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Research Report

Immunohistochemical characterization of 5-HT3A receptors inthe Syrian hamster forebrain

Maria Carrillo, Lesley A. Ricci, Jared J. Schwartzer, Richard H. Melloni⁎

Behavioral Neuroscience Program, Department of Psychology, 125 Nightingale Hall, Northeastern University, 360 Huntington Avenue,Boston, MA 02155, USA

A R T I C L E I N F O

⁎ Corresponding author. Program in Behavior360 Huntington Avenue, Boston, MA 02155, U

E-mail address: melloni@research.neu.ed

0006-8993/$ – see front matter © 2010 Publisdoi:10.1016/j.brainres.2010.02.038

A B S T R A C T

Article history:Accepted 9 February 2010Available online 4 March 2010

The Syrian hamster (Mesocricetus auratus) has been extensively used as an animal model toinvestigate neuronal networks underlying various behaviors where 5-HT3A receptors havebeen found to play a critical role. To date, however, there is no comprehensive description ofthe distribution of 5-HT3A receptors in the Syrian hamster brain. The current studyexamined the localization of 5-HT3A receptors across the neuraxis of the Syrian hamsterforebrain using immunohistochemistry. Overall, 5-HT3A receptors were widely andheterogeneously distributed across the neuraxis of the Syrian hamster brain. Notably, themost intense 5-HT3A immunolabeling patterns were observed in the cerebral cortex andamygdala. In addition, high variability in receptor density and expression patterns (i.e.,perikarya, fibers and/or neuropilar puncta) was observed within the majority of brain areasexamined, indicating that the role this receptor has in the modulation of a particular neuralfunction differs depending on brain region. In some regions (i.e., nucleus accumbens)differences in the immunolabeling pattern between rostral, medial and caudal portionswere also observed, suggesting functional heterogeneity of this receptor within a singlebrain region. Together, these results and the localization of this receptor to brain areasinvolved in the regulation of sexual behavior, aggression, circadian rhythm, drug abuse andanxiety implicate 5-HT3A receptors in the modulation of various behaviors and neuralfunctions in the Syrian hamster. Further, these results underscore the importance ofevaluating 5-HT3A receptors as a pharmacological target for the treatment of variouspsychopathological disorders.

© 2010 Published by Elsevier B.V.

Keywords:Syrian hamster5-HT3A

ImmunohistochemistryNeuroanatomy

1. Introduction

In the mammalian brain, the serotonergic system has beenimplicated in the modulation of a variety of behaviorsincluding sleep, mood, memory, cognition, anxiety, andaggression. The diverse activity of this indolamine has beenshown to be, in part, dependent on the serotonin-3 receptor

al Neuroscience, DepartmSA. Fax: +1 617 373 8714u (R.H. Melloni).

hed by Elsevier B.V.

subtype (5-HT3) (Costall and Naylor, 1992). 5-HT3 is uniqueamong 5-HT receptors as it is the only excitatory, fast-actingligand-gated cation channel (Derkach et al., 1989; Maricq et al.,1991; Sugita et al., 1992). Similar to other members of theligand-gated ion-channel superfamily (i.e., nicotinic acetyl-choline, γ-aminobutyric acid type A and glycine polymericreceptors) 5-HT3 receptors have a pentameric structure

ent of Psychology, 125 Nightingale Hall, Northeastern University,.

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composed of five monomers surrounding a central cation-permeable pore with each subunit composed of four trans-membrane domains and an extracellular N-terminus domainwhich contains the ligand binding site (Barnes and Sharp,1999; Boess et al., 1995). To date, five 5-HT3 subunits have beenidentified (5-HT3A–5-HT3E) however 5-HT3A appears to be themost widely expressed across the mammalian brain and hasthe greatest functional diversity. Interestingly, the pharma-cological profile of 5-HT3A receptors depends on whether itis expressed as a homomeric (i.e., only the A subunit) orheteromeric receptor (i.e., A subunit co-expressed with B)(Dubin et al., 1999). In contrast, no significant functionaldifferences between the two 5-HT3A splice variants i.e., long(5-HT3A–L) vs short (5-HT3A–S), have been reported.

The distribution of 5-HT3 receptors in the brain was firstcharacterized using radioligand binding in the rat, followed bymapping studies in rats and humans (Barnes et al., 1989;Laporte et al., 1992). In the central nervous system (CNS),5-HT3 receptor binding has been examined using [3H]-meta-chlorophenylbiguanide (Steward et al., 1993;Wong et al., 1993a),[3H]-quipazine (Wong et al., 1993b,c), [3H]-zacopride, [3H]-(S)-zacopride, and [125I]-zacopride in rats (Barnes et al., 1990b;Gehlert et al., 1993b; Laporte et al., 1992), in addition to [3H]-granisetron (Bufton et al., 1993) and [3H]-GR65630 in rats(Kilpatrick et al., 1987, 1988b, 1989a) and humans (Barnes etal., 1989; Marazziti et al., 2001). The localization of 5-HT3

