Anxiolytic-like effects after vector-mediated overexpression ofneuropeptide Y in the amygdala and hippocampus of miceSH Christiansen MV Olesen CR Goslashtzsche DPD Woldbye Laboratory for Neural Plasticity Laboratory of Neuropsychiatry Psychiatric Centre Copenhagen Department of Neuroscience and Pharmacology Universityof Copenhagen Denmark

A R T I C L E I N F O

Article historyReceived 17 January 2014Accepted 8 September 2014Available online

KeywordsrAAV-NPYAnxietyDepressionAmygdalaHippocampus

A B S T R A C T

Neuropeptide Y (NPY) causes anxiolytic- and antidepressant-like effects after central administration inrodents These effects could theoretically be utilized in future gene therapy for anxiety and depressionusing viral vectors for induction of overexpression of NPY in specific brain regions Using a recombinantadeno-associated viral (rAAV) vector we addressed this idea by testing effects on anxiolytic- and depression-like behaviours in adult mice after overexpression of NPY transgene in the amygdala andor hippocampustwo brain regions implicated in emotional behaviours In the amygdala injections of rAAV-NPY causedsignificant anxiolytic-like effect in the open field elevated plus maze and lightndashdark transition tests Inthe hippocampus rAAV-NPY treatment was associated with anxiolytic-like effect only in the elevatedplus maze No additive effect was observed after combined rAAV-NPY injection into both the amygdalaand hippocampus where anxiolytic-like effect was found in the elevated plus maze and lightndashdark tran-sition tests Antidepressant-like effects were not detected in any of the rAAV-NPY injected groups Immobilitywas even increased in the tail suspension and forced swim tests after intra-amygdaloid rAAV-NPY Takentogether the present data show that rAAV-NPY treatment may confer non-additive anxiolytic-like effectafter injection into the amygdala or hippocampus being most pronounced in the amygdala

copy 2014 Elsevier Ltd All rights reserved

1 Introduction

The widely distributed peptide transmitter neuropeptide Y (NPY)has been implicated in several neuropsychiatric disorders includ-ing anxiety and depression (Heilig et al 1989 Koefoed et al 2012Kormos and Gaszner 2013 Melas et al 2013 Mickey et al 2011Morales-Medina et al 2010 2012 Slawecki et al 2005 Soslashrensenet al 2004 Wu et al 2011) Thus NPY knockout mice display in-creased anxiety-like behaviour (Karl et al 2008 Verma et al 2012)and central administration of NPY causes anxiolytic-like effects(Heilig et al 1989 1993) Flinders-sensitive line rats a rodent modelof depression have lower levels of NPY in the hippocampus(Jimeacutenez-Vasquez et al 2000) and depressed human patients showlower levels of NPY in cerebrospinal fluid (CSF) (Gjerris et al 1992Heilig et al 2004 Widerloumlv et al 1988) Moreover antidepres-sant treatment causes increases in NPY in several limbic brain regionsof rodents and CSF of human patients (Bolwig et al 1999 Caberlotto

et al 1998 Husum et al 2000 2001 Mathe 1999 Mikkelsen andWoldbye 2006 Mikkelsen et al 1994 Nikisch and Matheacute 2008Nikisch et al 2005 Stenfors et al 1989 Wahlestedt et al 1990)and central administration of NPY has antidepressant-like effect(Husum et al 2000 Redrobe et al 2002 Stogner and Holmes 2000)

More specifically NPY appears to influence anxiety- anddepression-like behaviours at least in part via the amygdala andhippocampus two brain regions centrally involved in mediatinganxiety and depression (Alvarez et al 2008 Heilig 2004 Sajdyket al 1999 Sehlmeyer et al 2009) Thus intra-amygdalar infu-sion of NPY agonists induces anxiolytic-like effect in rodents via Y1receptors (Deo et al 2010 Fendt et al 2009 Gutman et al 2008Heilig et al 1993) while amygdaloid Y2 receptors appear to mediateanxiogenic- and prodepressive-like effects (Sajdyk et al 2002a2002b Tasan et al 2010) In the hippocampus infusion of NPY pro-duces antidepressant-like effect (Ishida et al 2007) most likely viaY1 receptors Likewise transgenic rats overexpressing NPY in hip-pocampal CA1 and CA2 fields display reduced anxiogenic responses(Thorsell et al 2000) Moreover Y4 receptor knockout mice havealso revealed a potential role for Y4 receptors in both anxiety- anddepression-like behaviours (Painsipp et al 2008)

Gene therapy with viral vectors is under investigation as a futuretreatment option for brain disorders particularly of neurologicalorigin (Alexander et al 2010 Hudry et al 2010 Kaplitt et al 2007Manfredsson and Mandel 2010 Mitra and Sapolsky 2010 Woldbye

Funding This work was supported by The Danish Medical Research Council(64750) Ivan Nielsen Foundation Psychiatric Basic Research Foundation Dr SofusCarl Emil Friis and His Wife Olga Doris Friisrsquos Foundation Lundbeck Foundation

Correspondence to Laboratory for Neural Plasticity Department of Neuroscienceand Pharmacology University of Copenhagen 3 Fruebjergvej Copenhagen DK-2100Denmark

E-mail address woldbyesundkudk (DPD Woldbye)

httpdxdoiorg101016jnpep2014090040143-4179copy 2014 Elsevier Ltd All rights reserved

Neuropeptides (2014) ndash

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Contents lists available at ScienceDirect

Neuropeptides

journal homepage wwwelseviercom locate npep

et al 2010) Nonetheless psychiatric disorders including anxietyand depression could also become a future focus for gene thera-peutic treatment Previous rodent studies indicate that NPY is apotential transgene candidate for regulating emotional behavioursThus in the amygdala anxiolytic-like effects have been shownfollowing Herpes simplex (Primeaux et al 2005) and Sindbis(Thorsell et al 2007) viral vector-induced NPY overexpression inrats Mouse studies have shown that recombinant adeno-associatedviral (rAAV) vector-induced overexpression of NPY or Y1 recep-tors but not Y5 receptors also confers anxiolytic-like effect in thehippocampus (Lin et al 2010 Olesen et al 2012a 2012b) Robustreciprocal connections have been shown between the amygdala andhippocampus in the rat (Pitkaumlnen et al 2000) In the mouse dataare more limited but it is generally believed that there are no majorspecies differences (Martiacutenez-Garciacutea et al 2012 Witter 2012) Thepotential importance of connections between amygdala andhippocampus at regulating emotional behaviours was recentlydemonstrated in a mouse study where optogenetic inhibition of theamygdaloid input to ventral hippocampus decreased anxiety-likebehaviour (Felix-Ortiz et al 2013) Thus gene therapeuticoverexpression of NPY in either amygdala or hippocampus is likelyto act both via local effects as well as by regulating input tohippocampus or amygdala respectively

