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Behavioural Brain Research 218 (2011) 288–295

Contents lists available at ScienceDirect

Behavioural Brain Research

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esearch report

mygdalar orexinergic–GABAergic interactions regulate anxiety behaviors of theyrian golden hamster

nnio Avolio, Raffaella Alò, Antonio Carelli, Marcello Canonaco ∗

omparative Neuroanatomy Laboratory of Ecology Department, University of Calabria, Ponte Pietro Bucci 4b, 87030 Arcavacata di Rende, Cosenza, Italy

r t i c l e i n f o

rticle history:eceived 5 July 2010eceived in revised form 22 October 2010ccepted 5 November 2010vailable online 11 November 2010

eywords:rexinABAnxietylevated plus mazeight–dark exploration testmygdala

a b s t r a c t

At present neurobiological interests are directing more attention towards the major role of the amyg-dalar GABAA receptor on orexin-dependent behaviors. This telencephalic region has been widely studiedespecially in view of its control on various psychiatric disorders such as anxiety and depression. Recently,cross-talking relationships between these two specific neuroreceptor systems of the central-corticalamygdalar complex has been considered an important element for anxiety type of behaviors. In thepresent study, we investigated the effects of central amygdalar infusions with orexin-A, orexin-B ± GABAA

receptor �2 subunit agonist (flunitrazepam) on elevated plus-maze and light–dark explorative behaviorsof the facultative hibernating Syrian hamster. In a first case, it seemed that doses of orexin administereddirectly into the central nucleus were responsible for greater anxiogenic type of effects as shown bymore time being spent both in the dark compartment and the closed arm of the elevated plus-maze,whereas, these effects were suppressed in the presence of flunitrazepam. At the cellular level, the effects

of orexin accounted for evident argyrophilic reactions (neurodegeneration phenomena) including alteredcell membrane and loss of cytoplasmic architecture in most amygdalar and hippocampal neuronal fields,while in the presence of flunitrazepam these reactions resulted to either be unappreciable or absent.Overall the actions of �2-dependent inhibitory signals tend to corroborate, for the first time, a neuropro-tective role against the over-excitatory orexinergic neurodegeneration reactions and thus its abnormalanxiety-like indications may prove to be therapeutically useful for orexin-dependent sleeping disorders.

. Introduction

The family of orexin (ORX) neuropeptides also known asypocretins consists mainly of two subtypes recognized as Orexin-

and -B (ORX-A and ORX-B), which derive from a commonrecursor prepro-orexin following post-translational proteolyticleavage [51]. Studies have largely shown that ORX producing neu-ons are localized in the lateral hypothalamic area (LHA) and inosterior portions of the hypothalamus [40]. Both neuropeptidesere first identified as novel peptide ligands of two orphan Grotein-coupled receptors and namely ORX1 receptor (ORX1R) andRX2 receptor (ORX2R). In the case of the former subtype it dis-lays a greater affinity for ORX-A (almost 50 times greater than) while ORX2R shows a comparable affinity for both neuropep-

ides [51]. At the brain level such ORX producing neurons widelyroject to various limbic regions among which the cerebral cor-ex, olfactory bulb, hypothamamic areas, hippocampus (HIP) andmygdala (AMY) [40]. The activities of these two orphan G protein-

∗ Corresponding author. Tel.: +39 0984 492974; fax: +39 0984 492986.E-mail address: canonaco@unical.it (M. Canonaco).

166-4328/$ – see front matter. Published by Elsevier B.V.oi:10.1016/j.bbr.2010.11.014

Published by Elsevier B.V.

coupled receptors appear to require the participation of severalother neuronal receptor systems such as serotonin [34], nora-drenaline [23], histamine [15], corticotrophin-releasing hormone(CRH) [55] and above all GABAA receptor (GABAAR)/glutamate sub-types [1,38]. As far as the major neuroinhibitory GABAAR complexis concerned, the benzodiazepines, which are a highly noted fam-ily of anxiolytics are capable of mediating their sedative, amnesicand anxiolytic actions through the binding of the � subunits ofthis receptor complex [28,29]. In particular, �2/�3 appear to bethe major subunits involved with the regulation of anxiety-likebehaviors [46]. In addition, it appears that the inhibitory GABAer-gic effects are mostly responsible for the blocking of AMY ORXergicfibers and consequently a decrease of anxiety state [26]. Consistentwith this, reduced central GABAergic activity has been reportedin subjects with panic disorder, and drugs like �2/�3 GABAergicsubunit agonist that are involved with the restoration of inhibitionresponses have proven to be clinically effective for similar disor-

ders. As a matter of fact, acute disruption of GABAergic signals inpanic-generating brain sites such as the dorsomedial–perifornicalhypothalamus, the AMY or the dorsal periaqueductal gray inrats, leads to panic-like behavior and increased cardiovascularresponses [45].

