 25), gluoortioids (Flón, 1999; enyssi et l., 21) nd teholmines (Mrtin nd Meissl, 1992; Smejim et l., 1994; Ekström nd Meissl, 1997). Cortisol is gluoortioid synthesized in the interrenl tissue of fish. It plys n importnt role in severl spets of fish physiology, inluding energy metolism, ioni nd osmoti regultion, growth, immune funtion nd stress response (Henderson nd Grlnd, 198; MCormik, 1995; Wendelr ong, 1997; Mommsen et l., 1999). Plsm ortisol levels disply irdin rhythm in teleost speies suh s ommon dentex, Dentex dentex (Pvlidis et l., 1999), ut suh dily pttern ppers to depend on the fish speies (Gri nd Meier, 1973; Pikering nd Pottinger, 1983; Pvlidis et l., 1999; Sito et l., 24; Eesson et l., 28). For rinow trout, mny studies hve reported inresed plsm ortisol t night, peking efore the light onset, then flling nd remining low during the dy (Rne et l., 1982; oujrd nd Letherlnd, 1992). Suh dily profile is lso influened y feeding time (oujrd nd Letherlnd, 1992), nd supports rhythmi ortisol seretion eing synhronized y oth photoperiod nd feeding tivity, with differenes mong sesons. sed on findings tht: (1) ortisol levels show dily vritions; (2) there is intertion within severl neurohumorl signls nd meltonin prodution; (3) ortisol irulting levels inrese right The Journl of Experimentl iology 147

2 The Journl of Experimentl iology (214) doi:1.1242/je List of revitions AANAT ryllkylmine N-etyltrnsferse 5-HIAA 5-hydroxyindoleeti id 5-HT 5-hydroxytryptmine (serotonin) fter fish re exposed to stress (Wendelr ong, 1997; Mommsen et l., 1999); nd (4) gluoortioids exert n inhiitory effet on AANAT tivity of ultured pinel orgns in trout (enyssi et l., 21; Ynthn nd Gupt, 27), we hypothesized tht stress negtively ffets meltonin synthesis in pinel orgn of rinow trout with ortisol mediting suh inhiitory effet, in the sme wy s hs een desried for other teleost speies suh s tipli, Oreohromis mossmius (Nikido et l., 21), nd tht previously suggested for trout (Lrson et l., 24). The im of the present study ws therefore to evlute the impt of stress on meltonin synthesis in rinow trout pinel orgn, nd to evlute the role of ortisol on suh effet. Thus, we evluted dy night vritions of plsm ortisol nd meltonin levels, pinel ontent of meltonin, serotonin (5-HT; n immedite meltonin preursor) nd its min oxidtive metolite, 5-hydroxyindoleeti id (5-HIAA), s well s AANAT2 enzyme tivity nd mrna undne in fish kept under norml housing onditions or exposed to different stressors or hving reeived ortisol implnts. An in vitro ssy of pinel orgns ws lso performed in order to orroorte the speifiity of the effet. RESULTS Stress ffets plsm ortisol nd meltonin levels The effet on plsm ortisol nd meltonin ontent of exposing trout to different stressors (hypoxi, hsing nd high stoking density; see Mterils nd methods) is shown in Fig. 1. Plsm ortisol vried signifintly (P=.4) in the dy nd night in ontrol fish (higher levels oserved t night) nd in fish under high stoking density (lower levels t night). Exposing nimls to different stress onditions signifintly inresed ortisol levels t oth middy nd midnight, reltive to the respetive ontrol nonstressed group. The inrese of ortisol levels ws greter in nimls exposed to ute stress, i.e. hypoxi nd hsing. Meltonin levels in the ontrol group showed dy night vrition, with higher levels eing oserved during the night (P<.1). The sme trend ws oserved for ll the stressed groups. However, signifint derese of plsm meltonin levels ws notied fter stressing nimls round middy (P=.47; P=.6 nd P=.12 for hypoxi, hsing nd high stoking, ompred with ontrols), wheres round midnight there ws derese in meltonin in the hsing nd high stoking density groups (P=.41 nd P=.8, respetively). Effet of stress on meltonin ontent, AANAT2 tivity nd mrna undne in trout pinel orgn Meltonin ontent, AANAT2 enzyme tivity nd mrna undne in the pinel orgn of trout exposed to different stressors re shown in Fig. 2. Similrly to tht found for plsm meltonin, dy night vrition of meltonin ontent in the pinel orgn ws oserved, with higher vlues ourring t night (P<.1 reltive to dytime). No effet of stress ws notied t middy, wheres meltonin levels signifintly deresed t midnight in fish exposed to high stoking density (P=.42 reltive to ontrol). AANAT2 enzyme tivity in trout pinel orgn showed ler dy night vrition in ontrol, hypoxi nd high stoking groups, with higher tivity notied t midnight. Stress did not ffet Cortisol (ng ml 1 ) Meltonin (pg ml 1 ) A Dy Dy AANAT2 tivity during the dy, ut signifintly deresed it t night (P=.2, P<.1 nd P<.1 reltive to ontrol t night for hypoxi, hsing nd high stoking). This effet ws more roust in fish exposed to hsing, in whih the dy night vrition disppered. A signifint dy night vrition of nt2 mrna undne ws oserved in the pinel orgn of ontrol fish nd those exposed to high stoking density, with higher vlues t midnight (P=.2 nd P=.29, respetively). Deresed noturnl nt2 mrna undne, reltive to the ontrol group, ws found in fish exposed to ute stress (hypoxi nd hsing), suh tht the dy night vrition of nt2 expression disppered in oth groups. 