In order to select the potentially most efficient strategy for futureNPY gene therapy (ie amygdala vs hippocampus or single regionvs both regions) the purpose of the present study was to directlycompare the effects of rAAV-induced overexpression of NPY in theamygdala andor hippocampus on anxiety- and depression-likebehaviours in adult mice

2 Materials and methods

21 Animals

Adult male NMRI mice (7 weeks old Taconic DK) were housedin cages with 2ndash4 animals in each on a 12-hr lightndashdark cycle (6PMndash6 AM) All experiments were performed in accordance withguidelines from the Danish Animal Experimentation Inspectorateand approved by the local ethical committee

21 Vector injections

Human preproNPY cDNA was subcloned into a chimeric sero-type 12 rAAV expression cassette consisting of the neuron-specific enolase (NSE) promoter woodchuck post-transcriptionalregulatory element (WPRE) and a bovine growth hormone polyA(bGHpA) signal flanked by AAV2 inverted terminal repeats (NSE-NPY-WPRE-bGHpA) (Genedetect Auckland New Zealand) (Goslashtzscheet al 2012 Richichi et al 2004 Woldbye et al 2010) The sameexpression cassette without the transgene (NSE-empty-WPRE-bGHpA) was used as control Mice were injected intraperitoneally(ip) with temgesic (006 mgkg Schering-Plough DK) rimadyl(5 mgkg Pfeizer APS DK) and baytril (10 mgkg Bayer AS DK)and were subsequently anaesthetized with ketamine (50 mgkg ipPfizer APS DK) and xylazine (20 mgkg ip Bayer AS DK) beforebeing placed in a stereotaxic frame (Kopf instruments USA) Holeswere drilled in the exposed skull and 1 μl of either rAAV-NPY orrAAV-empty was injected bilaterally using a 10 μl Hamilton syringeat a rate of 01 μlmin The viral stocks were diluted 11 with sterilephosphate-buffered saline (PBS) to a final solution for vector injec-tion containing 05 times 1012 genomic particlesml Mice receiving rAAV-NPY were divided into three groups One group was injected in theamygdala (n = 12) another in the hippocampus (n = 11) and a thirdgroup was injected in both the amygdala and hippocampus (n = 12)

Mice receiving rAAV-empty were likewise divided into three groupsthat were injected in the amygdala (n = 5) hippocampus (n = 5) orboth regions (n = 5) A group of non-injected age-matched naiumlve miceserved as an extra control group (n = 11) The injection coordi-nates for amygdala (one site in each hemisphere) were (fromBregma) anteroposterior (AP) ndash14 mm mediolateral (ML) plusmn25 mmdorsoventral (DV) ndash41 mm (Paxinos and Franklin 2001) The in-jection coordinates for hippocampus (two sites in each hemisphere)were AP ndash29 mm ML plusmn 29 mm DV ndash22 mm and AP ndash29 mmML plusmn 29 mm DV ndash30 mm Animals were monitored post-surgeryuntil recovery from anaesthesia The following 3 days the animalsreceived one daily ip injection containing rimadyl and baytrilas above Mice were left for 3ndash4 weeks counting from the day ofoperation before behavioural testing

22 Behavioural tests

Since pilot experiment (insert of Fig 3A) indicated thatcombined rAAV-NPY injections targeting both amygdala and hip-pocampus might lead to increased body weight we decided to foodrestrict the rAAV-NPY-injected mice Mice injected with rAAV-empty vector received standard rodent chow ad libitum while rAAV-NPY or naiumlve mice were fed the average amount of food ingestedby the rAAV-empty group as determined on the previous dayBecause pilot data indicated that rAAV-NPY-treated mice might showweight gains compared to control mice even when fed the sameamount of food we further food restricted the animals in the fol-lowing way When the average weight gain of the rAAV-NPY groupwas bigger (or smaller) than that of rAAV-empty mice the formergroup would then receive less (or more) food the following day equalin gram to this weight difference between the groups Naiumlve micewere subjected to the same procedure although rarely necessaryAll mice received tap water ad libitum

The same mice were used in all behavioural tests Testing started3ndash4 weeks after vector injections in all groups and was carried outin the following order open field test lightndashdark transition test el-evated plus maze test tail suspension test and forced swim testThere was an interval of 7 days between the last two tests whilethere were only 3 days between the other tests One hour prior totesting mice were transported to the testing room for habitua-tion All experiments were performed from 8 AM to 4 PM

221 Open field testMice were tested in the open field (Olesen et al 2012a Schmidt

et al 2011) placed in the centre of a darkened room with 4 times 60 Wincandescent light bulbs placed in each corner of the room Fourmice were tested at a time in four separate open fields each thesize of 40 times 40 times 80 cm At the beginning of each trial the mice wereplaced in the centre of the field and left undisturbed in the roomfor the remaining period of the trial A trial lasted 30 min and wasrecorded using a Logitech ultra vision web camera located onem above the open field boxes Using Ethovision (Noldus ver 5) theopen fields were following divided into a rim zone constitutingthe outer 6 cm of the field and a centre zone including the rest ofthe field Parameters measured were total distance moved time spentin the centre and total centre entries After each trial the fields werecleaned with 70 ethanol and dried using paper towels

222 Elevated plus maze testMice were tested in the elevated plus maze placed 50 cm above

the floor in the centre of a darkened room with 4 times 60 W incan-descent light bulbs placed in each corner of the room (Olesen et al2012a) One mouse was tested at a time for the duration of 10 minEach open arm of the elevated plus maze measured 27 times 7 cm

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Each closed arm measured 27 times 7 times 135 cm At the beginning of eachtrial the mice were placed in the centre of the elevated plus mazefacing one of the closed arms and left undisturbed in the room forthe remaining time of the trial The mice were recorded using aLogitech ultra vision web cam located one metre above the mazeUsing Ethovision (Noldus ver 5) the centre open and closed armsof the elevated plus maze was subsequently divided into separatezones Parameters measured were time spent on open arms entriesinto open arms and total entries into open and closed arms Aftereach trial the maze was cleaned with 70 ethanol and dried usingpaper towels