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Fig. 1. Dorsal views of the skull of a 150 g male Syrian golden hamsters. The positionof the guiding cannula between the horizontal planes passing through lambda lead-

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Recent behavioral studies have displayed that stressful con-itions tend to alter ORXergic-dependent awakening processes2,26]. Indeed, this specific neuropeptide is known to control somef the major circadian rhythms including feeding and sleep-wakeycle underlining that ORXergic fibers are widely associated withhe maintenance of homeostasis [4,33,48]. Interestingly, ORXer-ic neurons have shown to be very sensitive as exhibited by theirbility to activate arousal promoting transmitters, including theorticotrophin releasing factor (CRF), a peptide involved in stressesponses [59]. The increased activity of ORX seems to cause areater frequency of awakening state that could contribute toeveral neuropsychological functions such as vulnerability drugeeking events [12]. Consequently, the blocking of ORX1 attenuatestress-induced reinstatement of extinguished cocaine [7] and alco-ol [47] seeking stimuli. ORX-synthesizing neurons have also beenhown to be involved in not only mobilizing sympathetic responseslus desensitizing parasympathetic mediated baroreflex stimuli,ut also a simultaneous increases of blood pressure and heart rate,hich are typical of panic attacks [26].

At the brain level, it seems that most of environmental-derivedtimuli are responsible for the activation of distinct encephalicRXergic fibers [52] very likely through the major limbic olfactory

arget and precisely AMY. This telencephalic region is composedf different anatomically interconnected amygdaloid nuclei thatnclude basolateral (BLA), cortico-medial and centromedial nuclearroups, [44,48] which are capable of influencing emotional andnemonic functions [17], vary likely through extensive visceral

hypothalamus and olfactory lobes) and autonomic-somatomotoronnections [50]. Even the central AMY nucleus (Ce) that is involvedith aversive learning, escaping responses and mnemonic capa-

ilities of stressful experiences [3,32] has been identified as aritical site for the mediation of responses to anxiogenic stimuli ortress-related behaviors [41,52] deriving from elevated-plus mazeEPM), shock probe burying, and social anxiety tests [53]. Curiously,umerous evidences have demonstrated that Ce is mostly inner-ated by ORX neurons as displayed by dense levels of ORX1R andRX2R fibers throughout this AMY nucleus [60]. As a consequencef this feature, plus Ce not only being largely responsible for stress-ul type of behaviors via its interaction with BLA [49] but also of iteing directly involved with learning, feeding and fear behaviors31] constitutes such an AMY nucleus as an important limbic siteor emotional events.

On the basis of these features it was our intention to evaluatehe role of intracerebroventricular (i.c.v.) ORX-A infusions alone orogether with �2 subunit agonist flunitrazepam, in specific AMYites on anxiety-like behaviors of a facultative hibernating rodenty using light–dark exploration (LDT) and EPM tests. These effectsere compared to animals treated with ORX-B especially since thiseuropeptide has recently shown to play a key role on synapticlasticity [5,54]. The results of predominating and distinct effectsxerted by both neuropeptides may supply further insights regard-ng the role of olfactory communicating channels operating viaMY stations on plasticity events that are known to be graduallyut constantly changing.

. Materials and methods

.1. Animals

In the present study, male Syrian golden hamster (Mesocricetus auratus; Charlesiver, Como Italy) weighing 130–160 g at the time of surgery were housed two

er cage, maintained under a 12:12 h light/dark cycle (lights on 07:00) at roomemperature (23 ± 1 ◦C) and had free access to food and water so that they werellowed to adapt to their new laboratory conditions for at least 2 weeks beforeurgery. Hamsters were handled about 3 min each day prior to behavioral testing.ll experiments were performed between 9:00 h and 15:00 h and each hamster

n = 5) was tested only once.

ing to the amygdalar target site, i.e. Ce (AP: +3.6 mm, ML: +3.6 mm, DV: −5.4 mm).Abbreviations: Ce, central amigdaloid nucleus; La, lateral amigdaloid nucleus; BL,basolateral amigdaloid nucleus; BLV, basolateral amigdaloid nucleus ventral; BM,basomedial amigdaloid nucleus; CoMe, corticomedial amigdaloid nucleus.

Animal maintenance and all experimental procedures were carried out inaccordance with Guide for Care and Use of Laboratory Animals issued by the Euro-pean Communities Council Directive of 24 November 1986 (86/609/EEC). Effortswere made to minimize animal suffering and reduce the number of experimentsused.

