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Neuropeptides

Expression of orexin A and its receptor 1 in the choroidplexuses from buffalo brain

Simona Tafuri a,b, Luigi M. Pavone a,b, Vincenzo Mastellone c, Anna Spina a,b,Luigi Avallone a, Alfredo Vittoria a, Norma Staiano a,b,*, Gaetano Scala a

a Department of Biological Structures, Functions and Technologies, University of Naples Federico II, Via F. Delpino 1, 80137 Naples, Italyb Department of Biochemistry and Medical Biotechnologies, University of Naples Federico II, Via S. Pansini 5, 80131 Naples, Italy

c Department of Experimental and Clinical Medicine ‘‘G. Salvatore”, University of Magna Graecia, Viale Europa, Germaneto, 88100 Catanzaro, Italy

Received 24 October 2008; accepted 24 January 2009

Abstract

The hypothalamic peptide orexin A, deriving from the proteolytic cleavage of the precursor molecule prepro-orexin, has a widerange of physiological effects including the regulation of feeding behaviour, neuroendocrine functions, sleep–wake cycle, and energyhomeostasis. Lowered excretion of orexin A into the cerebrospinal fluid (CSF) plays a pathological role in animal and human nar-colepsy. Altered levels of orexin A into the CSF have been also found in numerous disorders of the central nervous system, includingParkinson’s and Huntington’s disease, dementia, and depressive disorders. While the localization of orexin A and its receptor 1,OX(1), has been elicited in many regions of the mammalian brain and in peripheral organs, there are no information on the expres-sion of the neuropeptide and its receptor 1 in the choroid plexuses (CPs) producing the CSF. In this study, we investigated theexpression of orexin A and OX(1) in the CPs from the brain of an adult mammalian species, Bubalis bubalis, by immunogold-label-ling in scanning electron microscopy. Both orexin A and OX(1) immuno-reactivity appeared to be widely distributed on the surfaceof choroid epithelium. Interestingly, a marked orexin A labelling was detected in the areas surrounding the CP blood capillaries. Theexpression of prepro-orexin and OX(1) mRNA transcripts of 200 and 300 bp, respectively, was assessed in the CPs by reverse-tran-scription polymerase chain reaction, while Western blotting analysis confirmed the presence of these two proteins in the tissue. Ourfindings provide the first evidence for orexin A and OX(1) expression in the CPs from mammalian brain, and suggest that the levelsof orexin A into the CSF are probably regulated by CP activity.� 2009 Elsevier Ltd. All rights reserved.

Keywords: Buffalo brain; Choroid plexuses; Immunogold-labelling in scanning electron microscopy; Orexin A; Orexin receptor 1

1. Introduction

Orexin A and orexin B are two peptides first isolatedfrom the rat hypothalamus (de Lecea et al., 1998; Saku-rai et al., 1998). Orexin A is a 33 amino acid peptide withN-terminal pyroglutamyl residue and two intra-chain

0143-4179/$ - see front matter � 2009 Elsevier Ltd. All rights reserved.

doi:10.1016/j.npep.2009.01.003

* Corresponding author. Address: Department of Biological Struc-tures, Functions and Technologies, University of Napoles Federico II,Via F. Delpino 1, 80137 Naples, Italy. Tel.: +39 081 2536108; fax: +39081 2536097.

E-mail address: staianor@unina.it (N. Staiano).

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disulphide bonds, whereas orexin B is a 28 amino acidlinear peptide. They both derive from the proteolyticcleavage of a common 130 amino acid precursor pep-tide, prepro-orexin. Orexins bind the two G(q)-coupledreceptors termed OX(1) and OX(2). While OX(1) ishighly selective for orexin A, OX(2) shows similar affin-ity for both the peptides (Sakurai et al., 1998; Smart andJerman, 2002).

