Journal of Vestibular Research 20 (2010) 401–406 401DOI 10.3233/VES-2010-0390IOS Press

Immunohistochemical and biomolecularidentification of 5-HT7 receptor in ratvestibular nuclei

Roza Khalmuratovaa,1, Young-Sool Hahb,1, Seong-Ki Ahna,∗, Sea-Yuong Jeona, Jin-Pyeong Kima andCarey D. BalabancaDepartment of Otolaryngology, School of Medicine, Gyeongsang National University, Jinju, South KoreabClinical Research Institute, School of Medicine, Gyeongsang National University, Jinju, South KoreacDepartment of Otolaryngology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA

Received 2 June 2010

Accepted 2 November 2010

Abstract. The association between migraine and balance disorder morbidities has been a topic of interest for many years, andserotonin (5-HT) receptor is known to be closely related with migraine and also to be associated with vestibular symptoms.However, the mechanism underlying the pathogenesis of migrainous vertigo and its association with 5-HT has not been elucidated.Of the many 5-HT receptors, 5-HT7 receptor has recently attracted attention in the context of migraine treatment. The purpose ofthis study was to investigate the localization and expression of 5-HT7 receptor in the rat vestibular nuclei by immunohistochemicalstaining and reverse transcriptase-polymerase chain reaction (RT-PCR). The present study might provide additional insight intothe role of 5-HT7 receptor in the pathogenesis of migraine-related vestibular symptoms.

Keywords: Migraine, vertigo, 5-HT7 receptor, vestibular nuclei

1. Introduction

The association between migraine and balancedisorders has been a subject of interest for manyyears [11]. Vestibular symptoms frequently occurin association with migraine, but the pathogenesis ofmigraine-related vestibular symptoms has not been de-termined. The activation or blockade of serotonin (5-hydroxytryptamine, 5-HT) receptors is known to beclosely related with migraine and its associated vestibu-lar symptoms [5]. However, the nature of this involve-ment has not been fully elucidated yet.

∗Corresponding author: Seong-Ki Ahn, M.D., Ph.D., Departmentof Otolaryngology, School of Medicine, Gyeongsang National Uni-versity, Jinju, 660-702, South Korea. Tel.: +82 55 750 8176; Fax:+82 55 759 0613; E-mail: skahn@gnu.ac.kr.

1These two authors contributed equally to this study.

The pathophysiology of migrainous vertigo is un-clear; however, there are several links between cen-tral vestibular pathways and proposed mechanisms in-volved in migraine. Such interactions may involve thevestibular nuclei, the trigeminal system and thalamo-cortical pathways. Reciprocal connections between theinferior, medial and lateral vestibular nuclei and thetrigeminal nucleus caudalis may provide a mechanismwhereby vestibular signals can influence trigemino-vascular pathways and that produces a tight linkage be-tween vestibular and trigeminal information processingduring migraine [5].

Several neurotransmitters that are involved in thepathogenesis of migraine (serotonin, calcitonin-generelated peptide, noradrenaline, dopamine) are knownto modulate the activity of vestibular neurons. Sev-eral lines of evidence in the literature have suggestedthat 5-HT7 receptors may be involved in the pathogen-

ISSN 0957-4271/10/$27.50 2010 – IOS Press and the authors. All rights reserved

402 R. Khalmuratova et al. / 5-HT7 receptor in rat vestibular nuclei

esis of migraine [16]. The 5-HT7 receptor, coupled toGs proteins and stimulating cAMP production [7], isthe most recently identified member of the family of5-HT receptors. Recently, a series of 5-HT7 receptorantagonists were developed, and studies on the selec-tive 5-HT7 receptor antagonists SB-269970-A and SB-656104-A suggest that this receptor plays a role in var-ious disorders, such as depression and migraine [1,20].The 5-HT7 receptor and its mRNA are found distribut-ed in meningeal tissues including middle meningeal ar-teries [18], trigeminal ganglion [17], and spinal trigem-inal nucleus [21], which indicates that 5-HT7 receptorsare located in the pathway of trigeminovascular system.The 5-HT7 receptors and their effect possibly includepain, hyperalgesia, and the facilitation of neurogenicinflammation [16].