2.2. Stereotaxic surgery and microinjections

All behavioral studies were handled on hamsters were anesthetized intraperi-toneally with ketamine hydrochloride (50 mg/kg) and xylazine (4 mg/kg) andplaced in a Stoelting stereotaxic instrument. The stainless steel guide cannula(CMA/Microdialysis AB, Stockholm, Sweden) was stereotaxically implanted bilat-erally directed towards and 1 mm above the dorsal border of the Ce (coordinatesrelative to lambda: AP: +3.6 mm, ML: +3.6 mm, DV: −5.4 mm; Fig. 1) according tothe hamster stereotaxic atlas [37]. It was then fixed to the skull with acrylic dentalcement and the animals were allowed 7 days to recover before the test. Three groupsof hamsters received a microinjection of 1 �l containing ORX-A (20 nM), or ORX-B(60 nM), or flunitrazepam (100 nM) alone while other hamsters received a double

treatment of ORX-A/B + flunitrazepam with respect to controls that received 1 �l ofsaline every day in the morning, 30 min before the first behavioral test. The innercannula was left in place for an additional 60 s to allow diffusion of the solution andto reduce the possibility of reflux. At the end of the study, injecting 1 �l/hamster of1% methylene blue solution and determination of the injected dye in left Ce, identi-fied and verified the injection site. Approximately 5–10 min after the last behavioral

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bservation, the animals were decapitated and their brain were removed, blockednd cut coronally throughout cannula placement.

.3. Drugs

The drugs used in the present study were ORX-A 20 nM, ORX-B 60 nM (Peptidenstitute Inc., Osaka, Japan), and GABAA agonist �2 subunit, flunitrazepam 100 nMSigma–Aldrich, Dorset, UK). All drugs were dissolved in sterile 0.9% saline. Controlsnly received sterile 0.9% saline. Peptide solutions were freshly prepared before allests.

.4. Behavioral tests

.4.1. Light–dark exploration test (LDT)In a first case, ORXergic-dependent behavioral effects were evaluated by using a

DT apparatus, which consisted of a box with two compartments: a first arena com-osed of a small and dark opaque familiar compartment (16 cm × 16 cm × 16 cm)ontaining a black glass while the second arena containing a large translucent andhite illuminated compartment (25 cm × 25 cm × 30 cm), considered the unfamiliar

nvironment. The floor of the white illuminated compartment is divided into 9 iden-ical squares to allow determination of exploratory behaviors. The compartmentsere connected by a communicating door (7 cm × 7 cm), which allowed hamsters to

reely move from one compartment to the other. A spotlight illuminated the appa-atus so that it was able to provide an aversive stimulus (250 lx), which was onlyisible from the light compartment. A camera (Logitech QuickCam Pro5000) wasositioned 1 m above the apparatus and connected to a computer to record hamsterehaviors during the test. The evaluations of anxiety were subsequently elaboratedt a computed-assisted video recorder.

Seven days after having implanted the cannula, hamsters are tested on LDT forconsecutive days in morning, noon and afternoon. Prior to the behavioral observa-

ions, animals were subjected to a brief period of habituation in the LDT apparatus.he following day, each animal of all experimental groups received 30 min prior tohe behavioral test 1 �l of drugs (ORX-A or ORX-B ± flunitrazepam) plus controls,ubsequently the hamster was placed in the center of the light compartment withheir head opposite to the opening and was allowed to explore the two compart-

ents for 5 min. During this period, the time the hamster spent in either the familiarr unfamiliar compartment, along with the number of crossings between the twonvironments were recorded. The advantage of using this apparatus is linked tohe presence of an unfamiliar environment so that the hamster is placed in frontf a natural conflict deriving from the necessity to explore a novel environmentspontaneous exploratory behavior) and the innate aversion to an open field andrightly illuminated arena. In this latter case, it was in the presence of light andot in darkness that were responsible for the induction of stress-like conditions

or hamsters, as shown by the percentage of time spent in the dark compartment.n addition, the exploratory behavior in the light compartment (time spent in theight compartment) was also used as an indicator of anxiety. Similarly the numberf crossings between compartments provides an estimation of the anxiety level ifeasured without changes of spontaneous locomotor activity [6].