Orexin A and orexin B are found throughout the cen-tral nervous system (CNS) due to extrahypothalamicprojections (Nambu et al., 1999). The receptor OX(1)is expressed mainly in the hypothalamus and locus

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coeruleus, as well as other brain regions and the spinalcord; OX(2) is expressed in the hypothalamus, cortex,spinal cord, and a few discrete brain nuclei (Trivediet al., 1998). However, both orexins and receptors arealso expressed in peripheral organs such as the gut andthe genital tract of mammals (Darnel et al., 2003; Kar-teris et al., 2004; Barreiro et al., 2005; Russo et al.,2008).

The orexins modulate feeding behaviour (Sakuraiet al., 1998) and energy homeostasis, as well as associ-ated drinking behaviours (Kunii et al., 1999). They alsoregulate arterial blood pressure and heart rate (Shira-saka et al., 1999), sleep/wake cycle (Piper et al., 2000),sexual behaviour and arousal (Gulia et al., 2003).Because of the presence of orexin A in the nervousand endocrine cells of the gastrointestinal wall, a rolefor orexin A in the regulation of gut motility, blood flowand epithelial secretion has been suggested (Kukkonenet al., 2002; Ehrstrom et al., 2003). Disruption of prep-ro-peptide expression or mutations in the gene encodingorexin receptors result in a narcoleptic phenotype in var-ious animal models, while several clinical studies havelinked disruption of the orexin system to narcolepsy inhumans (Chemelli et al., 1999; Baumann and Bassetti,2005). Recent evidence demonstrated that the loss oforexin neurons or impaired orexin neurotransmissionmay exist as a part of the neurodegeneration associatedwith several diseases of the CNS (Fronczek et al., 2007).In particular, altered levels of orexin A into the cerebro-spinal fluid (CSF) have been correlated to many neuro-degenerative diseases (Petersen et al., 2005; Brundinet al., 2007).

The CSF is produced by the choroid plexuses (CPs),leaf-like highly vascular structures lying in the ventricu-lar system of the brain which form one of the interfacesbetween the blood and the CNS (Abbott, 2005). Besidetheir main function of producing CSF, CPs are alsoinvolved in numerous exchange processes that eithersupply the brain with nutrients and hormones, or cleardeleterious compounds and metabolites. Furthermore,CPs greatly contribute to maintaining brain homeostasisbeing involved in neurohumoral brain modulation andneuroimmune interactions (Strazielle et al., 2005).

Although the regional distribution of orexin A andOX(1) in the brain and other peripheral tissues of differ-ent animal species has been extensively investigated, nodata on their localization in the CPs are available. In thepresent study, we report the detection of orexin A andOX(1) in the CPs from the brain of an adult mammalianspecies, Bubalus bubalis, using immunogold-labelling inscanning electron microscopy (SEM). In addition, theexpression of prepro-orexin and OX(1) mRNAs andthe proteins in the CPs from buffalo brain was demon-strated by reverse-transcription polymerase chain reac-tion (RT-PCR) and Western blotting analysis,respectively.

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2. Materials and methods

2.1. Animals

Twelve heads were collected immediately afterslaughter from adult water buffalos (Bubalus bubalis).Buffalo breeding is very common in our country(South Italy). In this study, we chose to use CPs frombuffalo brain because of the similarity of the encepha-lon for all mammals (Abbott, 2005; Strazielle et al.,2005) and the lacking of data related to this animalspecies.

The heads were perfused through the maxillary arterywith 0.1 M phosphate buffered saline (PBS), pH 7.3. TheCPs were collected from the brain, and processed in adifferent way depending on the type of analysis to beperformed.