receptors in the brain has also been examined in severalspecies using immunohistochemistry (Miquel et al., 2002;Morales et al., 1998b; Mukerji et al., 1996; Spier et al., 1999).Together, the results from these studies show a wide andheterogeneous distribution of this receptor across the mam-malian brain. Overall, the highest 5-HT3 receptor levels havebeen localized to the hindbrain and spinal cord, including thenucleus of the solitary tract, the vagus and trigeminal nervesand the superficial layers of the dorsal horn, while moderate tolow levels have been observed in forebrain and midbrainstructures (i.e., hippocampus, amygdala, cortex) (Barnes et al.,1990a; Gehlert et al., 1991; Laporte et al., 1992). Notably, thedistribution of 5-HT3 receptors in the CNS is in agreement withthe role this receptor plays in the modulation of a variety ofbehaviors and neurobiological functions. For example, 5-HT3

receptors have been implicated in the modulation of socialbehaviors (Newman, 1999; Ricci et al., 2004b; Rudissaar et al.,1999b), circadian rhythm (Graff et al., 2007), anxiety (Costall etal., 1989, 1993b; Hensler et al., 2004) and drug addiction (Carboniet al., 1989a; Chen et al., 1991a; Costall and Naylor, 2004; Ricci etal., 2004d), suggesting that its functional influence in brainareas associated with the control of these behaviors isconsiderable.

During the past decade the use of the Syrian hamster(Mesocricetus auratus) as an animal model to investigate theneurobiology of various behaviors has significantly increased.Of particular interest, is the frequent use of the Syrian hamsterto investigate behaviors where 5-HT3 receptors have beenfound to play a critical role including, sexual behavior (Petrulisand Eichenbaum, 2003; Petrulis, 2009), social communication(Albers and Prishkolnik, 1992; Albers and Bamshad, 1998; Ferriset al., 1996), aggression (Cervantes and Delville, 2009; Harrisonet al., 2000; Melloni et al., 1997), circadian rhythm (Agostino etal., 2004; Carr et al., 2003; Lungwitz and Gannon, 2009), drug

addiction (DiMeo and Wood, 2006; Triemstra et al., 2008) andanxiety (Moise et al., 2008; Yannielli et al., 1996). To date,however, 5-HT3A receptor expression in the hamster brain hasnot been characterized. Results from several studies support-ing considerable differences in 5-HT3A receptor expressionbetween species (Kilpatrick et al., 1989b; Peroutka, 1988)underscore the importance of examining the distribution ofthis receptor in the Syrian hamster. In this study we presentthe first characterization of 5-HT3A receptor localization acrossthe neuraxis of the Syrian hamster brain using immunohisto-chemistry. In particular, we focus on examining the distribu-tion of 5-HT3A receptors in neural networks underlying themodulation of social behavior (i.e., sexual behavior andaggression), circadian rhythm, anxiety and drug abuse.

2. Results

2.1. 5-HT3A-ir in the Syrian hamster forebrain

Significant heterogeneity in reaction product density (RPD)and expression pattern (i.e., cellular localization; neuropilarpuncta, fibers or perikarya) of 5-HT3A receptors was observedacross the neuraxis of the Syrian hamster forebrain (Table 1,Fig. 1). Intense 5-HT3A staining patterns were detected in themajority of regions/nuclei of the cerebral cortex and amygda-la. In contrast, the ependyma and subependymal layers, thecaudate putamen (CPu), globus pallidus (GP), hippocampus,substantia nigra (SN), dorsal raphe (DR) and periaqueductalgrey (PAG) exhibited moderate to low 5-HT3A-ir. Notably,differences in 5-HT3A labeling patterns were observed withinthe majority of brain areas allowing identification of discretenuclei/subregions. Moreover, some brain nuclei exhibiteddifferences in 5-HT3A RPD (referred to as heterogeneous inTable 1) and expression patterns between the anterior, medialand/or posterior portions (heterogeneous; Table 1). Lastly,although the majority of brain regions showed 5-HT3A-ir fiberand/or neuropilar puncta, few brain regions/nuclei exhibitedcell body immunolabeling. Specifically, cell body staining wasobserved in all regions of the anterior olfactory nucleus (AON),cerebral cortex and hippocampus.

2.1.1. Olfactory nucleiIn the olfactory system 5-HT3A-ir was weak to non-existent(Table 1, Fig. 1). In general, most areas lacked 5-HT3A-ir, exceptfor the AON, wheremoderately stained cell bodies (i.e., lateral,medial and dorsal regions), neuropilar puncta (i.e., lateral anddorsal regions) and fibers (i.e., lateral region) were observed.

2.1.2. Cerebral cortexThe cerebral cortex exhibited some of the most intense 5-HT3A-ir staining (Figs. 1 and 2, Table 1). However, heterogene-ity in 5-HT3A staining patterns was detected across corticallayers and regions. In all cortical layers (i.e., I–VI) throughoutthe rostrocaudal extent of motor (M; Fig. 2A) and cingulate (Cg;Fig. 2B) cortices, 5-HT3A-ir was observed as intensely stainedmultipolar or pyramidal-like cell bodies, neuropilar punctaand/or fibers. Rostral sections of the orbital (ORB; Fig. 2C),prefrontal (PFC; Fig. 2D) and piriform (Pir) cortices had few butintensely stained 5-HT3A-ir cell bodies, in addition to fibers in

Table 1 – Qualitative and semi-quantitative distribution of 5-HT3A receptors in the hamster brain. Reaction product density[RPD] was characterized as weak (1), moderate (2), dense (3) or heterogeneous (H; RPD changed throughout the rostrocaudalextent). In addition, the immunolabelingpatternwas described as 5-HT3A-ir cell bodies (¤), fibers (|) and/orpunctate (°). 5-HT3A-irwas characterized for each brain region along its rostrocaudal extent as indicated byMorin andWood (2001) Hamster Atlas. Inthe table, rostral portions reflect brain portions located between4.6 and 2.9 mm frombregma,medial portions showbrain areaslocated between 2.4 and −0.1 mm from bregma and caudal regions show regions located between −0.3 mm and −4.9 mm frombregma.