.4.2. Elevated plus-maze testIn the other behavioral study using EPM, it was possible to evaluate in a greater

anner anxiety responses, as previously described by Pellow and File [42]. EPM is aooden, cross-shaped maze, consisting of four arms arranged in the shape of a plus

ign. Two of the arms have no side or end walls (open arms; 50 cm × 10 cm). Thether two arms contain lateral and end walls, but are open at the top (closed arms;0 cm × 10 cm × 40 cm). A square platform (10 cm × 10 cm) is located at the pointf intersection of the four arms intersect. The apparatus was elevated to a heightf 50 cm above the floor and illuminated by white lamps (4 × 60 W). Seven daysfter having implanted the cannula, hamsters are tested on EPM for 3 consecutiveays in morning, noon and afternoon. Prior to the behavioral observations, animalsere subjected to a brief period of habituation in the EPM apparatus. The followingay, each animal of all experimental groups as above received 30 min prior test�l of drugs and subsequently their anxiety plus locomotor bouts were tested inPM. A camera (Logitech QuickCam Pro5000) placed 1 m above the apparatus andonnected to a computer was used to record hamster behaviors during the entireest. The number of entries into open arms, the number of entries into closed armsnd the total time spent in either the open or closed arms were measured. Entryas defined as the amount of times each hamster placed all four paws in the arms.

he percentage of open arm entries and open arm times that are used as standardnxiety indexes were calculated as follows: (a) %OAT (the time ratio spent in thepen arms to total time spent in any arm × 100); (b) %OAE (the ratio of entries intopen arms to total entries × 100); (c) total closed arm entries were measured as aelative pure index of motor activity.

.5. Neurodegeneration study

The possibility that the different behavioral and molecular alterations wereaused by some ORXergic or GABAergic dependent neurodegeneration events washecked by amino cupric silver stain (ACS) method. This staining technique isargely used for detecting both necrosis as well as apoptotic-like degeneration [9].

esearch 218 (2011) 288–295

Indeed ACS method is a valuable tool since it allows a selective analysis of bothearly and semi-acute neurodegeneration events in which not only advanced dam-aged cell bodies, dendrites, axons and terminals are readily visualized but also therecruitment of new structures in progressive pathologic processes against a clearbackground [13]. For ACS method some of the hamsters belonging to the sametreatment of the behavioral study, were sacrificed by decapitation after the finalbehavioral observation session. Subsequently, the brain was quickly removed andstored at −40 ◦C according to common cryostat procedures for unfixed brains [10].Brains were mounted on a freezing stage of a sliding cryostat (Microm-HM505E;Zeiss, Wallford, Germany), and a serial set of representative coronal sections (30 �m)was selected at an interval of 240 �m for ACS procedures in which an exposi-tion period (25 min) to neutral red for the different brain sections was adapted forour animal model. Afterwards, sections were rinsed with distilled H2O, placed intodishes containing a pre-impregnating solution (silver nitrate [AgNO3], distilled H2O,d,l-alanine, copper nitrate [Cu(NO3)2], cadmium nitrate [Cd(NO3)2], lanthanumnitrate [La(NO3)2], neutral red, pyridine triethanolamine, isopropanol), heated in amicrowave oven 45–50 ◦C) for 50 min, and cooled at room temperature for 3 h. Thesections were then rinsed in distilled H2O, and after a quick rinse in acetone theywere placed in an impregnating solution AgNO3, distilled H2O, ethanol, acetone,lithium hydroxide (LiOH), ammonium hydroxide (NH4OH)] for 50 min, followed bya 25 min fixation in a reducer solution (formalin, citric acid monohydrate, ethanol,distilled H2O) at a temperature range of 32–35 ◦C. These sections were left overnightin distilled H2O, while the next day they were placed in a first bleaching solution[potassium ferricyanide in potassium chlorate solution, lactic acid] for 60 s at roomtemperature and left in a second bleaching solution (potassium permanganate, sul-furic acid) for 60 s and rinsed in distilled H2O. For the stabilization phase, sectionswere transferred in sodium thiosulfate solution and rinsed again in distilled H2O.Finally, they were immersed in a rapid fixer solution for 5 min and counterstainedwith 0.5% neutral red solution (Carlo Erba, Milan, Italy) for 25 min, dehydrated inethanol (50–100%) and xylene, and mounted with DPX (p-xylene-bis[N-pyridiniumbromide]; Sigma, Milano, Italy) for observations with a bright-field Dialux EB 20microscope (Leitz, Stuttgart, Germany). For the estimation of the percentage of dam-aged neuronal fields in diencephalic and extra-diencephalic areas, it was necessaryto calculate the volume (defined as Vref) of areas such as the habenular nucleus, AMY,and HIP, using the following formula:

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In this case Nv represents the number of stained damaged neurons; N is the numberof damaged neurons in a single section; Vsection is the volume of a single section; nis the number of sections; Vref is the total volume of above brain regions [20].

2.6. Statistical data analysis

All anxiety and motor activities (means ± S.E.M.) were evaluated by two-wayanalysis of variance (ANOVA) and subsequently, when appropriate (p < 0.05), aNewman–Keul’s multiple range post hoc analysis was performed for intra-specificvariations when p < 0.05. In the case of the neurodegeneration reaction the effectsof both ORX-A and ORX-B ± flunitrazepam, expressed as a percentage with respectto controls, were estimated and compared among the different treatment groups byalso using ANOVA followed a post hoc Newman–Keul’s multiple range test. *,ap < 0.05,**,bp < 0.01, ***,cp < 0.001.