223 Lightndashdark transition testThe lightndashdark transition test apparatus (Olesen et al 2012a)

consisted of a rectangular box divided into two equally sized boxes(20 times 20 times 42 cm) by a black Plexiglas partition (black on one sideand white on the other) with a hole at the bottom (5 times 5 cm) Fourboxes were placed in a square with the light boxes lining each otherThe boxes were lit by 2 times 60 W incandescent light bulbs placed 75 cmabove the boxes At the beginning of each trial the mice were placedin the dark box and a lid quickly attached Four mice were testedat a time for the duration of 10 min The mice were recorded usinga Logitech ultra vision web camera located 75 cm above the boxesand scored by an observer blinded to the treatment Parameters mea-sured were time spent in the light box and entries into the lightbox After each trial the boxes were cleaned with 70 ethanol anddried using paper towels

224 Tail suspension testThe tail suspension test was previously described (Christiansen

and Woldbye 2010 Christiansen et al 2011) In brief mice werefastened with adhesive tape by the tip of their tails (1ndash2 cm) to aflat metal bar hanging 50 cm above the ground The metal bar wassecured inside a container with black walls isolating the mice fromvisual distractions Four mice were tested simultaneously being re-corded with a Logitech ultra vision web camera Mouse immobilitywas defined as the absence of limb movement and was scored overa period of 6 min by an observer blinded to the treatment

225 Forced swim testThe forced swim test (Olesen et al 2012b) was a slightly modi-

fied version from that developed by Porsolt et al (1977) Briefly micewere placed in glass cylinders (10 times 25 cm) containing 10 cm of water(maintained at 25 degC plusmn 05 degC) Six mice were tested at a time for theduration of 6 min and were scored by an observer blinded to thetreatment The mice were considered immobile when they floatedin an upright position making only small movements to keep theirheads above water

23 NPY mRNA in situ hybridization

To confirm the induction of NPY transgene overexpression byrAAV-NPY treatment mouse brains were removed immediately afterthe last behavioural experiment was conducted frozen on dry-iceand stored at ndash80 degC until sectioning on a cryostat (Shandon Inc)using Cryo-embed (Ax-Lab AS) thaw-mounting onto Superfrost Plusslides (VWR International ApS) and gently drying on a hotplateCoronal serial sections (15 μm) were cut through the amygdala andhippocampal formation [bregma ndash100 mm rarr ndash316 mm] and storedat ndash80 degC until further processing (Paxinos and Franklin 2001) Oneglass slide from each animal was defrosted for 10 min at room tem-perature (RT) and then hybridized for NPY mRNA All sections wereprocessed in the same experiment The subsequent in situ hybrid-ization procedure was described previously (Christensen et al 2006

Christiansen and Woldbye 2010) The slides were fixed for 5 minin ice-cold 4 paraformaldehyde The following synthetic oligo-nucleotide DNA antisense probe were used (MWG-BiotechGermany) NPY mRNA 5prime-GTC CTC TGC TGG CGC GTC CTC GCC CGGATT GTC CGG CTT GGA GGG GTA-3prime (Mikkelsen and Woldbye 2006)The probe was labelled at the 3prime-end with [35S]-ATP (AmershamBiosciences) using terminal deoxynucleotidyl transferase (RocheDiagnostics Mannheim Germany) and probe activity was in therange of 50000ndash300000 cpmμl Labelled probe (12 μl) was addedto 120 μl hybridization buffer containing 50 formamide (vv) 4 times SSCbuffer (20 times SSC 3 M NaCl 03 M Na3C6H5O7middot2H2O pH 70) and 10dextran sulphate The sections were covered with hybridization buffer(120 μlper slide) by placing a Parafilm slip over the sections Theywere then incubated at 42 degC overnight and subsequently rinsedbriefly in 1 times SSC at RT followed by 30 min stringent wash in 1 times SSCat 60 degC Finally the sections were dehydrated in a series of ethanolrinses dried and exposed together with 14C-microscales to a phos-phor imaging plate (Fujifilm Bas-TR2040) at RT for 10 days andscanned with a Fujifilm FLA-9000 (Starion) scanner Region specif-ic injection was assessed qualitatively by manual delineationbilaterally in the hippocampus and amygdala In addition since vari-able degree of spread of vector-induced labelling to the lateralhypothalamus was observed in mice that received rAAV-NPYinjections targeting the amygdala we quantified NPY mRNA levelsin the lateral hypothalamus and compared it to those parts of theamygdala where highest levels of labelling were found central andmedial amygdala In control experiments the specificity of theantisense probe was confirmed by addition of correspondingunlabelled antisense probe A person blinded to the treatment ofthe animals performed all measurements

24 NPY immunohistochemistry

The slides were defrosted for 10 min and subsequently fixed in4 paraformaldehyde for 20 min Hereafter the slides were washedtwice in TBS for 5 min and in incubation buffer (5 horse serum1 bovine serum albumin 03 Triton X-100 in TBS) for 30 min Afteradding rabbit anti-NPY antibody (1500 N9528 Sigma-Aldrich DK)in incubation buffer to the slides they were incubated overnight at4 degC The slides were subsequently washed three times in washingbuffer (1 bovine serum albumin 03 Triton X-100 in TBS) for 5 minincubated with Alexa-conjugated 568 goat anti-rabbit antibody(1200 Invitrogen Taastrup DK) in incubation buffer for 1 hour andwashed twice in TBS for 5 min The slides were then mounted withanti-fade mounting medium (DAKO Glostrup DK) and photo-graphs were obtained using an Olympus BX-51 microscope andVisiopharm Integrator System software (Visiopharm Hoslashrsholm DK)

25 Statistical analysis

Data were analysed using one-way ANOVA followed by NewmanndashKeuls post-hoc test Data points more than two standard deviationsabove or below the mean were excluded as outliers P-values below005 were considered statistically significant Data are presented asmeans plusmn SEM

3 Results

31 Transduction efficiency

Using in situ hybridization (Fig 1) and immunohistochemistry(Fig 2) it was verified that rAAV-NPY treatment induced pro-nounced overexpression of transgene NPY in the amygdala andorhippocampus In hippocampus-injected mice NPY overexpressionwas observed to be equally strong in both dorsal and ventral parts

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Fig 1 rAAV-mediated NPY mRNA overexpression in the amygdala andor hippocampus as revealed by in situ hybridization Pictures are displayed in inverted black andwhite colours White indicates NPY mRNA labelling Mice were injected with rAAV-NPY (DJ) in the amygdala (AMY) (EK) in the hippocampus (HIP) or (FL) in both regionssimultaneously (AMY + HIP) Control mice were not injected with vector (AG) or received rAAV-empty vector (BH) C and I show brain regions of A B DndashF and G H JndashLrespectively as depicted in the atlas of Paxinos and Franklin (2001) Magnification bar = 1 mm