5-HT nd 5-HIAA ontent in the pinel orgn of stressed trout Dy night vritions of 5-HT nd its min metolite, 5-HIAA, nd the rtios of 5-HIAA to 5-HT nd meltonin to 5-HT re shown in Fig. 3. Serotonin ontent in the pinel orgn ws signifintly lower t midnight in ll the experimentl groups. Stress did not signifintly ffet 5-HT levels during the dy, wheres signifint inrese ws oserved t midnight, only in fish exposed to high stoking (P=.1, P=.2 nd P=.8, reltive to the other groups). *, Hypoxi Chsing *,, *, High stok *, *, Fig. 1. Dy night vritions of plsm ortisol nd meltonin in trout sujeted to vrious stressors. (A) Cortisol nd () meltonin levels in rinow trout dpted to norml housing onditions or exposed to different stressors: hypoxi, hsing nd high stok density. Animls were smpled t either middy (N=8 1) or midnight (N=8 1). Dt re mens ± s.e.m. *Signifintly ffeted (P<.5) ompred with the dy vlues for the sme tretment within the sme group. Different letters indite signifint differenes (P<.5) etween groups t the sme time point. *, The Journl of Experimentl iology 148

3 The Journl of Experimentl iology (214) doi:1.1242/je Meltonin (pg pinel orgn 1 ) AANAT2 tivity (pmol pinel 1 h 1 ) nt2 mrna reltive fold hnge A C Hypoxi Chsing High stok Dy Dy Dy Similrly to tht desried for serotonin, 5-HIAA dy night hnges were found in ll the experimentl groups, with signifintly higher levels t middy. During the night, signifint derese of 5- HIAA ontent ws found in trout exposed to hypoxi, reltive to tht of ontrol (P=.34) nd high stoking groups t this time period. The 5-HIAA/5-HT rtio only showed dy night vritions in the hypoxi-exposed group, nd ws higher t night (P=.4). In *, *, *, *, *,, *, *, *,, Fig. 2. Meltonin ontent, AANAT2 enzyme tivity nd nt2 mrna undne in the pinel orgns of stressed or non-stressed trout t middy or midnight. (A) Meltonin ontent, () AANAT2 enzyme tivity nd (C) nt2 mrna. Dt re mens ± s.e.m. of smples tken t either middy (N=8 1) or midnight (N=8 1). For mrna undne mesurements, eh vlue is the men ± s.e.m. of four pools of pinel orgns (n=3 pinels/pool) nd dt show reltive fold hnge to tht mesured in ontrol group during the dy. *Signifintly ffeted (P<.5) ompred with dytime vlues for the sme tretment within the sme group. Different letters indite signifint differenes (P<.5) mong groups t the sme time point. *, ddition, exposing fish to high stoking density tended to derese the 5-HIAA/5-HT rtio t night, ut this effet did not reh signifine when ompred with the ontrol t night. A ler dy night vrition of the meltonin/5-ht rtio ws found in ll the experimentl groups, with higher vlues ourring t night. During the dy, hypoxi nd high stoking density signifintly deresed the meltonin/5-ht rtio, ompred with ontrol nd hsing groups. In ontrst, only the high stoking density signifintly deresed the rtio t midnight, reltive to ontrol, hypoxi nd hsing groups. Plsm ortisol nd meltonin levels fter ortisol intrperitonel dministrtion Plsm ortisol nd meltonin levels were mesured fter intrperitonel dministrtion of ortisol in oonut oil or oonut oil lone (Fig. 4). Dt otined from the ontrol non-implnted group re not shown ut remined quite similr to those of the ontrol implnted fish. Although no signifint dy night vrition of plsm ortisol levels ws found in the ontrol group, tendeny to higher noturnl hormone levels persisted (P=.82) in the sme wy s tht oserved in the previous experiment (see Fig. 1). As expeted, signifint inrese of ortisol levels ws found in trout smpled 5 h or 48 h fter the intrperitonel dministrtion, ompred with the ontrol group t oth times. The only signifint dy night vrition ws found t 5 h fter ortisol injetion, with higher levels t night, reltive to the sme tretment group during the dy. Plsm meltonin levels in the ontrol group showed the sme dy night vrition s desried ove, with higher levels t night (P<.1). The dministrtion of ortisol enhned levels of meltonin fter 48 h during the dy nd deresed it t night, in oth ses, reltive to the ontrol group (P=.16 nd P=.38, respetively), resulting in derese in the mplitude of the dy night vrition derese in the 48 h ortisol-implnted trout. Meltonin ontent, AANAT2 tivity nd mrna undne in the pinel orgn of ortisol-implnted trout Fig. 5 shows meltonin ontent, AANAT2 enzyme tivity nd mrna undne in the pinel orgn of implnted trout. Dt for non-implnted fish (not shown) were onsistent with those tken from ontrol-implnted trout for the three prmeters ssessed. A signifint dy night vrition ws found for meltonin ontent in ll the experimentl groups, with higher levels ourring t midnight. The intrperitonel ortisol dministrtion signifintly redued noturnl meltonin levels reltive to tht found in ontrols t night (P=.11 nd P=.27 for 5 h nd 48 h, respetively). No effets of ortisol dministrtion were found during the dy. AANAT2 tivity displyed signifint dy night vrition in ontrol trout, with higher levels t midnight. In ontrst, ortisol signifintly inhiited the enzyme tivity only t night (P=.18 nd P=.22 for 5 h nd 48 h, respetively), leding the typil dy night vrition of the enzyme tivity to dispper t oth 5 h nd 48 h post-injetion. This inhiitory effet of ortisol ws not oserved t middy. The nlysis of nt2 mrna undne in pinel orgn of rinow trout reveled signifintly higher expression t night in ontrol trout, reltive to tht mesured t middy. Cortisol dministrtion showed time-dependent effet. Thus, nt2 expression ws signifintly enhned during the dy t 5 h postortisol injetion (P=.41), ut deresed fter 5 h nd 48 h t night (P=.42 nd P=.6 for 5 h nd 48 h, respetively), with the effet eing greter fter 48 h. This inhiitory effet of ortisol The Journl of Experimentl iology 149