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Table 1 (continued)

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the PFC, puncta in the ORB and puncta and fibers in the Pircortex. Throughout the rostrocaudal extent of the somato-sensory cortex, 5-HT3A-ir was observed in all six layers asweakly stained puncta. Moreover, layers I–III of the visualcortex displayed lightly immunostained neuropilar punctalocalized only in the caudal region of the brain. Further, thislayer exhibitedweak somatodendritic staining in layers IV andV and fiber labeling in layers I–IV. The rostral and medialportions of the dorsal endopiriform nucleus were character-ized by intense cell body, puncta and/or fiber staining whileonly puncta and fiber immunostainingwas observed in caudalportions. In contrast, only neuropilar puncta was detected incaudal portions of the lateral endopiriform nucleus.

2.1.3. Septal nuclei5-HT3A-ir in the septum was detected in the anterointermedi-ate, laterodorsal, lateroventral, medial and hippocampalnuclei (Table 1, Figs. 1 and 3). In these regions, 5-HT3A-ir wascharacterized by light to moderately stained neuropilarpuncta and/or fibers. Interestingly, a slight decrease in RPDwas detected in the lateroventral nucleus, with rostral regionshaving a stronger reaction density compared to the morecaudal portions.

2.1.4. Hippocampal formationHeterogeneity in 5-HT3A immunolabeling density and stainingpatterns was observed across hippocampal structures (i.e.,Cornu Ammonus [CA] 1–3 and dentate gyrus [DG]; Table 1,Figs. 1 and 4). For instance, weakly labeled neuropilar puncta

and cell bodies were detected in rostral portions of CA1 andCA2 while solely fibers were observed in rostral portions ofCA3 and DG. However, a comparable 5-HT3A immunolabelingpattern was detected in caudal portions of CA1–3 and DG,where weakly stained neuropilar puncta and cell bodies wereobserved. In addition, 5-HT3A-ir cell bodies, puncta and fiberswere observed in the subiculum and lateral entorhinal cortex.

2.1.5. Basal gangliaIn the Syrian hamster, distinct 5-HT3A-ir patterns wereobserved throughout basal ganglia structures (Table 1, Figs. 1and 5). Noticeably, within the nucleus accumbens (NAc) well-defined 5-HT3A-ir patterns allowed identification not only ofcore and shell regions but also of the five discrete subregionsof the NAc shell (Todtenkopf et al., 2000). The NAc coreexhibited a weaker staining pattern compared to the shell andcontained scattered puncta and sparse fibers throughout therostrocaudal extent. Within the NAc shell, all subregions (i.e.,vertex, arch, cone intermediate zone and ventrolateral region)contained weak to dense 5-HT3A-ir fibers and/or neuropilarpuncta, in addition to cell body staining detected in theintermediate zone.

5-HT3A-ir throughout the rostral andmedial portions of theCPu and GP consisted of weakly stained punctate and fiber.However, only lightly stained fibers were observed in caudalportions of the CPu and GP. Moreover, rostrocaudal changes in5-HT3A staining pattern were detected in the ventral pallidum(VP) with rostral sections containing solely fibers, medialsections characterized by the presence of fibers and neuropilar

Fig. 1 – Schematic images showing the distribution of 5-HT3A receptors (black shaded areas) in coronal sections throughout therostrocaudal extent of the Syrian hamster brain. Plates were adopted from the hamster atlas by Morin and Wood (2001) andreflect specific position in the rostrocaudal plane (i.e., distance in mm from bregma to the plane of section at the skull surface).

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puncta and caudal sections containing only neuropilarpuncta. In addition, an overall rostrocaudal increase in RPDwas observed in the VP (i.e., rostral sections exhibiting aweaker staining pattern compared to more caudal portions).Notably, no 5-HT3A-ir was observed within the internal orexternal capsules of the VP.

2.1.6. AmygdalaOverall, the amygdala exhibited one of the most intensedensity patterns (Table 1, Figs. 1 and 6). 5-HT3A-ir puncta andfibers were observed in the anterior cortical nucleus (ACo) andin the anterior, anterodorsal, anteroventral and posterodorsalportions of the medial amygdala (MeA). Within the centralnucleus (CeA), differential 5-HT3A immunolabeling patternsallowed identification of the capsular (CeC) and medial (CeM)regions. The CeC contained strongly labeled cell bodies inanterior portions andpuncta, fibers and cell bodies in posteriorportionswhile the CeMonly contained 5-HT3A-ir fibers.Withinthe basomedial nucleus (BMA) rostrocaudal changes in immu-noreactive patternwere detected.While rostral sections of thisnucleus solely contained intensely stained cell bodies, poste-rior portions contained neuropilar puncta, fibers and perikar-

ya. Lastly, heavily stained cell bodies, fibers and neuropilarpuncta were detected in the basolateral nucleus (BLA).