3. Results

3.1. Anxiogenic-like behaviors promoted by ORX-A and -B

These facultative hibernating Syrian hamsters that receiveda microinjection of 1 �l of ORX-A (20 nM) or ORX-B (60 nM) inthe amygdalar Ce every morning 30 min before first behavioraltest, exhibited differentiated anxiogenic-like behaviors. In a firstcase, a very great (p < 0.001) reduction of hamsters entering intoOA [F(2;11) = 3.97] resulted to be preferentially evident for ORX-A-treated animals over controls (−91%) as shown by animals enteringEPM apparatus at a very low frequency above all with respect toORX-B-treated animals (−127%; Fig. 2A). When both neuropep-tides were next tested on temporal permanence in OA of EPMapparatus [F(2;11) = 4.02, p < 0.05], it seemed that ORX-B-treatedhamsters spent moderately less time (−43%) in the open-arms

with respect to both controls, whereas this effect turned out tobe of a very great nature (−115%) with respect to ORX-A-treatedanimals (Fig. 2B). It is worthy to note that neuropeptide B wasstill responsible for consistently increased effects of motor activ-ity [F(2;11) = 3.91; p < 0.05] as displayed by hamsters spending a

E. Avolio et al. / Behavioural Brain R

Fig. 2. Effects of Ce AMY i.c.v. injection of hamsters (n = 5) with ORX-A and -B. Forthis part animals were injected with either ORX-A (1 �l/hamster; 20 nM) or ORX-B(1 �l/hamster; 60 nM) and tested in both EPM (A–C) and LDT (D). The test was per-formed 30 min after i.c.v. injection. Each bar represents % mean ± S.E.M. of OAE (A),OAT (B), back- and forward motor activities (C) or time spent in the dark box (D) withrespect to controls that received saline solution. The differences were evaluated byANOVA plus a post hoc Newman–Keul’s test, *,ap < 0.05, **,bp < 0.01 and ***,cp < 0.001.

esearch 218 (2011) 288–295 291

great (+75%) amount of time passing from open to closed armswith respect to controls, while ORX-A treatment only accountedfor a moderately great (+58%) of time passing through both arms(Fig. 2C). Conversely, it was ORX-A that preferentially operatedon animals maintained in LDT [F(2;11) = 7.40, p < 0.01] as shown byhamsters remaining a greater (p < 0.01) amount of time (+75; +85%)in the dark compartment when compared to both their controls(Fig. 2D) and to ORX-B-treated animals, respectively, while ORX-B-treated hamsters supplied a weaker response as displayed by amoderate permanence (+40%) in this dark compartment.

3.2. Effects of flunitrazepam on ORX-A induced anxiolyticbehaviors

Surprisingly, when the effects of ORX-A were tested in thepresence of �2 GABAAR agonist (100 nM), the anxiogenic effectswere notably attenuated in hamsters evaluated in all behavioraltests. Conversely, the permanence of hamsters in OAE [F(3;38) = 4.11,p < 0.05] appeared to greatly increase (+65) following the sequen-tial administration of flunitrazepam to ORX-A-treated animals(Fig. 3A). This highly selective benzodiazepine agonist appearedto rather induce anxiolytic effects as indicated by a notable %increase of OAT [F(3;38) = 4.11, p < 0.05] and thereby causing ham-sters to spend moderately more time (+48%) in the open-armswith respect to their control (Fig. 3B), while ORX-A alone inducedanimals to remain for a very less amount of time (−91%) inOAE (Fig. 3A). Similarly, even back- and forward movements[F(3;38) = 4.09, p < 0.05] were modified by this same treatment asindicated by great and moderately great increases in ORX-A (+61%)and ORX-A + flunitrazepam (+37%) treated hamsters, respectively,not only when compared to controls but also to animals thatreceived a double administration of ORX-A + flunitrazepam. Thelowering effects of �2 agonist appeared to preferentially play amajor role on motor activities as shown by moderate decreases(−35%) registered when hamsters only received flunitrazepam(Fig. 3C). Subsequently, the preference of ORX-A-treated animals[F(3;38) = 4.47, p < 0.01] to remain in LDT apparatus for a greateramount of their time (+78%) in the dark compartment with respectto controls resulted to be very greatly reduced (−110%) followingtreatment with flunitrazepam (Fig. 3D).