4 The Journl of Experimentl iology (214) doi:1.1242/je HT (pg pinel orgn 1 ) 5-HIAA (pg pinel orgn 1 ) A C Fig. 3. Dy night vritions in 5-HT 5-HIAA Dy Dy Hypoxi Chsing High stok *, *, *, *, *, *, *, *,, 5-HIAA/5-HT MEL/5-HT D 5-HIAA/5-HT Dy MEL/5-HT Dy *,,, *, *, *, *, 5-HT nd 5-HIAA in the pinel orgns of stressed or nonstressed trout. (A D) 5-HT (A) nd 5-HIAA () levels, nd the rtios of (C) 5-HIAA/5-HT nd (D) Mel/5-HT otined from pinel orgn of stressed nd non-stressed rinow trout. Dt re mens ± s.e.m. for nimls smpled t middy (N=8 1) or midnight (N=8 1). *Signifintly ffeted (P<.5) ompred with dytime vlues for the sme tretment within the sme group. Different letters indite signifint differenes (P<.5) etween groups t the sme time point. on mrna expression did led to the dispperne of the dy night vrition in nt2 expression in oth ortisolimplnted groups. 5-HT nd 5-HIAA ontent in the pinel orgn of implnted nimls Fig. 6 shows the dily vrition of serotonin nd 5-HIAA levels, nd the 5-HIAA/5-HT nd meltonin/5-ht rtios. The ontrol group showed signifint dy night vrition in 5-HT ontent in the pinel orgn, with higher levels t middy. Wheres ortisol dministrtion for 5 h hd no effet on 5-HT ontent (reltive to ontrol group) the 48 h dministrtion signifintly deresed the diurnl 5-HT (P=.24 nd P=.38 reltive to ontrol nd 5 h dytime smple) nd inresed the noturnl 5-HT ontent (P=.5 nd P=.11 reltive to ontrol nd 5 h dytime smple). Thus, the pinel 5-HT ontent ws higher t night nd lower during the dy, whih ws the opposite profile thn tht oserved in the ontrol. No dily vritions of 5-HIAA ontent in the pinel orgn were oserved in either the ontrol group or the group dministered ortisol for 5 h. However, noturnl signifint inrese of the metolite ws oserved in nimls dministered ortisol for 48 h (P=.49 reltive to ontrol). Then 5-HIAA ontent in trout pinel orgn displyed signifint dy night vrition only in nimls implnted with ortisol for 48 h, with higher 5-HIAA levels ourring t night (P=.4 reltive to dytime vlues). The 5-HIAA/5-HT rtio did not signifintly hnge within groups during the dy nd the night. Dy night signifint vritions of the rtio were found only in trout intrperitonelly dministered ortisol for 5 h, with higher vlues t night (P=.7 reltive to dy). All the experimentl groups displyed signifint dy night vrition in the meltonin/5-ht rtio, with higher vlues t midnight. Cortisol dministrtion signifintly deresed the rtio only t night, ompred with ontrol group (P<.1 nd P=.7 for 5 nd 48 h reltive to ontrol), with the more importnt effet eing oserved fter 48 h. No suh effet ws found during the dy, t oth 5 nd 48 h post-injetion. Effet of ortisol tretment on meltonin prodution in vitro Meltonin prodution in ultured pinel orgns in the presene or sene of light ws ompred mong experimentl groups (Fig. 7). Meltonin ws deteted in the ulture medium of smples olleted under eh lighting ondition. There were no group-speifi differenes in sl meltonin relese in oth light nd drk. However, the ddition of medium ontining ortisol resulted in signifint derese in meltonin prodution in drkness, reltive to the drk ontrol (P=.37) nd to tht oserved efore ortisol ddition within the sme group. This inhiitory effet of ortisol ws prevented y the ortisol ntgonist RU486 (P=.32, reltive to ortisol) when oth hemils were dded together. DISCUSSION In the present study, different stressors were evluted, i.e. hypoxi, hsing nd high stoking density, tht mimi those potentilly stressful situtions to whih fish n e exposed when rered. The response to stress in fish involves the tivtion of the hypothlmus symptheti nervous system hromffin tissue xis nd the hypothlmus pituitry interrenl tissue xis, followed y rpid inrese in teholmine nd ortisol levels in plsm, whih indue metoli nd funtionl ltertions (Iwm et l., 26), nd ffet fish physiology (rton et l., 22). Little is known regrding the effets of those hormones in the trout pinel orgn, ut previous studies hve not found ny effet of teholmines in this tissue lotion, in ontrst to tht in other teleosts for whih regultory role hs een proposed (Flón et l., 1991). Meltonin synthesis in trout pinel orgn ws reported to e influened y gluoortioid hormones (enyssi et l., 21), with ortisol eing serious ndidte s meditor of suh n effet. The Journl of Experimentl iology 141