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of hippocampus In amygdala-injected mice NPY overexpression inthe amygdala was most prominent in the central and medial partsAdditionally in mice receiving rAAV-NPY injections targeting theamygdala NPY overexpression of variable intensity was observedin the adjoining lateral hypothalamus and occasionally in the mostcaudal parts of the caudatendashputamen However quantification oflabelling showed that rAAV-NPY induced NPY overexpression in theamygdala- and amygdalahippocampus-injected groups at a muchhigher level in the amygdala than in the lateral hypothalamus ThusNPY mRNA levels in the rAAV-NPY amygdala-injected group in thecentral and medial amygdala were respectively 285 plusmn 76(P lt 000005 paired t-test) and 205 plusmn 34 (P lt 0000001) higher thanin the lateral hypothalamus (0 plusmn 17) Similarly in the central andmedial amygdala of rAAV-NPY amygdalahippocampus-injected miceNPY mRNA levels were respectively 199 plusmn 71 (P lt 001) and177 plusmn 43 (P lt 000001) higher than in the lateral hypothalamus(0 plusmn 15)

In the rAAV-NPY hippocampus-injected group quantificationrevealed modest spread to the lateral hypothalamus in two out of11 mice (36 and 29 lower than mean labelling in rAAV-NPY

amygdala and amygdalahippocampus groups respectively) whilelevels were close to zero in the rest of the group

32 Body weight and food intake

Body weight did not differ between the groups during the courseof the experiment as evidenced by non-significant treatment effectin repeated measures two-way ANOVA (P = 0291) confirming thatfood restriction was efficient (Fig 3A) The free-feeding rAAV-empty control group increased the mean body weight by 18 within4 weeks In contrast the free-feeding mice in the pilot study dis-played dramatic weight gain (64) within the same period (insertof Fig 3A) All three groups of food-restricted rAAV-NPY injectedmice displayed a significant decrease in the daily amount of foodnecessary to maintain stable body weight during the study com-pared to naiumlve or rAAV-empty injected mice (Fig 3B P lt 0001NewmanndashKeuls post-hoc test after significant one-way ANOVAF(449) = 6769 P lt 00001 five outliers excluded and two missingdata points) 68 (rAAV-NPY in amygdala or amygdalahippocampus)or 82 (rAAV-NPY hippocampus) that of free-feeding rAAV-emptymice The decrease in food needed for the mice injected with rAAV-NPY in the hippocampus was significantly lower than the decreasein food intake for those injected in the amygdala or amygdalahippocampus (Fig 3B P lt 0001) Food intake did not differ betweennaiumlve and rAAV-empty injected mice

33 Anxiolytic-like responses and motility

No significant differences between rAAV-empty injected mice andnaiumlve mice were observed in any of the behavioural tests and theywere consequently pooled into one single control group In the openfield test injection of rAAV-NPY into the amygdala increased thetime spent in the centre of the open field by 31 as compared tothe control group (Fig 4A P lt 0001 NewmanndashKeuls post-hoc testafter significant one-way ANOVA F(353) = 456 P = 0007 four out-liers excluded) while no effect was observed after injection of rAAV-NPY into the hippocampus alone or into both the amygdala andhippocampus Intra-amygdaloid rAAV-NPY-injected mice simulta-neously displayed a 20 decrease in total distance moved comparedto control mice while no locomotor effect was observed for miceinjected into the hippocampus or both regions together (Fig 4BP lt 005 after significant ANOVA F(356) = 360 P = 0019 one outlierexcluded) In the elevated plus maze the time spent by theamygdala-injected mice on the open arms was increased 60 com-pared to control mice (Fig 4C P lt 005 after significant ANOVAF(355) = 330 P = 0027 two outliers excluded) No effect was seenin mice injected in the hippocampus and amygdalahippocampusalthough a trend towards increased time spent on the open armswas observed All three groups of rAAV-NPY injected mice made sig-nificantly more entries into the open arms (Fig 4D amygdala 76P lt 001 hippocampus 38 P lt 005 amygdalahippocampus 61P lt 001 ANOVA F(356) = 768 P = 00002 one outlier excluded)No significant effect was observed on total entries into the open andclosed arms (Fig 4E) nor on closed arm entries (data not shown)In the lightndashdark transition test the control mice spent approxi-mately an equal amount of time in either of the boxes (Fig 5A) Incomparison the amygdala and amygdalahippocampus injected micespent 66 and 70 respectively of the test time in the light box(Fig 5A P lt 0001 for both vs control after significant ANOVAF(356) = 907 P lt 00001 one outlier excluded) No effect was ob-served in mice receiving intrahippocampal rAAV-NPY There wereno group differences with respect to the total number of entries(Fig 5B)

To determine whether spread of NPY transgene overexpressionto the lateral hypothalamus might contribute to the observedbehavioural effects in mice receiving injections targeting the

CeA

BLA MeA

ic

A B

C D

E F

rAAV-NPY inAMY+HIP

rAAV-empty inAMY+HIP

Fig 2 rAAV-mediated overexpression of NPY as revealed by immunohistochem-istry Picture shows NPY-like immunoreactivity (white colour) in a mouse injectedin the amygdala and hippocampus (AMY + HIP) with rAAV-empty (ACE) or rAAV-NPY (BDF) In the amygdala overexpression was found in the basolateral amygdala(BLA) but was most pronounced in the central (CeA) and medial (MeA) parts of theamygdala Magnification bars = 05 mm ic = internal capsule

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amygdala we analysed whether animals with high levels of NPYmRNA labelling in the lateral hypothalamus (amygdala- or amygdalahippocampus-rAAV-NPY 6 or 5 mice out of 12 per grouprespectively) behaved differently than those with low levelsHowever no significant differences were found in any of the anxietytest parameters analysed between high and low level lateral hy-pothalamus NPY mRNA labelling neither in the amygdala- (Studentrsquost-tests P = 045ndash086) nor in the amygdalahippocampus-rAAV-NPY group (P = 025ndash087) (data not shown) Likewise excluding themice with high levels of NPY mRNA expression from the analysesdid not change the obtainment of significance in any of the anxietytests in the amygdala- or amygdalahippocampus-rAAV-NPY groups(data not shown) Finally in the rAAV-NPY hippocampus-injectedgroup excluding the two mice with moderate spread of NPY mRNAlabelling to the lateral hypothalamus also did not influence obtain-ment of significance in the anxiety testing