3.3. The effect of flunitrazepam on ORX-B induced anxiolyticbehavior

Similarly to ORX-A-treated animals, even the addition of the�2 subunit agonist to ORX-B-treated animals appeared to atten-uate, aside OAE parameter, anxiogenic effects for most tests. Inthis case, it appeared that hamsters treated concomitantly withORX-B + flunitrazepam [F(3;38) = 4.37, p < 0.01] showed a moder-ately great amount of time (+39%) entering into OA, in spiteof the great amount of time (+65%) caused by flunitrazepamalone (Fig. 4A). Of particular interest was the moderate increaseof amount of time spent entering into OA by hamsters treatedwith ORX-B + �2 subunit agonist, which turned out to be of verygreat entity (+203%) when compared to the moderately reduced(−40%) time spent by hamsters treated with ORX-B with respectto controls (Fig. 4A). Similarly, a moderate amount of time (+48%)spent in OA was only reported for flunitrazepam-treated ham-sters [F(3;38) = 4.41] with respect to controls, while the combinedtreatment still continued to exhibit a tailing down of time spentin OA although not in a significant manner with respect to the

moderately reduced (−41%) amount of time detected for ORX-Btreated hamsters (Fig. 4B). In line with the above results, even% locomotor activities tended to increase [F(3;38) = 4.50, p < 0.01]as exhibited by greater (+75%) ORX-B-dependent back- and for-ward movements while flunitrazepam accounted for a moderately

292 E. Avolio et al. / Behavioural Brain Research 218 (2011) 288–295

Fig. 3. Effects of Ce i.c.v. injection with ORX-A, flunitrazepam alone or in combina-tion (ORX-A + flunitrazepam) on hamsters (n = 5) in order to establish if such drugtreatments motivated animals to prefer transiting from the closed arm chamberto the open arm of EPM apparatus or spent more time in LDT dark compart-ment. For this part hamsters received either a single infusion [ORX-A, 1 �l/hamster(20 nM) or flunitrazepam, 1 �l/hamster (100 nM)] or a combination of both ORX-A + flunitrazepam volumes and compared to control (animals infused with onlysaline solution. The test was performed 30 min after i.c.v. injection. Each bar repre-sents % mean ± S.E.M. of OAE (A), OAT (B), back- and forward motor activities (C) ortime spent in the dark box (D) with respect to controls that received saline solution.The variations induced by above treatments were evaluated in a same manner tothat in Fig. 2.

Fig. 4. Effect of Ce i.c.v. injection of hamster (n = 5) with ORX-B or flunitrazepam ordouble treatment with ORX-B + flunitrazepam. Animals were injected with eitherORX-B (1 �l/hamster; 60 nM) or flunitrazepam (1 �l/hamster; 100 nM) or ORX-B + flunitrazepam (1 �l/hamster, 60 nM; 1 �l/hamster, 100 nM) and tested in bothEPM (A–C) and LDT (D). The test was performed 30 min after i.c.v. injection and eachbar represent % mean ± S.E.M. of %OAE (A), %OAT (B), % motor activites (C) or timespent in the dark box (D) with respect to controls that received saline solution. Thedifferences induced by above treatments were evaluated in a same manner to thatin Fig. 2.

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ecreased (−35%) activity. However, such a reducing type of effectf this �2 subunit agonist proved to be responsible for a moderatelyreater (+44%) locomotor activity for animals that received ORX-+ flunitrazepam, which in any case supplied a greatly reduced

−70%) activity with respect to hamsters that were given ORX-BFig. 4C). On the other hand, in spite of ORX-B having influenced theehavior of hamsters maintained in LDT [F(3;38) = 4.50, p < 0.01] asisplayed by hamsters spending a moderate amount of time (+38%),his permanence was then very strongly reduced (−150%) whenamsters were treated with ORX-B + flunitrazepam (Fig. 4D).

.4. Neurodegeneration analysis

The varying behavioral events of both ORXergic neuropeptideseemed to be strongly correlated to altered neuronal responses asupported by evident neurodegeneration activities in telencephalicreas that are involved with the execution of motor and emo-ional behaviors. Indeed, by using ACS approaches it was possibleo highlight distinct damages of neuronal fields, via argyrophiliceactions in both ORX-A and -B-treated animals (Fig. 5). In partic-lar it was possible to observe, despite a mild neurodegenerationf most telencephalic areas, consistently dark perikarya, dendritesnd axons after treatment with ORX-A and -B with respect toontrols above all in lateral habenular nucleus, HIP (Fig. 5b–e)nd AMY (Fig. 5g–j). It was interesting to note that a significantlyvident neurodegeneration response [F = 3.97] was obtained