2.1.7. ThalamusOverall, thalamic nuclei displayed light to moderate 5-HT3A-irstaining across the rostrocaudal extent (Table 1). The ante-roventral, anterodorsal, anterior paraventricular and reuniensthalamic nuclei were predominantly characterized by finefibers and scarce punctate labeling in the neuropil (Figs. 1and 6B). Caudally, the medial and posteromedial portions ofthe ventral nuclei contained few moderately stained bipolar-like cells in addition to 5-HT3A-ir fibers in the medial portion.In contrast, the posterolateral portion of the ventral nucleuscontained some fibers and neuropil puncta. Moreover, theposterior triangular nucleus was characterized by the pres-ence of lightly stained fibers throughout the rostrocaudalextent.

2.1.8. HypothalamusNotably, 5-HT3A-ir in all hypothalamic nuclei of the Syrianhamster brainwas characterized by the presence of fibers and/or neuropil punctate labeling with only the paraventricular

Fig. 2 – 5-HT3A receptor immunostaining in the cortex. Brightfield photomicrographs showing 5-HT3A receptor labeling inrostral portions of the (A) motor, (B) cingulate, (C) orbital and (D) and prefrontal cortices. Cortical cell body labelingwas observedthroughout layers I–VI. Scale bars=10 µm in A, B, C and D.

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nucleus (PVN) exhibiting cell body staining (Table 1, Figs. 1and 6). Nonetheless, differences in RPD betweenhypothalamicnuclei were noticed, with some regions exhibiting light (e.g.,nucleus circularis [NC], periventricular nucleus [PeVN], ven-tromedial hypothalamus [VMH] and ventrolateral hypothala-mus [VLH]), moderate (e.g., supraoptic nucleus [SON],suprachiasmatic nucleus [SCN], medial preoptic area [MPOA],

Fig. 3 – 5-HT3A receptor immunostaining in the septum. Brightfieshowing 5-HT3A immunopositive varicose axonal or dendritic fibtraversing into the intermediate LS. Scale bars=40 µm in A and 2

lateral preoptic area [LPOA] and medial preoptic nucleusmPON) or dense (e.g., anterior hypothalamus [AH] and arcuatenucleus [Arc]) immunolabeling patterns. Moreover, a hetero-geneous density pattern was observed in some hypothalamicbrain regions (lateral hypothalamus [LH], PVN, and posteriorhypothalamic area [PH]), where the RPD pattern variedthroughout the rostrocaudal extent.

ld photomicrographs at (A) low and (B) high magnificationer course through the dorsal portion of the lateral septum (LS)0 µm in B.

Fig. 4 – Brightfield photomicrographs of 5-HT3A receptor immunolabeling in the hippocampus. (A) 5-HT3A immunoreactivity inthe hippocampuswas characterized as fine varicose fibers and cell body staining patterns. (B) Fine varicose fibers and cell bodylabelingwere observed throughout the granule cell layer in CA1. (C) Small diameter fibers in rostral portions of CA3 (D) mediumsize cell bodies and varicose fibers were detected in the polymorph and molecular layers of the dentate gyrus (DG). Scalebars=100 µm in A, 20 µm in B and C and 10 µm in D.

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2.1.9. Bed nucleus of stria terminalis (BNST)Light to moderately stained fibers and scattered neuropilarpuncta were observed in the anterointermediate and poster-ointermediate portions of the BNST (Table 1, Figs. 1 and 6D).This immunolabeling pattern was observed throughout therostrocaudal extent of the BNST. A similar 5-HT3A-ir stainingpattern was observed in the rostral and medial portions of theposteromedial region. However, only fibers were detected incaudal portions. Further, a rostrocaudal reduction in RPD wasdetected in this region. Lastly, solely neuropilar punctalabelingwas detected in the anteromedial portion of the BNST.

2.1.10. Brainstem structuresOverall, weak 5-HT3A receptor labeling was observed in rostralportions of the brainstem (Table 1, Fig. 1). Few regions haddistinct immunoreactive patterns including, the deep grey layerof the superior colliculus (DpG), the SN, the deepmesencephalicnucleus (DpMe), the red nucleus (RN), the DR and the PAG. Ingeneral, 5-HT3A-ir in these structures was observed as light tomoderately stained fibers and neuropil puncta.

3. Discussion

5-HT3A receptor distribution in the central nervous systemhasbeen characterized in several species since its identification asthe only ligand-gated ion-channel subtype among serotoner-gic receptors. The Syrian hamster has been extensively usedas an animal model of several behaviors where 5-HT3A

receptors have been observed to play a critical role. To date,

however this receptor has not been characterized in the Syrianhamster. The current report provides the only comprehensivebrain characterization of 5-HT3A receptor distribution in theSyrian hamster brain. In addition, it specifically describes thedistribution of this receptor in neural circuits underlying theregulation of sexual behavior, aggression, circadian rhythm,anxiety and drug abuse. The results from the present reportreplicate findings in other species localizing 5-HT3A receptorexpression/activity to cortical structures, olfactory system, theamygdalar complex, hippocampal formation, thalamus, hy-pothalamus, basal ganglia and some brainstem areas inseveral species using immunohistochemistry, in situ hybrid-ization, and in situ receptor autoradiography (Bufton et al.,1993; Gehlert et al., 1993a; Kilpatrick et al., 1987; Laporte et al.,1992; Morales et al., 1998b; Tecott et al., 1993).