34 Depression-like behaviour

As no significant difference between rAAV-empty injected miceand naiumlve mice was observed in any of the following tests they wereconsequently pooled into a single control group The amygdala in-jected mice showed an increased immobility of 57 in the tailsuspension test as compared to control mice (Fig 5C P lt 001NewmanndashKeuls post-hoc test after significant one-way ANOVAF(357) = 463 P = 00057) No effect was observed in hippocam-pus and amygdalahippocampus injected mice In the forced swimtest only the amygdala-injected mice likewise responded with asignificant increase of 14 in total immobility compared to thecontrol group (Fig 5D P lt 005 after significant ANOVAF(355) = 372 P = 0017 two outliers excluded) Further analysisshowed that animals with high levels of NPY mRNA labelling in thelateral hypothalamus did not behave differently from those with lowlevels neither in the amygdala- (Studentrsquos t-tests P = 007ndash051) norin the amygdalahippocampus-rAAV-NPY group (P = 025ndash087)

(data not shown) indicating that spread to the lateral hypothala-mus also did not affect the response in the two depression testsSimilarly excluding two mice with moderate spread to the lateralhypothalamus from the hippocampus rAAV-NPY group also did notaffect obtainment of significance

4 Discussion

The present study explored the potential anxiolytic-like effectsof rAAV-mediated NPY overexpression in the amygdala and hip-pocampus of adult mice Consistent with previous data with viralvector-induced NPY overexpression in the rat amygdala (Primeauxet al 2005 Thorsell et al 2007) and mouse hippocampus (Lin et al2010) we found significant anxiolytic-like effect after rAAV-NPYinjection in both studied murine brain regions However effectsappeared more robust in the amygdala showing significance in allthree anxiety tests as opposed to only in the elevated plus mazeafter rAAV-NPY treatment in the hippocampus The previous mousestudy only found significant anxiolytic-like effects after hippocam-pal NPY transgene overexpression in the open field test and not inthe elevated plus maze or lightndashdark tests (Lin et al 2010)This appears to confirm that anxiolytic-like effects after rAAV-NPYin the hippocampus are less robust and underline the importanceof not relying on one anxiety test for detecting anxiolytic-likeeffects

No additive effects were observed after injection of rAAV-NPYinto both the amygdala and hippocampus This did not appear tobe due to a ceiling effect of rAAV-NPY treatment Thus combinedregional treatment with rAAV-NPY even appeared to decrease theanxiolytic-like effect observed after rAAV-NPY in the amygdala aloneas evidenced by absent anxiolytic-like effect measured as percent-age time in the centre of the open field test or percentage time onopen arms of the plus maze test after rAAV-NPY injection into bothamygdala and hippocampus It is not clear why effects of NPYtransgene overexpression were not additive however it might

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Fig 3 Mean body weight and food intake following rAAV-NPY or rAAV-empty injection in naiumlve mice Mice were injected in the amygdala (AMY) hippocampus (HIP) orin both regions simultaneously (AMY + HIP) (A) Two-way repeated measures ANOVA did not reveal significant treatment effect on mean body weight indicating that thefood restriction regimen prevented changes in body weight between the groups during the study Insert shows pilot experiment where prominent body weight increaseswere seen 4 weeks after rAAV-NPY injection in both the amygdala and hippocampus in male NMRI mice (n = 11) that were not food restricted (B) Mean daily food intakeacross the experiment expressed in mg food pellets per g weight of mice Data are means plusmn SEM sectsectsectP lt 0001 vs naiumlve or rAAV-empty daggerdaggerdaggerP lt 0001 vs HIP NewmanndashKeulspost-hoc test following significant one-way ANOVA

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Please cite this article in press as SH Christiansen MV Olesen CR Goslashtzsche DPD Woldbye Anxiolytic-like effects after vector-mediated overexpression of neuropeptide Y inthe amygdala and hippocampus of mice Neuropeptides (2014) doi 101016jnpep201409004

6 SH Christiansen et alNeuropeptides (2014) ndash

be relevant that an inhibitory interaction between activity in theamygdala and hippocampus may occur as evidenced with electri-cal kindling in rats (Haas et al 1992)

NPY has been shown to reduce depression-like behaviour aftercentral administration in rodents (Ishida et al 2007 Redrobe et al2002 Stogner and Holmes 2000) It was therefore expected thatoverexpression of NPY in the amygdala hippocampus or in both areaswould have antidepressant-like effects However surprisinglyamygdala-injected mice in the present study displayed increasedimmobility in both the tail suspension test and forced swim test

Increased immobility might indicate that vector treatmentinduces pro-depressive effects (Cryan and Holmes 2005 Cryan et al2005 Porsolt et al 1977) However it is also recognized that resultsobtained in the forced swim and tail suspension tests with a po-tentially sedative agent should be interpreted with caution (Lim et al2005 Schmidt et al 1991 Steru et al 1985) Thus mice with NPYoverexpression in the amygdala were less active in the open fieldtest (ie reduced total distance moved) Using tests that are moreresistant to sedative effects like the sucrose preference test(Overstreet 2012) might shed further light on this issue The factthat amygdala rAAV-NPY-injected mice needed less food than rAAV-empty control mice to maintain the same body weight suggests thatNPY overexpression could be associated with reduced metabolic

activity which is a previously reported effect of NPY (Levine et al2004) This might indicate that effects observed in the depressiontests could be related to reduced energy expenditure and not pro-depressive-like effects per se Nonetheless vector-mediatedoverexpression of NPY in the hippocampus and combined amygdalahippocampus showed no effect on immobility even though they alsoappeared to have decreased metabolic activity based on their meanfood intake

One previous study also did not find antidepressant-like effectafter rAAV-NPY in the hippocampus unlike the present study theyeven found increased immobility (Lin et al 2010) Likewise no effectson immobility were observed after overexpression of Y1 or Y5 re-ceptors (Olesen et al 2012a 2012b) It remains to be seen whetherrAAV-mediated overexpression of NPY transgenes could be effi-cient in rodents that express depression-like behaviour eg Flinders-sensitive line rats chronic mild stress learned helplessness(Overstreet 2012)

In the pilot study we observed prominent weight gain withinfour weeks after rAAV-NPY administration targeting the amygdalahippocampus Since increased body weight might influencebehavioural testing mice in the present study were subjected tofood restriction keeping similar body weights in all three rAAV-NPY groups and naiumlve mice as compared to the free-feeding

50

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ed (c

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Fig 4 Anxiolytic-like effects after rAAV-NPY injections in mice tested in the open field and elevated plus maze tests Mice were injected with rAAV-NPY in the amygdala(AMY) hippocampus (HIP) or in both regions simultaneously (AMY + HIP) The control group consisted of pooled rAAV-empty vector injected mice and naiumlve mice Param-eters measured in the open field test (A) time spent in centre and (B) total distance moved parameters measured in the elevated plus maze (C) time spent on open arms(D) entries into open arms and (E) total entries into open and closed arms Data are means plusmn SEM P lt 005 P lt 001 vs control P lt 005 P lt 001 vs AMYNewmanndashKeuls post-hoc test following significant one-way ANOVA