(2;11)n the habenular nucleus as shown by a great level in animalsreated with ORX-B (90%) while a moderate level was typical ofRX-A (48%) treated hamsters (Fig. 5b, c, and k) with respect to

he few if any signals in controls (Fig. 5a and f). Similarly for

ig. 5. Neurodegeneration processes in some brain areas of the hamster. Representativndicate damaged neurons showing the different levels of neurodegeneration as reportedg) and -B (h; n = 5) along with habenular nucleus, HIP (d and e) and AMY (i and j) of ORX-o (a and f) controls (n = 5). Scale bar of (a, b, and g) = 60 �m; scale bar of (f, c–e, and h–j)f Mesocricetus Auratus exposed to ORX-A, -B, flunitrazepam and double injection of ORXeuronal damages was also handled in hamsters treated with ORX-A or -B with respect toeurons ± S.E.M. The statistical analysis was conducted applying ANOVA plus post hoc tes

esearch 218 (2011) 288–295 293

AMY (Fig. 5g–j and k), it was ORX-B treated animals that stillsupplied a very great neurodegeneration activity (122%) whilea great reaction was detected in animals that received ORX-A(75%), whereas only a moderate reaction was reported for HIP(ORX-A = 56%; ORX-B = 68%; Fig. 5b, c, and k) when compared tothe lower signals supplied by controls (Fig. 5a, f, and k). How-ever, when these neuropeptides were injected concomitantly withflunitrazepam in the same brain areas that revealed strong neu-rodegeneration processes, this benzodiazepine agent exerted anevident neuroprotective activity as shown significantly lower ACSsignals [F(3;38) = 4.50]. Even in this case it was AMY neuronal fieldsof ORX-B + flunitrazepam treated hamsters that provided a verygreat reduction (−240%) of ACS signals with respect to a somewhatnumerically lesser density of positive ACS neurons (−105%) in thesame neuronal fields of ORX-A + flunitrazepam treated hamsters(Fig. 5i, j, and k). The same trend was also detected in the habenu-lar nucleus of ORX-B + flunitrazepam treated hamsters (−78%) thatdisplayed a great reduction of ACS signals while only a moderatereduction was reported for ORX-A + flunitrazepam (−32%) treatedhamsters (Fig. 5d, e, and k). In the case of HIP neuronal fields,only ORX-B in the presence of flunitrazepam provided a very greatreduction of neurodegeneration processes (−115%) as compared toonly a greater reduction (−70%) when flunitrazepam was added toORX-A-treated hamsters (Fig. 5d, e, and k).

e photograms of ACS (a–j) in which arrows point to dark neuronal perikarya thatfor habenular nucleus and HIP of ORX-A (b) and -B (c; n = 5) plus for AMY of ORX-AA + flunitrazepam and ORX-B + flunitrazepam (n = 5) treated hamsters with respect= 40 �m. Estimation of neurodegeneration (%) in habenular nucleus, AMY and HIP-A/-B + flunitrazepam with respect to controls. The evaluation of ORX-dependentORX-A/-B + flutirazepam and data expressed as the percentage (%) of degenerated

t (Newman–Keul’s test) when p-value ≤ 0.05. *,ap < 0.05, **,bp < 0.01 and ***,cp < 0.001.

The findings of the present study strongly underlie a distinctinteraction of Ce ORXergic and GABAergic neuronal systems oper-ating on both anxiogenic plus anxiolytic responses, respectively, of

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ur hibernating rodent model. The induction of anxiogenic statesvoked by ORXergic fibers of the Ce seems to be in good agreementith this same behavioral activity obtained for non-hibernating

odents [11]. It is worthy to note that anxiogenic-like effects foroth ORXs tested in the two separate apparatus are specificallyelated to ORXergic-dependent responses at least for our rodentodel. In line with such conditions, other works have already

emonstrated that both neuropeptides are capable of modifyingpontaneous motor activity or sleep–wake behavior in anotheribernating hamster like the Djungarian hamster [22] despite ORX-rather than ORX-A resulting to be particularly more effective on

hese continuously similar spontaneous motor states [57].The prevalent role of the former neuropeptide is further sup-

orted by a constant awakening state exhibited by hamstershroughout the various observation sessions. Consequently, itppears that ORX-B more than ORX-A exerts anxiogenic-likeffects, which bring us closer to the unraveling of the spe-ific ORX responsible for abnormal behavioral states such as theecent panic anxiety responses linked to dorsomedial–perifornicalypothalamic neuronal activities [26]. Such a condition fits nicelyith ORX-induced anxiety-like behaviors being also mediated byypothalamic related products such as CRH [24] since the hypotha-

amic periventricular nucleus of spontaneously hypertensive rats35], which releases this hyper-responsive hormone is densely pop-lated by ORX2R sites [11,39].