2.2. Immunogold-labelling in SEM analysis

Immunogold-labelling in SEM analysis was per-formed accordingly to our previously described proce-dure (Pavone et al., 2007). Briefly, the CP samplesremoved from the brain of six animals were immersedin PBS for 1 h. The tissues were incubated for 2 hwith a solution containing normal goat serum (X0907, Dako Italia s.p.a., Milano, Italy) diluted 1:10in PBS, and next with a primary goat polyclonalanti-orexin A (sc-8070), or anti-OX(1) (sc-8073) anti-bodies (Santa Cruz Biotechnology, Santa Cruz, CA,USA) diluted 1:1000 in PBS, overnight at 4 �C. Afterwashing in PBS, the samples were incubated withgold-conjugated rabbit anti-goat IgG (Aurion,Wageningen, The Netherlands) diluted 1:200 in PBS,for 1 h at room temperature. The secondary antibodywas conjugated with gold particles of 6 nm size. Afterwashings in PBS, samples were fixed by 2.5% glutar-aldehyde in 0.1 M cacodylate buffer containingCaCl2, pH 7.2, for 30 min, washed with distilledwater, and subjected to silver enhancement (BritishBioCell International, Cardiff, Wales, UK). Theenhancement process enables the use of antibodiesconjugated to small (1–5 nm) gold particles, preserv-ing the advantage of fast penetration and high label-ling efficiency. Next, samples were dehydratedthrough an ethanol series, and dried to the criticalpoint. The specimens, mounted on stubs, were exam-ined under a LEO 435 VP scanning electron micro-scope at variable pressure (80–120 Pa) in thebackscattered electron mode which allows to detectgold particles associated to cells even if they arelocated intra-cellularly (McMenamin et al., 2003).The samples had not been coated by gold–palladium,so that the only conjugated gold deriving from theimmunocytochemical reaction was observed bySEM, and photographed.

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2.3. RNA extraction and RT-PCR

Total RNA was extracted from the CPs collectedfrom the brain of three animals by using TRIzol solu-tion (Invitrogen, Carlsbad, CA, USA). The RNA wasre-suspended in 50 ll diethyl pyrocarbonate treatedwater, and stored at �80 �C until used. The synthesisof cDNA for the detection of prepro-orexin or OX(1)mRNA was performed by using a reverse-transcriptionsystem (Promega, Madison, WI, USA). The primersused for RT-PCR amplification were: forward 50-ATCCTTCCTCTACAAAGGTCTCC-30 and reverse50-CTTGCCCAGCGTGAGGAT-30 for prepro-orexin;forward 50-AGGCTGCGGTCATGGAAT-30 andreverse 50-TTCCTGACCAGGGCTGAC-30 for OX(1).The PCR products were separated on a 2% agarose geland visualized by ethidium bromide using a 1 kb DNAladder to estimate the band sizes. As a negative controlfor all reactions, distilled water was used in place ofcDNA. The used primers amplified a fragment of 200-bp for prepro-orexin and a fragment of 300-bp forOX(1). The bands were cut off from the gel, purifiedusing GFX PCR DNA and Gel Purification Kit (code27-9602-01, Amersham Biosciences, Little Chalfont,UK), and sequenced [GenBank accession no.:AB084625 for bovine prepro-orexin, and AB092488for bovine OX(1)].

As internal control for reverse transcriptase (RT) andreaction efficiency, amplification of a 400-bp fragment ofb-actin mRNA was carried out in parallel in each sam-ple, using the primer pair: forward 50-GGCACCCAGCACAATGAAGAT-30 and reverse 50-CCTT-CACCGTTCCAGTTTTTA-30.

As positive control, the same RT-PCR procedure wasperformed on samples from the whole brain of micewhich express high levels of prepro-orexin and OX(1)(Chen and Randeva, 2004). The primer used in themouse brain sample analysis were: forward 50-ACCACTGCACCGAAGATACC-30 and reverse 50-GCGAGGAGAGGGGAAAGTTA-30 for prepro-orex-in; forward 50-GCTCCCTCCCCTCTCCTC-30 andreverse 50-CGCGATGAGAACCCACTC-30 for OX(1).The used primers amplified a fragment of 200-bp forprepro-orexin and a fragment of 300-bp for OX(1).The bands were cut off from the gel, purified andsequenced [GenBank accession no.: AF041242 formouse prepro-orexin, and NM198959 for mouseOX(1)].