5 The Journl of Experimentl iology (214) doi:1.1242/je Cortisol (ng ml 1 ) Meltonin (pg ml 1 ) A Cortisol 5 h Cortisol 48 h Dy, Dy Dy night vritions of ortisol nd meltonin levels in plsm, the pinel orgn ontent of 5-HT, 5-HIAA nd meltonin, nd the AANAT2 tivity nd mrna undne t the pinel level were evluted in non-stressed, stressed nd ortisol-implnted trout. Those fish rered under norml housing onditions showed signifint (experiment 1) or pprent (experiment 2) dy night vritions of plsm ortisol with night vlues eing higher thn those mesured during the dy, in greement with previous studies in the sme speies (Rne et l., 1982; oujrd nd Letherlnd, 1992) nd others suh s the rown trout (Pikering nd Pottinger, 1983) nd tilpi (Mrtínez-Chvez et l., 28; Nikido et l., 21). Our results show tht ortisol levels were higher in those utely stressed groups (hypoxi nd hsing), wheres they were lower in the high-density group in prtiulr during the night. The inrese in ortisol ws higher in those fish stressed during the dy ompred with the sme groups t night, independent of the stress ondition. This result suggests tht the integrted response to stress ould e influened y the time of the dy in whih the stressor is present. Also the ft tht the higher ortisol inrese ws oinident with the time of dy in whih trout re more tive suggests tht reltionship etween ehviorl omponents nd the response to stress might exist. Further reserh should e rried out. *, *, *, Fig. 4. Dy night vritions of plsm levels of ortisol nd meltonin in trout implnted with dditionl ortisol. (A) Cortisol nd () meltonin levels in rinow trout implnted with oonut oil lone or ontining ortisol (5 mg kg 1 ody mss), nd smpled 5 or 48 h post-injetion. Dt re mens ± s.e.m. for nimls smpled t either middy (N=8 1) or midnight (N=8 1). *Signifintly ffeted (P<.5) ompred with dytime vlues for the sme tretment within the sme group. Different letters indite signifint differenes (P<.5) etween groups t the sme time point. *, Meltonin (pg pinel orgn 1 ) AANAT2 tivity (pmol pinel 1 h 1 ) nt2 mrna reltive fold hnge A C Cortisol 5 h Cortisol 48 h Dy Dy Dy, Plsm meltonin levels were negtively ffeted y stress, in tht the highest redution of dy night vrition of hormone levels ws oserved in fish exposed to long-term stress (high stoking density), rther thn those utely stressed (hsing, hypoxi), whih in ft lso showed deresed plsm meltonin levels, wheres pinel orgn meltonin levels remined unltered. This *, *, *, *, *, Fig. 5. Meltonin ontent, AANAT2 enzyme tivity nd nt2 mrna undne in the pinel orgn of trout implnted with dditionl ortisol. (A) Meltonin ontent, () AANAT2 enzyme tivity nd (C) nt2 mrna undne in trout implnted with oonut oil lone or ontining ortisol (5 mg kg 1 ody mss), nd smpled 5 or 48 h post-injetion. Dt re mens ± s.e.m. for nimls smpled t either middy (N=8 1) or midnight (N=8 1). For mrna undne eh vlue is the men ± s.e.m. of four pools of pinel orgns (n=3 pinels/pool) nd is the reltive fold hnge to tht in the ontrol group t middy. *Signifintly ffeted (P<.5) ompred with dytime vlues for the sme tretment within the sme group. Different letters indite signifint differenes (P<.5) etween groups t the sme time point. The Journl of Experimentl iology 1411

6 The Journl of Experimentl iology (214) doi:1.1242/je HT (pg pinel orgn 1 ) 5-HIAA (pg pinel orgn 1 ) A C Fig. 6. Dy night vritions in 5-HT 5-HT 5-HIAA Dy Dy, *, * Cortisol 5 h Cortisol 48 h *, *, 5-HIAA/5-HT MEL/5-HT D 5-HIAA/5-HT Dy MEL/5-HT Dy *, *, *, *, nd 5-HIAA in the pinel orgns of trout implnted with dditionl ortisol. (A D) 5-HT (A), 5-HIAA (), nd the rtios of 5-HIAA/5-HT (C) nd Mel/5-HT (D) otined from pinel orgn of trout implnted with oonut oil lone or ontining ortisol (5 mg kg 1 ody mss), nd smpled 5 or 48 h post-injetion. Dt re mens ± s.e.m. for nimls smpled t either middy (N=8 1) or midnight (N=8 1). For mrna undne, eh vlue is the men ± s.e.m. of nimls smpled t either middy (N=8 1) or midnight (N=8 1). *Signifintly ffeted (P<.5) ompred with dytime vlues for the sme tretment within the sme group. Different letters indite signifint differenes (P<.5) etween groups t the sme time point. indites tht time periods longer thn 1 5 min (of hypoxi nd hsing) might e required for signifint redution in meltonin ontent to e oserved in the pinel orgn. In ontrst, hnges ffeting AANAT2 enzyme tivity nd mrna undne our immeditely, s our results indite. However, one might hypothesize orreltion etween stress durtion nd the mgnitude of the inhiition of the nighttime meltonin prodution in the pinel orgn, whih is supported y the existene of similr effets in oth pinel orgn nd plsm meltonin ontent. In ontrst, dytime hnges in pinel meltonin fter ny stress were minor, refleting the redued levels of the hormone in lood of stressed trout during the dy, whih might involve ltertions in meltonin lerne rtes or, lterntively, tht hormone synthesis in other tissues ws lso defetive, i.e. retin nd the gstrointestinl trt, whih hve een suggested to ontriute to lood meltonin levels during the dy (Lepge et l., 25; Muñoz et l., 29). Further reserh is needed to lrify this. The effet of stress on the dy night meltonin seretion pttern hs een studied in severl fish speies with ontrditory results. Higher nighttime meltonin nd ortisol levels in soilly suordinted rinow trout reltive to the dominnt fish hve een reported previously (Lrson et l., 24). Similr results were oserved in our lortory in trout exposed to inresed