The ability of 5-HT3 receptors to facilitate various cellularresponses has vast implications for 5-HT function. Forexample, 5-HT3 receptors have been implicated in the directstimulation of vesicle release (Fozard and Kalkman, 1992),modulation of neurotransmitter release (i.e., dopamine,GABA, glutamate, acetylcholine, cholecystokinin, substance P)(Costall et al., 1987; Funahashi et al., 2004; Greenshaw andSilverstone, 1997;McNeish et al., 1993; Steckler andSahgal, 1995)and expression of immediate early genes (i.e., c-Fos, c-jun andATF-1) (Genova and Hyman, 1998). Moreover, pharmacologicalagents regulating 5-HT3 activity and/or function have beenshown to modulate neural mechanisms underlying variousbehaviors including, social behavior (Ricci et al., 2004c; Ricciet al., 2005a,b; Rudissaar et al., 1999b), anxiety (Costall andNaylor, 1992; Jonesetal., 1988) andaddiction (Kingetal., 1999a,b,

Fig. 5 – 5-HT3A receptor immunolabeling in the ventral forebrain. (A) Schematic representation of 5-HT3A immunopositivestaining patternswhich revealed both core and shell divisions of the nucleus accumbens (NAc) aswell as the 5 subregions of theshell. Brightfield photomicrographs showing neuropilar punctate and fine varicose fibers in the cone subdivision of theaccumbens shell (B) and intense cell body labeling in the intermediate zone (C). Scale bars=20 µm in B and 10 µ in C.

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2000; Matell and King, 1997; Ricci et al., 2004a) as well as neuralfunctions, such as circadian rhythm (Graff et al., 2007). Duringthe past decade the Syrian hamster has been increasingly usedto investigate theneuralnetworksunderlying themodulationofthese behaviors and neural functions. Thus, localization of 5-HT3, and specifically 5-HT3A receptors, to brain areas importantfor the regulation of social behaviors, circadian rhythm, anxietyand addiction in the Syrian hamster will help predict possiblefunctional roles of pharmacological agents targeting discreteaspects of the serotonergic system.

3.1. Social network (sexual and aggressive behavior)

Evidence from various studies supports a modulatory role for5-HT3 receptors in variousparameters of sexual and aggressivebehaviors. For example, 5-HT3 receptors are involved in theactivation of the hypothalamo-hypophyseal-testicular com-plex in males (Amstislavskaya and Popova, 2004), sexualreceptivity (Mendelson and Gorzalka, 1990), frequency andquality of lordosis (Maswood et al., 1998), copulatory behavior(Mendelson and Gorzalka, 1990; Tanco et al., 1994), impulsivity(Cervantes and Delville, 2009) and cocaine-(Ricci et al., 2004c)andapomorphine- (Rudissaar et al., 1999a) inducedaggression.These behavioral results are in agreement with the immuno-

histochemical localization of 5-HT3A receptors to the majorityof brain regions modulating sexual and aggressive behaviors(i.e., AON, LS, MeA, CeA, BNST,MPOA, PVN, AH, VMH, VLH, andPAG). The MPOA and AH function as key areas involved in theintegration of sexual- and aggression-related chemosensoryinformation, critical for initiation,motivation and consumma-tion of these behaviors (Delville et al., 2000; Ferris et al., 1984,1989; Grimes et al., 2006, 2007; Liu et al., 1997; Newman, 1999;Powers et al., 1987; Ricci et al., 2007; Simerly and Swanson,1986, 1988; Van De Poll and Van Dis, 1979;Wood and Newman,1995). In these brain regions, evidence indicates that theserotonergic system has an inhibitory effect on both sexualand aggressive behaviors and that the activity of this neuralsystem likely occurs through interactionswith dopamine [DA],glutamate [Glu] and/or γ-aminobutyric acid [GABA] systems(Lee et al., 2008; Lorrain et al., 1999; Ricci et al., 2009; Schwartzeret al., 2009; Yonezawa et al., 2009). For example, while agonismof either DAD2 or 5-HT2c receptors has no effect on ejaculation,simultaneous administration of both DA D2 and 5-HT2c

receptors results in a significant increase in ejaculationfrequency and amount (Yonezawa et al., 2009). Moreover,electrophysiological results indicate that in thehypothalamus,5-HTpresynaptically diminishesGABAergic and glutamatergicsynaptic activity (Lee et al., 2008). Together, these results

Fig. 6 – Brightfield photomicrographs of 5-HT3A receptor immunolabeling in hypothalamic and amygdaloid nuclei. (A) Theposterodorsal portion of the medial amygdala was characterized by fibers, neuropil puncta (thin arrows) and cell body labeling(thick arrows). (B) The anterior hypothalamus (AH) showed one of the strongest 5-HT3A immunoreactive patterns of thehypothalamic nuclei. This nucleus was characterized by the presence of small diameter axodendritic processes (thin arrows)and clusters of punctate staining (thick arrows). (C) Few intensely stained cell bodies (thick arrows) and neuropilar punctate(thin arrows) were observed in the paraventricular (PVN) nucleus. (D) The bed nucleus of the stria terminalis (BNST) exhibitedimmunopositive labeling for the 5-HT3A receptor that appeared as varicose axodendritic processes (thin arrows) and fewneuropil puncta (thick arrows). Scale bars=20 µm in A and C and 10µm in B and D.