However, no attempt has been made to investigatethe localization and expression of 5-HT7 receptor investibular nuclei. Therefore, the aim of this study wasto investigate the localization and expression of 5-HT7

receptor in rat vestibular nuclei by immunohistochemi-cal staining and reverse transcriptase-polymerase chainreaction (RT-PCR).

2. Methods

A total of 16 male Sprague-Dawley rats (300–400 g;Central Laboratory Animal Inc., Seoul) were used inthis study. All experimental protocols used compliedwith the Guidelines of the National Institute of Healthand the Declaration of Helsinki, and were approvedby the Committee on the Use and Care of Animals atGyeongsang National University.

2.1. Immunohistochemical staining of 5-HT7 receptorin the four major vestibular nuclei

The ten rats for the immunohistochemistry studywere deeply anesthetized with sodium pentobarbi-tal (100 mg/kg, i.p.) and perfused transcardiallywith phosphate-buffered saline (PBS, 0.9% NaClin 50 mM phosphate buffer, pH 7.3) followed byparaformaldehyde-lysine-periodate (PLP) fixative [10].Heads were removed, skinned, and brains were careful-ly removed and placed in 30% sucrose-PBS at 4◦C for48–72 h. Sections (35µ) were cut on a freezing-slidemicrotome, and sets of every sixth section were placedin PBS containing 30% sucrose and 30% ethylene gly-col and stored at−20◦C until required. After wash-ing with distilled water followed by PBS, free-floating

sections were treated for 1h with blocking buffer (PBScontaining 2% bovine serum albumin (BSA)) and in-cubated with 5-HT7 antibody (1:250, raised in rabbits,ImmunoStar, Hudson, WI, USA) for 48 h at room tem-perature. The sections were then washed in PBS andincubated with a secondary antibody (biotinylated goatanti-rabbit IgG; Vector Laboratories, Burlingame, CA,USA) for 1 h at room temperature. They were then pro-cessed using immunoperoxidaseprocedures using 3.3’-diaminobenzidine tetrahydrochloride (DAB) as a chro-mogen (Vectastain ABC Elite Kit, Vector Laboratories)to visualize the cellular localizations of 5-HT7 recep-tor in the vestibular nuclei. The sections were rinsedwith distilled water and mounted on gelatin/chromealum-subbedslides. After air-drying overnight, the sec-tions were dehydrated through a graded ethanol se-ries, cleared with xylene, and coverslipped with DPXMountant (Fluka, Milwaukee, WI, USA). The stain-ing was visualized with a Nikon Eclipse Ti microscope(Nikon, Tokyo, Japan). The images obtained were ad-justed for brightness and contrast, and then croppedusing Adobe Photoshop 7.0 (Adobe system, Inc., SanJose, CA, USA). Specificity for 5-HT7 receptor wasconfirmed by omitting the primary antibody, and pro-cessed sections using the immunoperoxidaseproceduredescribed above.

2.2. RT-PCR for 5-HT7 receptor mRNA expression

Another 6 animals were sacrificed for RT-PCR bydecapitation under deep anesthesia. Brains were rapid-ly isolated and mounted on a rat brain matrix in ice-cold PBS. Coronal sections were cut to isolate the brainstem at 2 mm and 4 mm rostrally from caudal marginof the 4th ventricle.

The location of the medial vestibular nucleus was themost easily recognized and therefore was selected forRT-PCR. Tissue containing the medial vestibular nucleiwas selectively isolated from brain stem slices using atissue punch (1.8 mm in ID: Fine Science Tools, USA).The landmark used for the medial vestibular nuclei wasthe lateral border of the 4th ventricle and the brown-colored borderline between the prepositus hypoglos-sal nucleus and the dorsal paragigantocellular nucleus.One tissue sample (1.8 mm in diameter) of each medi-al vestibular nucleus was isolated in the caudo-rostraldirection [4]. The trigeminal ganglion was also har-vested for comparison with the vestibular nuclei. TotalRNA was extracted from dissected tissues using TRIzolreagent (Gibco BRL, Gaitherburg, MD, USA). Briefly,each tissue sample was homogenized in 0.3 ml of TRI-