slinity (López-Ptiño et l., 211), ut this ws in ontrst to tht reported for Europen se ss (López-Olmed et l., 29). Inresed irulting meltonin levels were lso found in gilthed se rem sujeted to high stoking density, with suh effet eing prevented y fsting (Mner et l., 28). Similr results hve lso een desried for rinow trout in whih fsting dereses pinel orgn meltonin synthesis t night (Ceinos et l., 28), nd disturne stress negtively ffets severl prmeters inluding meltonin (Kulzykowsk, 21), whih is onsistent with our dt herein reported for trout exposed to different stressors. euse different speies pper to respond speifilly to ny stressor, we might Meltonin (pmol ml 1 ) Dy efore tretment After tretment CONT RU486 CORT RU486+ CONT RU486 CORT CORT, * RU486+ CORT Fig. 7. The inhiitory effet of ortisol on meltonin relese y ultured pinel orgns of rinow trout is prevented y the gluoortioid reeptor generl ntgonist, RU486. After 3 h in ulture in the presene or sene of light (respetively: N=4 groups; n=8 pinels/group), drugs (1. μgml 1 RU486, 1 ng ml 1 ortisol, or RU486+ortisol) or vehile were dded y repling ulture medium with fresh medium ontining eh drug. The ultures were then inuted in the light or drk for 3 hours. Meltonin ontent in the ulture medium is lso provided (efore tretment) in order to demonstrte the sene of ny differenes in sl meltonin relese. *Signifintly ffeted (P<.5) ompred with sl vlue within the sme group. Different letters indite signifint differenes (P<.5) etween groups t the sme time point. The Journl of Experimentl iology 1412

7 The Journl of Experimentl iology (214) doi:1.1242/je speulte tht there is speies-speifi effet of stress on meltonin synthesis in the pinel orgn. ering in mind tht: (1) pinel meltonin synthesis is differentilly regulted in teleosts in reltion to the environmentl signls, i.e. light drk yle (the trout meltonin rhythm-generting system seems to lk funtionl lok, in ontrst to tht of most non-slmonids); nd (2) the different soil ehviors nd physiologil dpttions to the quti environments in whih the fish live, it is proly not surprising tht there re speies-speifi responses to stress. In ddition, the nture nd the durtion of the stressor pper to lso influene the response of the pinel orgn, s reveled y our study. Stress lso diminished the AANAT2 enzymti tivity nd mrna undne in the pinel orgn t night. It is generlly epted tht the noturnl inrese in AANAT2 enzyme tivity is minly responsile for the dily rhythm of meltonin synthesis. In trout, light, y diretly ting on pinel photoreeptors, exerts n inhiitory influene on oth AANAT2 gene expression (López- Ptiño et l., 211) nd enzyme tivity (Flón, 1999; Ceinos et l., 25), with immedite onsequenes on pinel meltonin ontent (Ceinos et l., 25). This is in ontrst to tht previously reported for AANAT2 tivity nd gene expression in trout pinel orgn, in whih the nt2 expression dily profile ws not oserved (égy et l., 1998; Coon et l., 1998). The resons for these disrepnies re not known, ut methodologil differenes or different trout strins might e the most plusile explntions, s we previously reported (López-Ptiño et l., 211). Further reserh is needed. Thus ording to our previous dt (López- Ptiño et l., 211) nd tht desriing light effet (Ceinos et l., 25), our present results indite tht meltonin synthesis in trout pinel orgn is inhiited y stress y speifilly ffeting AANAT2 tivity, whih is proly onsequene of the inhiition oserved in nt2 mrna expression. In ddition, our dt showing inresed noturnl levels of 5-HT, ut not its min oxidtive metolite 5-HIAA, in pinel orgns of stressed trout lso support n inhiitory role for stress on meltonin synthesis. Therefore, it is likely tht, when stressed, the N-etyltion pthwy of 5-HT is inhiited euse of deresed AANAT2 enzyme tivity nd expression, leding to deresed meltonin levels nd intrellulr umultion of 5-HT, whih is oxidized to 5-HIAA. A previous in vitro study on the hormonl regultion of fish pinel meltonin synthesis reveled the presene of gluoortioid reeptors in trout pinel orgn, nd when ssyed in vitro with the gluoortioid nlogue dexmethsone, deresed AANAT2 tivity ws oserved (enyssi et l., 21), whih is onsistent with our results from the pinel orgn in vitro ssy showing deresed meltonin relese in pinel orgns exposed to ortisol in drkness. Also, previous reserh found deresed AANAT tivity in ultured pinel orgn of the North Afrin tfish (Clris griepinus) fter millimolr ortioid tretment, n effet similr to tht reported reently in ultured pinel orgns of tilpi with nnomolr ortisol onentrtions (Nikido et l., 21; Ynthn nd Gupt, 27). Therefore, gluoortioid regultion of pinel meltonin prodution is possile under physiologil onditions, nd this might our when ortisol levels re elevted s result of stress. On the sis of these studies, n in vivo experiment ws onduted in trout to evlute whether or not ortisol tretment might result in hnges in plsm nd pinel prmeters similrly to those eliited y stress. Our results show tht ortisol implnts for 48 h inrese plsm meltonin levels during the dy, wheres oth plsm (t 48 h) nd pinel meltonin (5 nd 48 h) levels deresed during the