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support the notion that 5-HT3A receptors likely modulatesexual and aggressive behaviors through interactions withother neural systems. Indeed, 5-HT3 receptors modulateneurotransmitter release including DA, GABA and Glu (Costallet al., 1987; Funahashi et al., 2004;McNeish et al., 1993). To date,however, very few studies have examined the specific neuralmechanisms throughwhich5-HT3A receptorsmodulate sexualand aggressive behaviors.

3.2. Circadian rhythm network

The Syrian hamster has been widely used to investigate theneural network modulating circadian rhythm. The circadianrhythm network is centered at the level of the SCN (i.e.,circadian pacemaker) which functions to synchronize biolog-ical and environmental rhythms. The SCN receives photic andnon photic information via three main pathways: 1) theretinohypothalamic tract (RHT; originates in the retinalganglion cells), 2) geniculohypothalamic tract (GHT; originatesin the IGL), and 3) directly from the MRN and indirectly fromthe DRN via IGL (Hay-Schmidt et al., 2003; Morin, 1994; Morinand Meyer-Bernstein, 1999). Results from behavioral andpharmacological studies strongly implicate the serotonergicsystem in the modulation of different aspects of the circadianrhythm (i.e., phase shifting, photic entrainment) (Challet et

al., 1998; Duncan et al., 2000; Pickard and Rea, 1997). Forexample, in the Syrian hamster pharmacological stimulationor inhibition of 5-HT3 receptors induces phase shiftingchanges in locomotor activity (Challet et al., 1998; Graff etal., 2007; Schuhler et al., 1998). In agreement, the current studyshowed 5-HT3A-ir fibers and neuropil puncta in the SCN andDRN, two critical regions for the modulation of circadianrhythm. Notably, to our knowledge this is the first studylocalizing 5-HT3A receptors to the SCN. Based on pharmaco-logical results, Graff et al., 2007 hypothesized that in the SCN5-HT3 receptors are likely presynaptically localized on RHTterminals and function to stimulate glutamate releasethrough activation of N-methyl-D-aspartic acid receptors(NMDA). Moreover, our results introduce the possibility that5-HT3A receptors likely participate in the regulation photope-riodic responses through modulation of 5-HT function in theDRN. In the Syrian hamster, the MRN provides indirectserotonergic input to the SCN via the IGL (Morin and Meyer-Bernstein, 1999). In fact, in the Syrian hamster electricalstimulation of the DRN as well as pharmacological activationof 5-HT receptors results in circadian phase-resetting andphase shifting, respectively (Glass et al., 2000; Grossman et al.,2004; Mintz et al., 1997). Thus, it is likely that serotonergicmodulation through 5-HT3A receptors in the MRN plays a rolein circadian rhythm control; however, studies investigating

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the colocalization of this receptor with other neural systemswithin the MRN are necessary to elucidate the specific rolethis receptor plays in the serotonergic control of circadianrhythms.

3.3. Anxiety network

The potential anxiolytic effect of 5-HT3 receptors has led togreat efforts to understand the neuralmechanisms underlyingthe role of this receptor in brain components of the anxietynetwork (Bill et al., 1992; Costall et al., 1993c; Costall andNaylor, 2004; Delagrange et al., 1999; Gargiulo et al., 1996;Olivier et al., 2000; Rodgers et al., 1995; Roychoudhury andKulkarni, 1997). To date, however, great controversy andquestions surround the localization as well as the functionalimportance of this receptor in brain areas that comprise theanxiety network. The current data indicate that in the Syrianhamster 5-HT3A receptors are widely expressed in the mainbrain regions belonging to the Papez circuit including the BLA,CeA, MeA, BNST, PFC, Cg AT, hypothalamus, septum, hippo-campus, PAG and DRN. Although the primary brain areainvolved in 5-HT3A-mediated regulation of anxiety behavior isstill unknown, converging evidence underscores the functionof certain brain areas (i.e., hippocampus, amygdala andhypothalamus) within the anxiety network as particularlyimportant for 5-HT3-dependent control of this behavior(Bhatnagar et al., 2004; Bhatnagar and Vining, 2004; Eggers,2007; Gargiulo et al., 1996; Hensler et al., 2004; Plaznik et al.,1996; Stefanski et al., 1993). In most of these brain regions, 5-HT3 receptors are not only highly co-localized with GABAergiccells but also serotonergic excitation of GABAergic cells isprimarily mediated through 5-HT3A receptors (Koyama et al.,2002; McMahon and Kauer, 1997; Morales and Bloom, 1997).For example, in the hippocampus, modulation of GABAergicactivity through 5-HT3A receptors is critical for long termpotentiation (LTP) (Gargiulo et al., 1996; Reznic and Staubli,1997; Staubli and Xu, 1995). Indeed, blockade of 5-HT3A

receptors results in reduced firing rates of hippocampalinterneurons, increased activity of pyramidal cells andaugmented LTP (Reznic and Staubli, 1997; Staubli and Xu,1995), and thus facilitating learning and memory. Theseresults are consistent with the punctate staining observedthroughout various hippocampal areas reported here. How-ever, in the hippocampus and most other areas of the anxietynetwork, 5-HT3A immunoreactive patterns were highly di-verse suggesting that 5-HT3A modulation of anxiety behavioris dependent on more than one signaling mechanism.