2.4. Western blotting

The CPs removed from the brain of three animalswere homogenized by an Ultraturrax L407 at 4 �C with5 ml/1.5 g tissue of buffer containing 50 mM Tris–HCl,pH 7.4, 150 mM NaCl, 1 mM EDTA, 10 mM NaF,0.5% deoxycholic acid, 0.1% sodium dodecyl sulphate

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(SDS), 1% Nonidet P-40, 1 mM phenylmethylsulphonylfluoride, 0.1 U/ml aprotinin, 10 lg/ml leupeptin, and1 mM Na3VO4. Homogenates were centrifuged at15,000g for 20 min at 4 �C. Supernatants were dividedinto small aliquots and stored at �80 �C until used.The amount of total proteins in each sample was deter-mined by the Bio-Rad DC protein assay (Bio-Rad Lab-oratories, Hercules, CA, USA). Samples containingequal amounts of proteins were boiled for 5 min inSDS sample buffer (50 mM Tris–HCl, pH 6.8, 2%SDS, 10% glycerol, 0.1% bromophenol blue, and 5%b-mercaptoethanol) and run on a 10% SDS/polyacryl-amide gel. After electrophoresis, the proteins were trans-ferred to nitrocellulose using a Mini trans-blotapparatus (Bio-Rad Laboratories) according to themanufacturer’s instructions. Membranes were blockedfor 1 h at room temperature with TBS-T buffer(150 mM NaCl, 20 mM Tris–HCl, pH 7.4, 0.1% Tween20) containing 5% milk. The blots were incubated over-night with a rabbit polyclonal anti-prepro-orexin anti-body (AB3096, Chemicon International Inc.,Temecula, CA, USA), or an anti-OX(1) antibody (sc-8073, Santa Cruz Biotechnology) diluted 1:1000 inTBS-T containing 2.5% milk. After incubation, themembranes were washed three times with TBS-T, andincubated for 1 h with horseradish peroxidase conju-gated anti-(rabbit IgG) Ig (Sigma Chem. Co., St. Louis,MO, USA) diluted 1:3000 in TBS-T containing 2.5%milk. The proteins were visualized by enhanced chemilu-minescence (Amersham Biosciences). To ensure specific-ity, pre-absorption of prepro-orexin antibody or OX(1)antibody with their respective blocking peptide [AG774,Chemicon International Inc., for prepro-orexin; sc-8073P, Santa Cruz Biotechnology for OX(1)] was performedbefore Western blotting. The same blots were strippedand re-probed using anti-tubulin monoclonal antibody(MAB1637, Chemicon International Inc.) to confirmequal loading of proteins in all lanes.

3. Results

3.1. Localization of orexin A and OX(1) in the CPs as

detected by immunogold-labelling in SEM

Observations by immunogold-labelling in SEM indi-cate a specific distribution of orexin A and OX(1)expression on the surface of the choroid epithelium inall the CP tissues studied herein, namely CPs from thelateral, the third and the fourth ventricles of buffalobrain (Figs. 1–5). The scanning electron micrograph ofCP epithelium shows orexin A immuno-reactive groupsof cells distributed on the typical folded structural orga-nization of the apical surface of choroid epithelium(Fig. 1). Interestingly, a prominent distribution of orexinA immuno-labelling is observed in the highly vascular-

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Fig. 1. Scanning electron micrograph of orexin A immuno-reactivecells on the surface of the choroid epithelium of a lateral ventricle frombuffalo brain. Immunogold-labelled particles of different sizes (1–5 lm)are clearly evident on the apical surface of the choroid epithelium. P:immunogold-labelled particles.

Fig. 2. Scanning electron micrograph of a transversal section of thechoroid plexus of a lateral ventricle from buffalo brain. A strong orexinA immuno-reactivity in the vascularised areas among the two choroidepithelium layers is observed. P: immunogold-labelled particles. S:choroid epithelium layer.