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Please cite this article in press as SH Christiansen MV Olesen CR Goslashtzsche DPD Woldbye Anxiolytic-like effects after vector-mediated overexpression of neuropeptide Y inthe amygdala and hippocampus of mice Neuropeptides (2014) doi 101016jnpep201409004

7SH Christiansen et alNeuropeptides (2014) ndash

rAAV-empty group Food restriction though has been shown to affectmurine behaviour inducing anxiolytic-like effect in the lightndashdarktransition and elevated plus maze tests and antidepressant-likeeffects in the forced swim test (Yamamoto et al 2009) Conse-quently just like weight gain could have influenced behaviouraloutcome food restriction could have influenced the results of thepresent study However for several reasons we do not believe thatfood restriction played a significant role in mediating behaviouraleffects observed Firstly the behavioural effects observed after foodrestriction in Yamamoto et al (2009) differed from the present studyin as far as increased not decreased immobility was observed inthe forced swim test although both studies showed anxiolytic-like effects Secondly food restriction in rAAV-NPY-treated mice ofthe present study was less pronounced compared to that inYamamoto et al (2009) ie 68 (amygdala or amygdalahippocampus) or 82 (hippocampus) compared to 50 Thirdly asmentioned above previous studies with free-feeding mice in-jected with rAAV-NPY or rAAV-Y1 into the hippocampus (Lin et al2010 Olesen et al 2012a) or free-feeding rats injected with rAAV-NPY into the amygdala (Primeaux et al 2005 Thorsell et al 2007)

did show anxiolytic-like effects suggesting that food restriction wasnot essential for inducing anxiolytic-like effects

It is not entirely clear why we observed weight gain in our pilotstudy and why the mice needed less food to maintain body weightduring food restriction after rAAV-NPY administration into the amyg-dala andor hippocampus However it could be due to unintendedspread from the amygdala to the lateral hypothalamus where othershave demonstrated rAAV-NPY-mediated weight gains (Lin et al2006a Tiesjema et al 2007) Thus we observed variable degreesof vector-mediated NPY overexpression in the lateral hypothala-mus in mice that received injections targeting the amygdalaHowever in the hippocampus-injected rAAV-NPY group the micealso needed less food than control mice to maintain body weightduring food restriction although this effect was less pronounced thanafter amygdala-targeting rAAV-NPY injections Excluding two micewith spread to the lateral hypothalamus from the hippocampusrAAV-NPY group still resulted in a modest yet significant reduc-tion in food intake as compared to rAAV-empty Consequently thereason why the mice needed less food to maintain body weight inthe hippocampus group does not seem to be related to spread tothe lateral hypothalamus No previous studies have reported weightgains after intrahippocampal rAAV-NPY administration in free-feeding rats and mice (Goslashtzsche et al 2012 Lin et al 2006b 2010Richichi et al 2004 Soslashrensen et al 2009) but these studies didnot display data on food intake that might address whether met-abolic rate could have been affected Further studies are needed toclarify this issue

From a potential therapeutic perspective it will be importantto establish in future studies whether overexpression of NPY in theamygdala and hippocampus may affect body weight and metabo-lism since these regions could be targeted in gene therapy forpatients suffering from intractable temporal lobe epilepsy (McCown2010 Riban et al 2009) Likewise it will be important to clarifyother potential brain effects of NPY gene therapy including effectson anxiety It remains to be seen whether NPY gene therapy willobtain a role in future treatment of anxiety disorders Nonethe-less the present study indicates that gene therapeutic overexpressionof transgene NPY will most likely not benefit from targeting bothamygdala and hippocampus as opposed to targeting each regionalone Moreover overexpression of NPY in the amygdala seemsmost promising since the anxiolytic-like effect was most consis-tent in this region

Acknowledgements

We thank Gitta Woumlrtwein for valuable suggestions regarding thebehavioural testing The technical assistance of Birgit H Hansen isgreatly appreciated

References

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Alvarez RP Biggs A Chen G Pine DS Grillon C 2008 Contextual fearconditioning in humans cortical-hippocampal and amygdala contributions JNeurosci 28 6211ndash6219

Bolwig TG Woldbye DPD Mikkelsen JD 1999 Electroconvulsive therapy as ananticonvulsant a possible role of neuropeptide Y (NPY) J ECT 15 93ndash101

Caberlotto L Fuxe K Overstreet DH Gerrard P Hurd YL 1998 Alterations inneuropeptide Y and Y1 receptor mRNA expression in brains from an animal modelof depression region specific adaptation after fluoxetine treatment Brain ResMol Brain Res 59 58ndash65

Christensen DZ Olesen MV Kristiansen H Mikkelsen JD Woldbye DP 2006Unaltered neuropeptide Y (NPY)-stimulated [35S]GTPgammaS binding suggestsa net increase in NPY signalling after repeated electroconvulsive seizures in miceJ Neurosci Res 84 1282ndash1291

Christiansen SH Woldbye DPD 2010 Regulation of the galanin system by repeatedelectroconvulsive seizures in mice J Neurosci Res 88 3635ndash3643

0

20

40

60

80A

Contro

lAM

YHIP

AMY+HIP

Tim

e in

ligh

t box

()

dagger dagger

0

20

40

60B

Tota

l ent

ries

Contro

lAM

YHIP

AMY+HIP

0

20

40

60

80

Imm

obili

ty ti

me

()

C

Contro

lAM

YHIP

AMY+HIP

0

20

40

60

80

Contro

lAM

YHIP

AMY+HIP

Imm

obili

ty ti

me

()

D

n=25

n=12

n=11

n=12

n=25

n=11

n=11

n=12

n=26

n=12

n=11

n=12

n=25

n=12

n=11

n=11

Fig 5 Effects of rAAV-NPY injections in mice tested in the lightndashdark transition (Atime spent in the light box B total entries between the light and dark box) tail sus-pension (C) and forced swim (D) tests Mice were injected in the amygdala (AMY)hippocampus (HIP) or in both regions simultaneously (AMY + HIP) Control mice con-sisted of rAAV-empty vector injected mice and naiumlve mice Data are means plusmn SEMP lt 005 P lt 001 P lt 0001 vs control daggerP lt 005 vs HIP P lt 005 vs AMYNewmanndashKeuls post-hoc test following significant one-way ANOVA

ARTICLE IN PRESS

Please cite this article in press as SH Christiansen MV Olesen CR Goslashtzsche DPD Woldbye Anxiolytic-like effects after vector-mediated overexpression of neuropeptide Y inthe amygdala and hippocampus of mice Neuropeptides (2014) doi 101016jnpep201409004