Interestingly, ORX-dependent anxiogenic effects declined fol-owing treatment with GABAAR �2 subunit agonist, above all forRX-B-treated animals. This type of interaction tends to go inn opposite direction to that of anxiolytical-dependent GABAer-ic interactions accounting for reduced ORXergic-related motorehaviors in non-hibernating rodents [21]. Similarly, hamstersreated with ORX-B and above all ORX-A spent more time in darkompartment of the LDT test with respect to that of animals treatedith only flunitrazepam, which seems to further corroborate a pre-ominance of GABAergic influences over ORXergic pro-anxiogenicffects as reported in other behavioral studies [25,26]. In thisontext, while these well known hypothalamic regions denselyopulated by GABAergic- and ORXergic-expressing neurons haveeen generally linked to anxiety conditions [36], it seems that anRX-B involvement in some behavioral paradigms such as panic

tates are mostly elicited when central GABAergic functions areecreased [26,56]. These relationships appear to be in line witheuronal disorders such as those of suicidal patients in which majornxiety difficulties characterizing them are tightly correlated toignificantly lower ORXergic products in the cerebrospinal fluidith respect to patients suffering from adjustment and dysthymiaisorders [8].

In order to determine if whether neuronal damages or dys-unctions characterized ORX-ergic enriched brain regions, theffects of combined treatments (ORX-A + flunitrazepam or ORX-+ flunitrazepam) on degeneration phenomenon in some majorotor-controlling areas and namely AMY, habenular neuronal

elds plus HIP were evaluated. For both single and combinedreated brain regions, the very few areas with signs of an argy-ophilic reaction of axonal processes and perikarya in the differenteuronal fields of animals treated also with GABAAR agonist tendo support the valuable indications supplied by ACS for a rapidetection of neurodegeneration processes in not only mammals19]. Surprisingly, these methods that are well known for theirbility to establish such a phenomenon, did not provide distinctilver staining differences in these brain areas of both ORX-A,-B

reated animals. A clear-cut distinction of ACS signals was insteadupplied when both neuropeptides were tested in the presencef �2 GABAAR agonist as shown by a notable reduction of dam-ged neuronal fields in all brain regions especially of ORX-B treatedamsters. ORX-B responses favorably exerting anxiogenic effects,

esearch 218 (2011) 288–295

very likely due to neurodegeneration events, appears to be in linewith other studies showing similar ORXergic-dependent responses[18]. Conversely, a reduction of these ORX-B-dependent neurode-generative events in the presence of the �2 GABAAR agonist, asshown by the few if any brain regions containing damaged neu-rons, tends to confirm the neuroprotective role of flunitrazepam[16,55]. This protective role of the GABAA �2 subunit should notbe surprising since a GABA-dependent protection against overex-citatory responses of limbic neuronal fields has been previouslydisplayed in conditions such as ischemia [61,62] as well as tonicimmobilization [14] and oxidative-related stressful states [30]. Inaddition, the scarce behavioral and neuronal alterations followingtreatment with flunitrazepam tends to assure, aside its neuropro-tective role, a defense against excitotoxic insults and hence thepromotion of physiological functions, i.e. feeding stimuli in animalsliving under extremely severe conditions such as hibernators [27].In this context it is tempting to propose, apart the participationof an ORXergic–GABAergic interaction, the involvement of cellu-lar factors like MAPK, which are involved with stressful-dependentischemic insults very likely during the awakening stages of hiber-nation [63].

Overall, the anxiogenic effects observed when hamster Cewas infused with ORX-B, seemed to account for modified motorbehaviors while the influences of this neuropeptide were stronglyreduced following the sequential treatment of ORX-B and fluni-trazepam. In particular the �2 GABAAR agonist, which has beenshown to promote anxiolytic effects and decreased motor activitiesin both behavioral tests provided consistently greater inhibitoryresponses in ORX-B treated animals. This specific relationship wasalso obtained for neurodegeneration phenomena in which a pro-tective role of the �2 GABAAR agonist was of a greater entity whenhamsters received ORX-B. Furthermore, the fact that a neuropro-tective activity is exerted following both neuropeptide-dependentbehavioral responses tend to support the participation of two dis-tinct of ORXR subtypes and precisely ORX1R and ORX2R that havedifferent affinities for the regionally two non overlapping ORX-ergic neuromodulators [35,51]. As a consequence, it is plausiblethat the different neuronal responses of ORX1R and ORX2R in thepresence of two neuropeptides may not only be strongly linked totheir distinct anxiogenic and anxiolytical properties but also to thenet neuroprotective measures exerted by the �2 GABAAR agonist.We are still at the beginning but differences in the roles of thesetwo neuroreceptor systems in anxiety-related behaviors may haveinteresting clinical bearings on motor deficits, which are typical ofnarcolepsy, Parkinson’s and Huntington’s diseases [43,58].

Acknowledgement

We gratefully acknowledge Research Ministry by MIUR whichhas co-financed this study (Italian University Research Ministry).

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