3.4. Drug addiction network (mesocorticolimbic system)

The current report highlights 5-HT3A immunopositive stainingpatterns in the mesocorticolimbic system (i.e., PFC, NAc,amygdala and VTA), suggesting that this receptor mediatesneural mechanisms underlying drug addiction. These resultsare consistentwith previous localization of this receptor to themesocorticolimbic system in humans and rats (Kalivas et al.,2001; Kilpatrick et al., 1987, 1988a; Laporte et al., 1992; Moraleset al., 1998b). Of particular interest is the clear and oftenstriking 5-HT3A-ir patterns observed in the NAc. Similar toprevious reports in the rat brain (Ricci et al., 2004d),

heterogeneous 5-HT3A receptor labeling within the hamster'sNAcwas observed, permitting the visualization not only of thecore and shell division but also subdivisions of the NAc shell,subregions previously identified using tyrosine hydroxylaseimmunoreactivity in the rat (Todtenkopf and Stellar, 2000).

The expression of 5-HT3A receptors within this brain regionis not surprising given that these receptors have beenimplicated in mechanisms underlying drug-induced with-drawal (Costall et al., 1990, 1993a), reward (Suzuki et al., 1992)and tolerance and sensitization (Ricci et al., 2004d). In addition,5-HT3A receptors have been shown to modulate dopamine-induced behaviors (Carboni et al., 1989b; Costall et al., 1987;Jiang et al., 1990; Reith, 1990) which are often associated withthe major reward pathway (i.e., the mesolimbic pathway).Indeed, local administration of 5-HT3 receptor agonistsincreases dopamine levels in the NAc (Campbell and McBride,1995; Jiang et al., 1990). Moreover, in vivo localization of 5-HT3

receptors within the NAc suggests that these receptors arepresynaptically expressed on dopamine terminals directlystimulating dopamine release (Chen et al., 1991b). In fact,studies employing selective 5-HT3 receptor antagonists (i.e.,ondansetron or (S)-zacopride) report significant reductions indorsal raphe-induced NAc-dopamine release further impli-cating a role of 5-HT3 receptors in the modulation of reward(De Deurwaerdere et al., 1998). In addition to 5-HT3A-dopamineinteractions in the NAc, it is likely that 5-HT3A receptors in thisbrain region also interact with the GABAergic system, giventhat medium spiny GABAergic cells comprise more than 80%of the NAc cell types (Morales et al., 1996b). Together, thesedata implicate 5-HT3A receptors in neuronal signaling under-lying drug addiction.

In summary, the current study replicates findings fromvarious neurobiological assays detailing the expression of 5-HT3A receptors across the neuraxis of several species. Notably,in the Syrian hamster, awide andheterogeneous expression ofthis receptor was observed in neuronal networks involved inthe regulation of social behaviors, circadian rhythm, anxietyand drug addiction. Nevertheless, considerable variability in 5-HT3A receptor density and expression patterns (i.e., perikarya,fibers and/or neuropilar puncta) was observed between brainregions part of each of the neuronal networks examined.Moreover, rostrocaudal changes in expression patterns and/orreceptor density were observed in some brain areas. Together,these data not only implicate 5-HT3A receptors in themodulation of several neurobiological functions but it alsoindicates a diverse functional capability of 5-HT3A receptorsacross the central nervous system. Implications for thefunctional significance of these findings may be relevant inseveral fields of research and underscore the importance ofexamining this receptor as a possible pharmacological targetfor the treatment of various psychopathological disorders.

4. Experimental procedures

4.1. Animals and tissue preparation

Adult male Syrian hamsters (n=10), were obtained fromCharles River Laboratories (Wilmington, MA). Hamsters (115–125 g) were deeply anesthetized with Ketamine/Xylazine

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(80 mg/12 mg) general anesthetic and transcardially perfusedwith a 21 °C saline rinse followed by fixative solutioncomprised of 4% paraformaldehyde, 0.2% picric acid and0.4% glutaraldehyde. Brains were removed, post-fixed for90 min in perfusion fixative and cryoprotected in 30% sucrosein distilled water at 4 °C overnight. Brains were then cut into35 µm sections using a freezing microtome in serial, coronalsections. This work was produced with an animal careprotocol approved by The Animal Care and Use Committee(IACUC) at Northeastern University and allmethods usedwereconsistent with the guidelines provided by the NationalInstitutes of Health for the scientific treatment of animals.