R. Khalmuratova et al. / 5-HT7 receptor in rat vestibular nuclei 403

zol. Chloroform was then added and the mixture wascentrifuged at 12000 g for 15 min at 4◦C. The RNAphase was precipitated with isopropanol, centrifugedat 12000 g for 8 min at 4◦C, and pellets were washedwith 75% ethanol, centrifuged at 12000 g for 5 min at4◦C, air-dried for 10 min, and dissolved in RNAse-freewater at 55–60◦C for 10 min. RNA concentrationswere determined using a UV-visible spectrophotome-ter (Optizen 3220 UV, Mecasys, Daejeon, Korea). Re-verse transcription was carried out in a reaction volumeof 20 A using iScript cDNA Synthesis Kits (Bio-RadLaboratories, Hercules, CA, USA). The extracted RNAwas reverse transcribed into cDNA using oligo-dT, andthe synthesized cDNA derived from total RNA wasthen amplified with specific primers for 5-HT7 recep-tor and glyceraldehydes-3-phosphate dehydrogenase(GAPDH). Two primers were designed from the clonedrat 5-HT7 receptor sequence (accession no. L22558)as follows; 5’-CTGACGATCGCAGGCAACTGCCT-3’(sense) and 5’-TGCCTGCAGCAGAGAGCTTC-CGGT-3’ (antisense) (964 base pairs). Two otherprimers (5’-TGCTGAGTAYGTCGTGGAGTC-3’ and5’-TTGGTGGTGCAGGAKGCATTGC-3’) (515 basepairs) of the cloned GAPDH sequence (accession no.M17701) were used as a positive control. All primersused in this study were obtained from Bioneer Co.(Chungwon, Korea). After cDNA synthesis, a PCRmixture was prepared containing; 2 A of cDNA, 5 A of10x PCR buffer, 4 A of 10 mM dNTP mix, 1.25 unitsof Taq polymerase (Takara Bio Inc, Otsu, Japan), 30pmole of each 5’- and 3’- primer for 5-HT7 receptorand GAPDH. PCR was performed using a thermocy-cler (Bio-Rad Laboratories, Hercules, CA) using; pre-denaturation at 94◦C for 3 min followed by 30 ampli-fication cycles (denaturation for 30 sec at 94◦C, an-nealing for 1 min at 58◦C, and extension for 1 min at72◦C). The PCR products obtained were separated byelectrophoresis in a 1.2% agarose gel and visualizedusing an UV illuminator in the presence of ethidiumbromide.

3. Results

3.1. Immunoreactivity of 5-HT7 receptor in the fourmajor vestibular nuclei

The presence of DAB immunopositive products investibular nuclei sections was taken to indicate im-munoreactivity for 5-HT7 receptor in the four majorvestibular nuclei. The nomenclature and boundaries

defined in the rat brain atlas of Paxinos and Watson [13]were utilized throughout. 5-HT7 receptor immunopos-itive neurons were found to be distributed throughoutthe four major vestibular nuclei. There appear to be twodistinct staining patterns in the vestibular nuclear com-plex, one pattern in the superior (Figs 1a–b) and medialvestibular nuclei (Figs 1e–g) and the other in the lateral(Figs 1c–d) and spinal vestibular nuclei (Figs 1e–f). Inthe former, there is a significant reticulum of neuropilstaining, whereas the immunolabeling in the lateral andspinal vestibular nuclei appears to be limited to cellbodies and their proximal dendrites. The other obser-vation is that there are somata in each of the vestibularnuclei that are clearly unstained. Some nuclear label-ing are also observed in the lateral, spinal, and medi-al vestibular nuclei. In control sections, which werenot treated with primary antibody, no neuronal stainingwas observed (data not shown).

3.2. Expression of 5-HT7 receptor mRNA in themedial vestibular nuclei

The presence of 5-HT7 receptor mRNA was inves-tigated by RT-PCR in the rat medial vestibular nucleiand trigeminal ganglia. The cDNA of the constitutivelyexpressed housekeeping gene GAPDH was also ampli-fied for control purposes. PCR products of the expectedsize (964 base pairs) for 5-HT7 receptor were consis-tently amplified in all samples (Fig. 2). Sequence anal-ysis of the selected PCR product confirmed its identitywith the published sequence of the corresponding rat5-HT7 receptor gene.

4. Discussion

It is being increasingly recognized that the symptomsof migraine and balance system disorders are interrelat-ed [5,19,20]. Recent studies have shown that dizzinessoccurs in 28–30% [3] and motion sickness in∼50% ofmigraine patients [8]. Furthermore, migrainous verti-go has been reported in 9% of migraine sufferers [12].However, the pathophysiology of migraine-associatedvestibular disorders is not well understood.