8 SH Christiansen et alNeuropeptides (2014) ndash

Christiansen SH Olesen MV Woumlrtwein G Woldbye DPD 2011 Fluoxetinereverts chronic restraint stress-induced depression-like behaviour andincreases neuropeptide Y and galanin expression in mice Behav Brain Res 216585ndash591

Cryan JF Holmes A 2005 The ascent of mouse advances in modeling humandepression and anxiety Nat Rev Drug Discov 4 775ndash790

Cryan JF Mombereau C Vassout A 2005 The tail suspension test as a model forassessing antidepressant activity review of pharmacological and genetic studiesin mice Neurosci Biobehav Rev 29 571ndash625

Deo GS Dandekar MP Upadhya MA Kokare DM Subhedar NK 2010Neuropeptide Y Y1 receptors in the central nucleus of amygdala mediate theanxiolytic-like effect of allopregnanolone in mice behavioral andimmunocytochemical evidences Brain Res 1318 77ndash86

Felix-Ortiz AC Beyeler A Seo C Leppla CA Wildes CP Tye KM 2013 BLAto vHPC inputs modulate anxiety-related behaviors Neuron 79 658ndash664

Fendt M Burki H Imobersteg S Lingenhohl K McAllister KH Orain D et al2009 Fear-reducing effects of intra-amygdala neuropeptide Y infusion in animalmodels of conditioned fear an NPY Y1 receptor independent effectPsychopharmacology (Berl) 206 291ndash301

Gjerris A Widerloumlv E Werdelin L Ekman R 1992 Cerebrospinal fluidconcentrations of neuropeptide Y in depressed patients and in controls JPsychiatry Neurosci 17 23ndash27

Goslashtzsche CR Soslashrensen AT Nikitidou L Olesen MV Soslashrensen G ChristiansenSH et al 2012 Combined gene overexpression of neuropeptide Y and itsreceptor Y5 in the hippocampus suppresses seizures Neurobiol Dis 45 288ndash296

Gutman AR Yang Y Ressler KJ Davis M 2008 The role of neuropeptide Y inthe expression and extinction of fear-potentiated startle J Neurosci 2812682ndash12690

Haas KZ Sperber EF Mosheacute SL 1992 Kindling in developing animals interactionsbetween ipsilateral loci Brain Res Dev Brain Res 68 140ndash143

Heilig M 2004 The NPY system in stress anxiety and depression Neuropeptides38 213ndash224

Heilig M Soderpalm B Engel JA Widerlov E 1989 Centrally administeredneuropeptide Y (NPY) produces anxiolytic-like effects in animal anxiety modelsPsychopharmacology (Berl) 98 524ndash529

Heilig M McLeod S Brot M Heinrichs SC Menzaghi F Koob GF et al 1993Anxiolytic-like action of neuropeptide Y mediation by Y1 receptors in amygdalaand dissociation from food intake effects Neuropsychopharmacology 8 357ndash363

Heilig M Zachrisson O Thorsell A Ehnvall A Mottagui-Tabar S Sjoumlgren M et al2004 Decreased cerebrospinal fluid neuropeptide Y (NPY) in patients withtreatment refractory unipolar depression preliminary evidence for associationwith preproNPY gene polymorphism J Psychiatr Res 38 113ndash121

Hudry E Van DD Kulik W De Deyn PP Stet FS Ahouansou O et al 2010Adeno-associated virus gene therapy with cholesterol 24-hydroxylase reducesthe amyloid pathology before or after the onset of amyloid plaques in mousemodels of Alzheimerrsquos disease Mol Ther 18 44ndash53

Husum H Mikkelsen JD Hogg S Mathe AA Mork A 2000 Involvement ofhippocampal neuropeptide Y in mediating the chronic actions of lithiumelectroconvulsive stimulation and citalopram Neuropharmacology 39 1463ndash1473

Husum H Vasquez PA Matheacute AA 2001 Changed concentrations of tachykininsand neuropeptide Y in brain of a rat model of depression lithium treatmentnormalizes tachykinins Neuropsychopharmacology 24 183ndash191

Ishida H Shirayama Y Iwata M Katayama S Yamamoto A Kawahara R et al2007 Infusion of neuropeptide Y into CA3 region of hippocampus producesantidepressant-like effect via Y1 receptor Hippocampus 17 271ndash280

Jimeacutenez-Vasquez PA Overstreet DH Matheacute AA 2000 Neuropeptide Y in maleand female brains of Flinders sensitive line a rat model of depression Effectsof electroconvulsive stimuli J Psychiatr Res 34 405ndash412

Kaplitt MG Feigin A Tang C Fitzsimons HL Mattis P Lawlor PA et al 2007Safety and tolerability of gene therapy with an adeno-associated virus (AAV)borne GAD gene for Parkinsonrsquos disease an open label phase I trial Lancet 3692097ndash2105

Karl T Duffy L Herzog H 2008 Behavioural profile of a new mouse model forNPY deficiency Eur J Neurosci 28 173ndash180

Koefoed P Woldbye DPD Hansen TVO Christiansen SH Mors O Kessing LVet al 2012 Association of the leucine-7 to proline-7 variation in the signalsequence of neuropeptide Y with major depression Acta Neuropsychiatr 2481ndash90

Kormos V Gaszner B 2013 Role of neuropeptides in anxiety stress and depressionfrom animals to humans Neuropeptides 47 401ndash419

Levine AS Jewett DC Cleary JP Kotz CM Billington CJ 2004 Our journey withneuropeptide Y effects on ingestive behaviors and energy expenditure Peptides25 505ndash510

Lim WC Seo JM Lee CI Pyo HB Lee BC 2005 Stimulative and sedative effectsof essential oils upon inhalation in mice Arch Pharm Res 28 770ndash774

Lin EJ Sainsbury A Lee NJ Boey D Couzens M Enriquez R et al 2006aCombined deletion of Y1 Y2 and Y4 receptors prevents hypothalamicneuropeptide Y overexpression-induced hyperinsulinemia despite persistenceof hyperphagia and obesity Endocrinology 147 5094ndash5101

Lin E-JD Young D Baer K Herzog H During MJ 2006b Differential actionsof NPY on seizure modulation via Y1 and Y2 receptors evidence from receptorknockout mice Epilepsia 47 773ndash780

Lin EJ Lin S Aljanova A During MJ Herzog H 2010 Adult-onset hippocampal-specific neuropeptide Y overexpression confers mild anxiolytic effect in miceEur Neuropsychopharmacol 20 164ndash175