Fig. 3. Scanning electron micrograph of orexin A immunogold-labelled cells in the zone surrounding a blood capillary of the choroidepithelium of the fourth ventricle. Single or grouped gold particles(1.5–2 lm) are visible on the external wall of the blood capillary. P:immunogold-labelled particles. BC: blood capillary.

Fig. 4. Scanning electron micrograph of OX(1) immuno-reactive cellson the surface of the choroid epithelium of a lateral ventricle frombuffalo brain. Immunogold-labelled particles of different sizes (1–5 lm)are clearly evident on the surface of the choroid epithelium. P:immunogold-labelled particles.

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ized areas among the choroid epithelium layers (Fig. 2).Many gold particles appear to be localized in proximityof the blood capillary walls (Fig. 3).

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A wide distribution of OX(1) immuno-labelled parti-cles on the apical surface is detected (Fig. 4). The scan-ning electron micrograph of CP epithelium allows toclearly distinguish either single or grouped immuno-

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Fig. 5. Scanning electron micrograph of OX(1) immuno-reactive cellson the surface of the choroid epithelium of the third ventricle frombuffalo brain. Grouped or single immunogold-labelled cells areabundantly localized on the surface of the choroid epithelium. PP:grouped immunogold-labelled particles.

Fig. 6. RT-PCR analysis for the expression of prepro-orexin andOX(1) mRNAs in the CPs from buffalo brain. A. Prepro-orexinmRNA transcripts from mouse brain tissue (lane 2), and from CPtissue (lane 3). Lane 1 corresponds to the DNA ladder. Negativecontrol is shown in lane 4. Bottom, expression of b-actin transcripts(internal control). B. OX(1) mRNA transcripts from mouse braintissue (lane 2), and from CP tissue (lane 3). Lane 1 corresponds to theDNA ladder. Negative control is shown in lane 4. Bottom, expressionof b-actin transcripts (internal control).

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positive cells on the surface of the choroid epithelium(Fig. 5).

No orexin A or OX(1) immuno-reactivity wasobserved in the CP samples treated with PBS in theplace of the primary antibody (negative controls). CPsfrom the lateral, the third and the fourth ventricles ofthe brains collected from different adult buffaloes fromdifferent breeding gave similar results.

3.2. Expression of prepro-orexin and OX(1) in the CPs

from buffalo brain: biochemical analyses

The expression of prepro-orexin and OX(1) mRNAsin the CPs was analyzed by RT-PCR. This analysisresulted in the amplification of specific DNA fragmentsof 200-bp for the prepro-orexin (Fig. 6A, top, lane 3)and of 300-bp for OX(1) (Fig. 6B, top, lane 3) in theCP samples as well as in a mouse brain tissue used asa positive control (Fig. 6, Panels A and B, top, lanes2). A 400-bp transcript was obtained from the amplifica-tion of b-actin cDNA in all tested samples (Fig. 6, Pan-els A and B, bottom).

The presence of prepro-orexin and OX(1) in the CPsfrom buffalo brain was confirmed by Western blotting,using, respectively, a rabbit polyclonal antibody raisedagainst a 17 amino acid peptide mapping near the C-ter-minus of mouse prepro-orexin, and a goat polyclonalantibody raised against a peptide mapping near the C-terminus of OX(1) of rat origin. The detected prepro-orexin showed a molecular mass of 16 kDa (Fig. 7A,upper blot, lane 2), while OX(1) a molecular mass of

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50 kDa (Fig. 7B, upper blot, lane 2). The specificity ofthe response was confirmed by pre-incubation of theprepro-orexin and OX(1) antibodies with their respec-tive blocking peptide. There was no expression of prep-ro-orexin and OX(1) in these preparations (Fig. 7,Panels A and B, upper blots, lanes 3), whereas the pres-ence of both proteins was detected in a mouse brainhomogenate which was used as positive control(Fig. 7, Panels A and B, upper blots, lanes 1). The strip-ping of the upper blots and their reprobing with a mousemonoclonal anti-tubulin antibody demonstrated equalloading of proteins in all lanes (Fig. 7, Panels A andB, lower blots).