night in ortisol-implnted trout. This differentil effet of ortisol on meltonin seretion seems to e independent of the ortisol inrese (i.e. ortisol levels in implnted fish were higher thn those of ontrols ny time). Thus, we might speulte tht inresed ortisol differentilly ffets the photoreeptor ell properties during the dy nd night (i.e. y ltering memrne permeility or light trnsdution pthwys) with meltonin synthesis eing ffeted. In greement with this hypothesis, we previously reported for rinow trout tht exposure to hemil stressor [i.e. polyyli romti hydrorons (Gesto et l., 29)] eliited similr effet to tht herein reported for pinel orgn meltonin synthesis. In ddition, ortisol implnts lso derese AANAT2 enzyme tivity nd mrna undne in the pinel orgn t night. These results lerly demonstrte the negtive effet exerted y ortisol on AANAT2 undne nd tivity. In ddition, the speifiity of the negtive effet of ortisol on pinel orgn meltonin synthesis hs een proved, s our results from the in vitro ssy with ortisol nd its ntgonist (RU486) lerly demonstrte. In support of this, we lso oserved tht trout reeiving ortisol implnts for 48 h hd inresed 5-HT levels t night. This effet ws similr to tht oserved for 5-HIAA, whih suggests tht the inhiitory effet of ortisol on meltonin synthesis leds to inresed 5-HT ontent, whih is then oxidized. A similr effet of ortisol ws not seen t middy, whih suggests tht those vritions oserved during the night might e minly due to deresed 5-HT utiliztion through the N-etyltion pthwy to synthesize meltonin during this time period. Thus we my disrd ny possile speifi effet of 5-HT synthesis in trout pinel orgn. Tken together, we onlude tht ortisol, through the tivtion of speifi gluoortioid reeptors, might e minly responsile for the influene of stress on teleost pinel orgn physiology, espeilly in those speies in whih teholmines might ply minor role (Flón et l., 1991). Therefore, the effets oserved in stressed, ortisol-implnted trout nd ultured pinel orgns tended to derese the dy night vrition of oth pinel nd plsm meltonin levels. It is likely tht the ortisol effet on pinel meltonin is meditted y either speifi gluoortioid reeptors s indited previously nd for other teleost speies (enyssi et l., 21; Nikido et l., 21), whih might in turn tivte gluoortioid-responsive elements t the AANAT2 gene promoter (enyssi et l., 21), nd/or other non-genomi tions t the ell surfe (Mommsen et l., 1999). Our results from ultured pinel orgns support this hypothesis. In fish, plsm ortisol levels re known to yle diurnlly (Hollowy et l., 1994; Reddy nd Letherlnd, 1995) nd to hnge through the sesons (MLeese et l., 1994), lthough the ortisol profile exhiits speiesspeifi dily nd sesonl ptterns ffeted y severl ftors suh s photoperiod (Pikering nd Pottinger, 1983) or feeding time (oujrd nd Letherlnd, 1992), mong others. In some speies, for exmple ommon dentex, dily flututions of plsm ortisol levels show endogenous rhythmi hrteristis (Pvlidis et l., 1999). This hormone is thought to e importnt in the output of the irdin lok system in vertertes (Lilley et l., 212). However, temporl reltionship etween physiologil loks nd plsm levels of ortisol nd meltonin hs not een estlished in fish. Tking into ount tht trout ppers to lk pinel irdin signl tht ontrols meltonin synthesis, one might not disrd the possiility tht dily hnges in ortisol ply modultory role in the light drk regultion of trout meltonin rhythm. In ft, ortisol vlues were shown to e espeilly high t the end of the night nd in the erly morning, when pinel meltonin synthesis hs deresed (Ceinos et l., 25). In ontrst, high meltonin levels hve een reported to redue ortisol seretion in goldfish (Azpelet et l., 21) nd to ountert the stress-indued ortisol inrese in The Journl of Experimentl iology 1413

8 The Journl of Experimentl iology (214) doi:1.1242/je Seneglese sole (López-Ptiño et l., 213). This hormonl orreltion is in support of our dt in trout showing n inverse reltionship etween high ortisol levels nd meltonin levels in stressed, ortisol-implnted fish nd in ultured pinel orgns. Therefore, further investigtion into flututions in ortisol nd its intertion with meltonin is likely to inrese our understnding of the regultion of pinel rhythmi physiology nd the irdin orgniztion whih is elieved to exist in trout (Sánhez-Vázquez nd Tt, 1998). In summry, our results provide evidene for n inhiitory effet exerted y ortisol on meltonin synthesis in the pinel orgn of rinow trout. This steroid proly medites the effets of different stressful situtions on the pinel orgn y tivting speifi gluoortioid reeptors. Suh tivtion seems to diretly influene AANAT2 enzyme tivity nd expression, whih re normlly inresed t night, llowing the meltonin seretory pek. Our results lso indite tht ortisol, either in non-stressful or stressful onditions, might hve modultory role on pinel rhythms, in prtiulr those relted to meltonin synthesis. Considering the pivotl role of meltonin in synhronizing rhythmi physiologil events to ylil environmentl hnges (minly the light drk yle), the effet of stress on meltonin synthesis might jeoprdize the response of the niml to suh flututions nd in onsequene, to ompromise its physiologil integrtion. MATERIALS AND METHODS Animls Immture rinow trout [Onorhynhus mykiss (Wlum 1792); 7.