4.2. Immunohistochemistry

The 5-HT3A receptor polyclonal antibody was raised in rabbitsimmunized against the rat amino acid sequence (SLEKRDEM-REVARD) and conjugated to KLH using an N-terminal cystine(5-HT3A receptor (AB-1) polyclonal Oncogene, Darmstadt,Germany). This antibody has been well characterized (Moraleset al., 1996a,c) and used previously in several extensiveimmunohistochemical studies of 5-HT3A receptor localizationin the rodent nervous system (Morales et al., 1996a,c, 1998a).

All immunohistochemical procedures were conducted at21 °C. Sections were washed 3×10min in 0.6% Triton-X in0.1 M phosphate buffered saline (PBS, pH 7.4) (PBSTx), thenpretreated with 4.5% H202 in distilled water for 8 min, andrinsed thoroughly with 0.1 M PBS. Sections were then pre-treated with 1% sodium borohydride in distilled water for5 min, rinsed thoroughly with 0.1 M PBS and incubated inantibody buffer comprising 10% normal goat serum and 1%bovine serum albumin (BSA) in 0.6% PBSTx for 90 min. Primaryantibody was prepared in antibody buffer diluted to finalconcentration of 1 µg/ml and incubation with free-floatingbrain sections was carried out overnight at 21 °C on a slowlyheating rotation wheel (final temperature=28 °C). Sectionswere then washed 3×10 min with 0.6% PBSTx, incubated for90 min in biotinylated secondary goat anti-rabbit IgG (VectorLaboratories, Burlingame, CA) in 0.6% PBSTx and 1% BSA,washed again 3×10 min in 0.6% PBSTx and incubated for90 min in Avidin–biotin complex (Vectastain ABC kit; VectorLaboratories, Burlingame, CA) with 1% BSA in 0.6% PBSTx. Theperoxidase reaction was revealed using 0.5% 3,3′-diamino-benzidine in distilled water as per manufacturers' recommen-dations (DAB kit; Vectastain; Vector Laboratories, Burlingame,CA). The sections were mounted on gel-coated slides, air-dried, dehydrated through a series of alcohol solutions,cleared with xylene and coverslipped with Cytoseal (StephensScientific, Kalamazoo, MI).

4.2.1. ControlsExperimental controls were performed on representativesections during the immunohistochemical procedure de-scribed above. These included omission and preadsorption ofthe primary antibody and omission of the secondary antibody.Briefly, the preadsorption control consisted of incubating the5-HT3A receptor peptide antigen (Anaspec Inc. San Jose, CA) at10×, 100× and 1000× molar excess of the primary antibodyat 4 °C for 90 min prior to use in the immunohistochemicalprotocol.

4.3. Light microscopy

The qualitative analysis of 5-HT3A immunoreactive (5-HT3A-ir)cell bodies, fibers, and puncta was determined in brain areasusing the BIOQUANT NOVA 5.0 computer-assisted microscopicimage analysis software package as previously described(DeLeon et al., 2000, 2002). The areas analyzed were selectedand identified using the Hamster Stereotaxic Atlas (Morin andWood, 2001). BIOQUANT NOVA 5.0 image analysis softwarerunningonaPentiumIIICSIOpenPCcomputer (R&MBiometrics,Nashville, TN, USA) was utilized to identify the brain regions ofinterest (ROI) at low power (4×) using a Nikon E600 microscope.

Then, under 20×magnification, 5-HT3A-ir cell bodies, fibers,and puncta were identified in each field and 5-HT3A-ir patterndescriptions were recorded. Descriptions at 20× continueduntil 5-HT3A-ir elements throughout the entire ROI wereexamined. This procedurewas used to verify staining patternsat 40× and oil immersion at 100× as needed.

4.4. Brain regions analyzed

Serial coronal sections of thehamster brainwere cut beginningat the anterior olfactory nucleus at the level of the ependymaand subependymal layer continuing through and concludingat sections containing rostral portions of the dorsal andmedialraphe nuclei. Two to four independent analyses for each brainarea were made from several consecutive sections from eachanimal. Descriptions of 5-HT3A-ir included in the analysiswereconsistent across animals.

4.5. Qualitative descriptions

5-HT3A-ir was qualitatively analyzed using three categoriesof description: (1) cell body labeling (2) fiber labeling and(3) punctate labeling (i.e., fiber terminals). Cell body labelingwas observed as either dense punctate signal comprising awell-defined outline of the perikarya or as opaque labeling ofthe cytoplasm sparing the nucleus. Fibers were observed aseither fine, smooth, hair-like structures or appeared moretextured or varicose in nature. The qualitative description ofpunctate immunoreactivity (ir) was used to describe thepresence of symmetrical round-shaped immunoreactive ele-ments in neuropil regions in the absence of any grossly visiblecell bodies or fibers associated with them. In addition to thequalitative description of 5-HT3A expression patterns, thecurrent report assigned a number to denote relative density ofreaction product for each area observed (Table 1). The numberone (1) indicated that the immunohistochemical localizationof the receptor appeared sparse, describing the lowest relativedensity of labeling patterns. The numbers two (2) and three(3) indicated comparatively more reaction product with(3) describing the most dense staining patterns.

Acknowledgments

This publication was made possible by Grant (RO1) DA10547from NIDA to R.H.M. Its contents are solely the responsibilityof the authors and do not necessarily represent the officialviews of NIDA.

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