Similar results were obtained by using CP tissuesfrom the lateral, the third and the fourth ventricles ofthe brains collected from different adult buffaloes fromdifferent breeding.

4. Discussion

Orexin A was originally isolated as a hypothalamicsecreted neuropeptide, but later its expression and thoseof its receptors were detected in other regions of thebrain and in peripheral tissues. Thus, in addition to itssignificant and relevant role in feeding behaviour andsleep–wakefulness, a broader picture of orexin A physi-ological roles including endocrine and metabolic actionshas been provided (Taylor and Samson, 2003; Heinonenet al., 2008).

Orexin A has been found in human plasma, althoughthe primary source of the peptide has not been eluci-dated; orexin A plasma levels vary in response to themetabolic state (Arihara et al., 2001). Significant

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Fig. 7. Expression of the proteins prepro-orexin and OX(1) in the CPs from buffalo brain. A. Western blotting analysis by using antiserum directedagainst prepro-orexin. B. Western blotting analysis by using antiserum directed against OX(1). Lanes 1, homogenate from a whole mouse brain(positive control); lanes 2, homogenate of CPs from buffalo brain; lanes 3, negative control (homogenates of CPs from buffalo brain treated with theantiserum directed against prepro-orexin or OX(1) pre-absorbed with their respective control peptide). The upper blots were stripped and re-probedwith an anti-tubulin monoclonal antibody to ensure equal loading of proteins in all lanes (A and B, lower blots). Molecular mass markers areindicated on the left. Similar results were obtained from four separate experiments of identical design.

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amount of orexin A is found in the CSF where its levelsare higher than in plasma (Heinonen et al., 2008). Arelationship between orexin A concentration in theCSF and pathological conditions has been well estab-lished. The levels of orexin A into the CSF are decreasedin patients affected by neurodegenerative diseases suchas Parkinson’s disease (Fronczek et al., 2007), Hunting-ton’s disease (Petersen et al., 2005), myotonic dystrophy(Martınez-Rodrıguez et al., 2003), and in patients withmajor depressive disorders (Brundin et al., 2007). Highlevels of orexin A were found into the CSF from patientsaffected by chronic migraine and medication overuseheadache (Sarchielli et al., 2008). In rats, CSF orexinA concentration was elevated transiently at 24 h afterbrain ischemia (Dohi et al., 2006).

Choroid epithelium lining the cerebral ventricles isresponsible for the production of CSF (Strazielle et al.,2005). The choroid epithelial cells form the blood–CSFbarrier. The CSF fills the ventricular system and sub-arachnoid space, and circulates around the brain andspinal cord before being reabsorbed into the blood cir-culation: it plays a major role in maintaining CNS envi-ronment homeostasis. A fine control of CSF volume andcomposition is necessary for normal neuronal functionand is essential for maintaining a physiological brainvolume and intracranial pressure (Abbott, 2005).Today, measurements to determine alterations in thecomposition of CSF are central in the differential diag-nosis of specific diseases of the CNS (Huhmer et al.,2006).

Beyond the role of producing CSF and forming theblood–CSF barrier, one of the broadest functions ofthe CPs is establishing and maintaining the extracellularmilieu throughout the brain and spinal cord, in part bysecreting numerous bioactive substances into the CSF(Abbott, 2005; Scala et al., 2007). In this study, weinvestigated the presence of orexin A and its receptorOX(1) in the CPs from mammalian brain because ofthe lacking of data on their localization in the CPs,

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and on the basis of the findings here summarized: (a)CSF contains orexin A whose source has not been elic-ited; (b) CSF is produced by CPs; (c) CPs secrete bioac-tive substances into CSF.