±.5 months old; 1±5 g ody mss] were otined from fish frm (Soutorredondo, Noi, Spin) nd trnsferred to our filities t the Vigo University. Animls were limted for 15 dys in 12 l tnks under our lortory onditions: 12 h:12 h light:drk photoperiod (lights on t 8: h, 4 lx intensity t the wter surfe), 14±1 C wter temperture, nd ontinuously renovted nd erted wter. During limtion fish were fed dily to stiety (1: h) with ommeril dry pellets (Diq-diproteg, Segovi, Spin). All the experimentl proedures nd niml mnipultion were designed ording to the Europen Union Counil (21/63/EU), nd the Spnish Government (RD 53/213) legl requirements. onsisting on 5 min of repeted hsing round the tnk followed y 15 min reovery. Then trout were netted nd nesthetized efore lood olletion nd killing. The high stoking nd the ontrol groups were exposed to norml hndling proedures, then netted nd rpidly trnsferred into new tnks where they were deeply nesthetized. One nesthetized, lood ws individully olleted nd fish were immeditely killed. All the mnipultions nd smple olletions t midnight were done under low intensity (<.4 lx) dim red light. In eh group (n=2), lood ws olleted from eight nimls for plsm ortisol nd meltonin quntifitions, nd their pinel orgns were ssyed for indole ontent nd AANAT2 tivity y high-pressure liquid hromtogrphy (HPLC). Additionlly, pools (N=4 per group) of pinel orgns (n=3 orgns per pool) from the remining 12 fish in eh group were proessed for mrna quntifition. In seond experiment, the effet of ortisol on pinel orgn metolism ws evluted during the dy nd night. Four groups of trout were divided etween two 12 l tnks (2 nimls per tnk). Following limtion, fish were nesthetized, weighed nd intrperitonelly dministered with slowrelese oonut oil implnts (ontrol group inluded) following proedures previously desried (Soengs et l., 1992). An dditionl ontrol group ws not implnted, ut ws sujeted to the sme mnipultion (shmimplnted). Implnts onsisted of oonut oil lone (1 μgg 1 ody mss) in ontrols, or the oil plus ortisol (5 mg kg 1 ody mss) ording to previous study in this speies (Vijyn et l., 23). After implnttion, fish were pled k into their respetive tnks. Fish from eh tnk were killed nd smpled t 5 h or 48 h post-implnttion, t middy (the first tnk of eh group) or midnight (the seond tnk). No mortlity ws oserved during the experiment. Animls killed t night were mnipulted s ove. Smples were proessed s indited (experiment 1). A third experiment ws performed to orroorte tht pinel orgn meltonin synthesis is inhiited y ortisol. Thus, individul pinel orgns were removed from fish nd immeditely pled in 96-well ulture pltes eh ontining 25 μl modified Hnks medium, ording to Yñez nd Meissl (Yñez nd Meissl, 1995), ut supplemented with.1 mmol l 1 tryptophn. Assys were performed under ontrolled temperture (16 C) nd in the presene or sene of light. After 3 h inution the ulture medium ws removed nd stored t 8 C, nd repled with 25 μl modified Hnks medium lone (ontrol group) or ontining ortisol (1 ng ml 1 ), the generl gluoortioid reeptor ntgonist, mifepristone (RU486; 1. μgml 1 ), or RU486+ortisol (n=8 orgns per group). After 3 h, medium ws removed nd stored t 8 C until ssyed. Meltonin ontent ws ssessed on eh medium frtion. Smple olletion Animls were deeply nesthetized y fst immersion in MS-222 (5 mg l 1 ) uffered to ph 7.4 with sodium ironte, nd lood ws olleted from the udl vein, using 1 ml heprinized syringes. Then, fish were srified y depittion nd pinel orgns were removed with sterilized implements nd pled into RNse-free 1.5 ml Eppendorf tues. Smples were immeditely frozen in liquid nitrogen, nd stored t 8 C until ssyed. Plsm ws otined y entrifuging lood t 9 g for 1 min t 4 C. Aliquots were immeditely frozen nd stored t 8 C until nlyzed. Experimentl designs Three experiments were designed to evlute the influene of stress on meltonin prodution in rinow trout pinel orgn. In first experiment, fish were rndomly distriuted in four groups: ontrol, or stressed y hypoxi, hsing, nd high stoking density (n=2 nimls eh). Following limtion, quntity of wter ws removed from the tnks ontining the high stoking density group until stressful high density ws rehed (7 kg fish mss m 3 ), where fish remined for 4 dys efore eing killed. Stoking density onditions remined unltered for the other groups (1 kg fish mss m 3 ). On the dy of smpling, nimls from the other stressed groups were exposed to different mnipultions nd then killed t middy or midnight s follows. Fish from the hypoxi group were normlly hndled nd netted ut remined in the net for 6 s efore eing deeply nesthetized in MS-222 solution, lood smpled nd killed. The hsing group ws sujeted to stndrdized hndling disturne Hormone nd metolite quntifition Plsm ortisol levels were mesured using ommerilly ville Enzyme Immunossy Kit (Cymn, Ann Hror, MI, USA), ording to the mnufturer s instrutions. Plsm meltonin levels were ssyed y HPLC with fluorimetril detetion s desried previously (Muñoz et l., 29). The resultnt residue fter the extrtion proedure ws dissolved in 1 μl moile phse nd filtered (.5 μm filter). An liquot (5 μl) of the filtrte ws injeted into the HPLC system. Dt from the nlysis re expressed s pg ml 1 plsm. Meltonin ontent in the ulture medium ws ssyed y diretly injeting 2 μl from eh olleted frtion (n=8 per group) into the HPLC system. For pinel meltonin quntifition, eh orgn (n=8 per group) ws homogenized y ultrsoni disruption in 1 μl of.2 mol l 1 phosphte uffer (ph 6.7) nd entrifuged t 16, g for 1 min. A 6 μl liquot ws ssyed for meltonin nd pinel indole ontent. The resulting 4 μl liquot ws immeditely ssyed for AANAT2 enzyme tivity. From the 6 μl liquot, pinel meltonin ontent ws mesured y diret injetion of 2 μl volume into the HPLC system, similrly to tht desried for plsm meltonin ssessment. Other onditions were s previously desried (Ceinos et l., 28). Pinel 5-HT nd its idi metolite, 5-hydroxindoleeti id (5- HIAA) were ssyed y HPLC with eletrohemil detetion from 1 μl liquot of the pinel homogentes s desried y Ceinos et l. (Ceinos et l., 25). AANAT2 enzyme tivity ws ssyed y in vitro inution of 4 μl of the ove mentioned smple homogentes with the sustrte, 4 μl of The Journl of Experimentl iology 1414

9 The Journl of Experimentl iology (214) doi:1.1242/je mmol l 1 tryptmine nd 4 μl of 1. mmol l 1 etyl-coa (finl onentrtions in ssy: 9 mmol l 1 tryptmine nd.5 mmol l 1 etyl- CoA). Assy onditions were s previously desried nd performed (Ceinos et l., 28) with modifition onsisting of 6 min inution period t 16 C (temperture within the optiml rnge for trout). A 2 μl smple of the finl solution were diretly injeted into the HPLC system in order to quntify the retion produt (N-etyl tryptmine; NAT) formed. The system ws HPLC pump (Gilson M11) with n Ultrsphere ekmn olumn (3 μm prtiles, 75 nd 4.6 mm internl dimeter) nd Jso FP-152 fluoresene detetor set t 285/36 nm exittion/emission wvelengths. All nlyses were performed t room temperture nd 1 ml min 1 flow rte. Smple pek res were quntified reltive to tht of suitle stndrds. Anlysis of pinel orgn nt2 mrna undne After dissetion, pinel orgns from fish reeiving the sme tretment were pooled (n=3 orgns per pool). Totl mrna ws extrted from eh pool (n=4 pools per group) using the TRIzol method (Gio RL, Githersurg, MD, USA) ording to mnufturer s instrutions. The isolted RNA qulity nd quntity were spetrophotometrilly determined. From eh smple, 2 μg RNA were onverted into DNA s desried previously (López-Ptiño et l., 211). A negtive ontrol for eh smple ws ssessed without reverse trnsriptse in order to e le to disregrd ny genomi ontmintion. Rel-time quntittive RT-PCR (qpcr) ws performed using MximTM SYR Green qpcr Mster Mix (K252, Ferments, urlington, ON, Cnd) nd io-rd (Herules, CA, USA) MyIQ reltime PCR system. The primers nd proes were sed on previously reported sequenes of rinow trout genes, nd otined from Sigm- Genosys (St Louis, MO, USA), inluding: nt2 (ession numer AF166.1) forwrd 5 -CATTCGTCTCTGTGTCTGGT-3, reverse 5 - TTTCTGGGATATGCTGGGT-3 ; nd β-tin (AJ438158) forwrd 5 - GATGGGCCAGAAAGACAGCTA-3, reverse 5 -TCGTCCCAGTTGGT- GACGAT-3. Gene expression ws normlized to tht of β-tin for eh smple. Reltive mrna expression ws lulted ording to the omprtive ΔC t method. For eh gene, smples olleted t the sme time point were proessed in prllel nd the expression ws mesured in triplite. Sttistil nlysis Differenes were evluted y two-wy ANOVA, with tretment nd time of dy s min ftors. When signifint effet ws identified within ftor, post ho omprisons were rried out within tht ftor using Student Newmn Keuls test. The signifine level ws set t P<.5. Aknowledgements We thnk L. Mnrique for her vlule orretions of the mnusript, nd S. González-Silv, A. enedetti nd S. Usndizg for exellent tehnil ssistne. Competing interests The uthors delre no ompeting finnil interests. Author ontriutions Coneption nd experiment design: M.A.L.-P., J.L.S. nd J.M.M. Exeution nd nlysis: M.A.L.-P., M.C.-S. nd M.G. Dt interprettion: M.A.L.-P., M.C.-S., M.G., J.L.S. nd J.M.M. Writing nd revisions: M.A.L.-P., M.G., J.L.S. nd J.M.M. Funding This work ws supported y the Spnish Ministerio de Cieni e Innovión nd Europen Fund for Regionl Development [grnt numer AGL C3-3 to J.L.S.]; nd Xunt de Glii (Consolidión e estruturión de uniddes de investigión ompetitivs no sistem universitrio de Glii [grnt numer CN 212/4 to J.L.S.]. M.A.L.-P. is n Isidro Prg Pondl Reserher (P.P. 3S 148). Referenes Azpelet, C., Mrtínez-Alvrez, R. M., Delgdo, M. J., Isorn, E. nd De Pedro, N. (21). Meltonin redues loomotor tivity nd irulting ortisol in goldfish. Horm. ehv. 57, rton,. A., Morgn, J. D. nd Vijiyn, M. M. (22). Physiologil nd onditionrelted inditors of environmentl stress in fish. In iologil Inditors of Aquti Eosystem Stress (ed. S. Mrshll Adms), pp ethesd, MD: Amerin Fisheries Soiety. égy, V., Vlotire, Y., Rvult, J. P., Collin, J. P. nd Flón, J. (1994). Detetion of estrogen reeptor mrna in trout pinel nd retin: estrdiol-17 et modultes meltonin prodution y ultured pinel photoreeptor ells. Gen. Comp. 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