5-HT receptors have been reported to be importantneuromediators of migraine development [9,14], andrecently, the link between 5-HT and migrainous vertigohas attracted renewed interest. Vestibular nucleus com-plex neurons express a number of different serotonergicreceptor subtypes, including 5-HT1A, 5-HT1F and 5-HT2A receptors [2,15]. Some studies have concluded

404 R. Khalmuratova et al. / 5-HT7 receptor in rat vestibular nuclei

Fig. 1. Photomicrographs of 5-HT7 receptor immunoreactive neurons in the rat vestibular nuclei. The photomicrographs in the right panel arehigh magnification views of those in the left panel (a+b, c+d, e+f and e+g). 5-HT7 receptor immunoreactive neurons were observed in thesuperior vestibular nucleus (SuVe), the lateral vestibular nucleus (LVe), the spinal vestibular nucleus (SpVe), and the medial vestibular nucleus(MVe). scp, superior cerebellar peduncle; icp, inferior cerebellar peduncle; g7, genu of facial nucleus; Pr, prepositus nuclei; DC, dorsal cochlearnucleus. Scale bars: 200µm (in e for a and c); 25µm (in g for b, d and f).

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Fig. 2. RT-PCR analysis of 5-HT7 receptor mRNA in rat medial vestibular nuclei (MVe) and trigeminal ganglion (TG) as visualized by gelelectrophoresis of PCR-amplified products obtained using selective oligonucleotide primers. Numbers indicate rat medial vestibular nuclei andtrigeminal ganglia samples, respectively. Level of GAPDH mRNA expression served as a loading control. TG1 and TG2 showed 5-HT7 receptorexpression in trigeminal ganglion as a positive control.

that 5-HT makes important contributions to the devel-opment of migraine-related vestibular symptoms [2,5].It has been reported that the vestibular nuclei receivemajor serotonergic projections from the dorsal raphenucleus and minor serotonergic and nonserotonergicprojections from the nuclei raphe pallidus and obscu-rus [6]. Thus, the presence of serotonin and its recep-tors in the vestibular pathway suggests a specific neuralbasis for the occurrence of migrainous vertigo.

The present study demonstrates that 5-HT7 receptormRNA is expressed in the rat medial vestibular nucleusand trigeminal ganglion, and the 5-HT7 receptor is ex-pressed in each of the four vestibular nuclei. The recip-rocal link between vestibular nuclei and the trigeminalnucleus caudalis implies that trigeminal and vestibularsignals are likely to be simultaneously activated dur-ing the development of migraine or migrainous vertigo.Given that 5-HT7 receptor is localized and expressed inrat vestibular nuclei, presumably a serotonergic mech-anism in vestibular pathways operates in parallel to thetrigeminal pathways involved in migraine. According-ly, a better understanding of the role of 5-HT7 receptoris likely to provide additional insight into the patho-genesis of migrainous vertigo.

It has been suggested that migraine may result fromsudden increases in the amounts of 5-HT released fromperivascular serotonergic and 5-HT-containing nora-drenergic fibers due to the activations of the dorsalraphe nucleus and the locus coeruleus, respectively, andthat these sudden releases lead to the stimulation of 5-HT7 receptor in large conduit vessels and to vasodilata-tion, which would lead to the activations of trigeminalsensory nerves [16]. In fact, the majority of migraineprophylactic 5-HT antagonists display relatively highaffinity for 5-HT7 receptor, and it has been reportedthat vasodilatation of the middle meningeal artery canbe blockedin vivo by SB-269970 (a selective 5-HT7

receptor antagonist) in the rat [18]. Furthermore, thetherapeutic efficacies of 5-HT7 receptor antagonists inmigrainous vertigo might be explained by the block-ade of 5-HT7 receptor, and thus, by its mediation ofcraniovascular vasodilatation.

In conclusion, the present study demonstrates, forthe first time, that 5-HT7 receptor is localized and ex-pressed in rat vestibular nuclei. This study may pro-vide new insights into the role of 5-HT7 receptor in thevestibular signal processing. Further studies are neededfor clarifying the role of 5-HT7 receptor in serotonergicneurotransmissions in normal as well as pathologicalstates.

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