By exploiting immunogold-labelling in SEM analysis,we observed a marked availability of OX(1) receptor onthe apical surface of the choroid epithelium. The apicalsurface of choroid epithelial cells contain elements mor-phologically specialized in controlling CSF homeostasisthrough absorption and secretion processes (Strazielleet al., 2005; Scala et al., 2007). Among other functions,the apical membrane of the choroid epithelium isinvolved in homeostasis of CSF calcium concentration(Borke et al., 1989; Stahl et al., 1992; Keep et al.,1999; Abbott, 2005). Both in neuronal and non neuronalcells orexin receptors activate Ca2+ influx and phospha-tidylinositol-specific phospholipase C pathway (Lundet al., 2000; Smart and Jerman, 2002; Uramura et al.,2001). Furthermore, several types of Ca2+ influx path-ways activated by G protein-coupled receptors havebeen described (Barritt, 1999). Thus, OX(1) might beactively involved in CP regulation of CSF calciumhomeostasis. However, other OX(1) responses to orexinstimulation may occur on the choroid epithelium,including an increase of neurotransmitter (i.e. serotonin)release, cAMP elevation, and coupling to protein kinasecascades involved in cell growth, differentiation anddeath (Kukkonen et al., 2002).

Orexin A immuno-labelled gold particles weredetected on the choroid epithelium surface. These find-ing is consistent with the reported detection of orexinA-immuno-reactive fibers in the CPs from rat brain(Cutler et al., 1999). Interestingly, orexin A labelled cellsappeared to be mostly localized in the highly vascular-ized areas among the two choroid epithelium layers,and some of them were found on the wall of the bloodcapillaries. Thus, although the possibility of external(brain and peripheral) orexin A arriving CPs cannotbe ruled out, the localization of orexin A on the choroid

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epithelium suggests that the peptide is locallysynthesized.

In addition to our immunogold-labelling in SEManalysis showing the specific localization of the orexinA and OX(1) on the choroid epithelium from buffalobrain, our RT-PCR analysis showed the presence ofmRNA transcripts for the orexin precursor moleculeprepro-orexin and OX(1) in the CP tissues. These resultsdefinitely demonstrate that mammalian CPs are able tosynthesize prepro-orexin and OX(1). Thus, the proteo-lytic processing of prepro-orexin originates the matureorexin A detected in the CPs by immunogold-labellingin SEM analysis.

Western blotting analysis confirmed the expression ofthe two proteins prepro-orexin and OX(1) in the CP tis-sue. This latter analysis also demonstrates that the pro-teins prepro-orexin, and OX(1) detected in the CPs frombuffalo brain are structurally similar to the commonlyknown proteins detected in the brain or other tissuesof other mammalian species (de Lecea et al., 1998; Sak-urai et al., 1998; Barreiro et al., 2005; Russo et al., 2008;Pavone et al., 2009).

The expression of orexin A and OX(1) in the CPs sug-gests a biological relevance for these peptides in the CPfunctions. On the basis of the known interactions exist-ing between the orexinergic system and either nitrergicor serotonergic neurotransmission (Cheng et al., 2003;Ehrstrom et al., 2003; Tao et al., 2006), and on the lightof our previous studies showing the expression of bothneuronal nitric synthase (nNOS) and serotonin trans-porter (SERT) in the CPs from buffalo brain (Scalaet al., 2007; Pavone et al., 2007), a direct and local actionfor orexin A in concert with the other two mediators isvery likely occurring in the mammalian CPs.

In conclusion, our findings provide the first evidencefor the expression of orexin A and its receptor OX(1) inthe CPs from mammalian brain, and highlight the needof further studies for elucidating the molecular mecha-nisms by which orexin A and its receptor OX(1) mayinfluence both physiological and pathological function-ing of the CPs.

Acknowledgements

This work was supported by a Grant (FISR 2005)from MIUR, Rome, Italy. The technical assistance ofMr. Antonio Calamo is gratefully acknowledged.

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