Manfredsson FP Mandel RJ 2010 Development of gene therapy for neurologicaldisorders Discov Med 9 204ndash211

Martiacutenez-Garciacutea F Novejarque A Gutieacuterrez-Castellanos N Lanuza E 2012 Piriformcortex and amygdala In Watson C Paxinos G Puelles L (Eds) The MouseNervous System London Academic Press

Mathe AA 1999 Neuropeptides and electroconvulsive treatment J ECT 15 60ndash75McCown TJ 2010 The future of epilepsy treatment focus on adeno-associated virus

vector gene therapy Drug News Perspect 23 281ndash286Melas PA Lennartsson A Vakifahmetoglu-Norberg H Wei Y Aberg E Werme

M et al 2013 Allele-specific programming of NPY and epigenetic effects ofphysical activity in a genetic model of depression Transl Psychiatry 3 e255

Mickey BJ Zhou Z Heitzeg MM Heinz E Hodgkinson CA Hsu DT et al 2011Emotion processing major depression and functional genetic variation ofneuropeptide Y Arch Gen Psychiatry 68 158ndash166

Mikkelsen JD Woldbye DP 2006 Accumulated increase in neuropeptide Y andsomatostatin gene expression of the rat in response to repeated electroconvulsivestimulation J Psychiatr Res 40 153ndash159

Mikkelsen JD Woldbye DPD Kragh J Larsen PJ Bolwig TG 1994Electroconvulsive shocks increase the expression of neuropeptide Y (NPY) mRNAin the piriform cortex and the dentate gyrus Brain Res Mol Brain Res 23317ndash322

Mitra R Sapolsky RM 2010 Gene therapy in rodent amygdala against feardisorders Expert Opin Biol Ther 10 1289ndash1303

Morales-Medina JC Dumont Y Quirion R 2010 A possible role of neuropeptideY in depression and stress Brain Res 1314 194ndash205

Morales-Medina JC Yvan D Charles-Etienne B Bastianetto S Flores G FournierA et al 2012 Role of neuropeptide Y Y1 and Y2 receptors on behavioral despairin a rat model of depression with co-morbid anxiety Neuropharmacology 62200ndash208

Nikisch G Matheacute AA 2008 CSF monoamine metabolites and neuropeptides indepressed patients before and after electroconvulsive therapy Eur Psychiatry23 356ndash359

Nikisch G Agren H Eap CB Czernik A Baumann P Matheacute AA 2005Neuropeptide Y and corticotropin-releasing hormone in CSF mark response toantidepressive treatment with citalopram Int J Neuropsychopharmacol 8403ndash410

Olesen MV Christiansen SH Goslashtzsche CR Nikitidou L Kokaia M WoldbyeDPD 2012a Neuropeptide Y Y1 receptor hippocampal overexpression via viralvectors is associated with modest anxiolytic-like and proconvulsant effects inmice J Neurosci Res 90 498ndash507

Olesen MV Christiansen SH Goslashtzsche CR Holst B Kokaia M Woldbye DP2012b Y5 neuropeptide Y receptor overexpression in mice neither affects anxiety-and depression-like behaviours nor seizures but confers moderate hyperactivityNeuropeptides 46 71ndash79

Overstreet DH 2012 Modeling depression in animal models Methods Mol Biol829 125ndash144

Painsipp E Wultsch T Edelsbrunner ME Tasan RO Singewald N Herzog Het al 2008 Reduced anxiety-like and depression-related behavior inneuropeptide Y Y4 receptor knockout mice Genes Brain Behav 7 532ndash542

Paxinos G Franklin K 2001 The Mouse Brain ndash in Stereotaxic Coordinates AcademicPress San Diego

Pitkaumlnen A Pikkarainen M Nurminen N Ylinen A 2000 Reciprocal connectionsbetween the amygdala and the hippocampal formation perirhinal cortex andpostrhinal cortex in rat a review Ann N Y Acad Sci 911 369ndash391

Porsolt RD Bertin A Jalfre M 1977 Behavioral despair in mice a primaryscreening test for antidepressants Arch Int Pharmacodyn Ther 229 327ndash336

Primeaux SD Wilson SP Cusick MC York DA Wilson MA 2005 Effects ofaltered amygdalar neuropeptide Y expression on anxiety-related behaviorsNeuropsychopharmacology 30 1589ndash1597

Redrobe JP Dumont Y Fournier A Quirion R 2002 The neuropeptide Y (NPY)Y1 receptor subtype mediates NPY-induced antidepressant-like activity in themouse forced swimming test Neuropsychopharmacology 26 615ndash624

Riban V Fitzsimons HL During MJ 2009 Gene therapy in epilepsy Epilepsia50 24ndash32

Richichi C Lin EJ Stefanin D Colella D Ravizza T Grignaschi G et al 2004Anticonvulsant and antiepileptogenic effects mediated by adeno-associated virusvector neuropeptide Y expression in the rat hippocampus J Neurosci 243051ndash3059

Sajdyk TJ Vandergriff MG Gehlert DR 1999 Amygdalar neuropeptide Y Y1receptors mediate the anxiolytic-like actions of neuropeptide Y in the socialinteraction test Eur J Pharmacol 368 143ndash147

Sajdyk TJ Schober DA Gehlert DR 2002a Neuropeptide Y receptor subtypesin the basolateral nucleus of the amygdala modulate anxiogenic responses inrats Neuropharmacology 43 1165ndash1172

Sajdyk TJ Schober DA Smiley DL Gehlert DR 2002b Neuropeptide Y-Y2receptors mediate anxiety in the amygdala Pharmacol Biochem Behav 71419ndash423

Schmidt C Gobaille S Hechler V Schmitt M Bourguignon JJ Maitre M 1991Anti-sedative and anti-cataleptic properties of NCS-382 a [gamma]-hydroxybutyrate receptor antagonist Eur J Pharmacol 203 393ndash397

Schmidt LS Thomsen M Weikop P Dencker D Wess J Woldbye DPD et al2011 Increased cocaine self-administration in M4 muscarinic acetylcholinereceptor knockout mice Psychopharmacology (Berl) 216 367ndash378

Sehlmeyer C Schoning S Zwitserlood P Pfleiderer B Kircher T Arolt V et al2009 Human fear conditioning and extinction in neuroimaging a systematicreview PLoS ONE 4 e5865

ARTICLE IN PRESS

Please cite this article in press as SH Christiansen MV Olesen CR Goslashtzsche DPD Woldbye Anxiolytic-like effects after vector-mediated overexpression of neuropeptide Y inthe amygdala and hippocampus of mice Neuropeptides (2014) doi 101016jnpep201409004

9SH Christiansen et alNeuropeptides (2014) ndash

Slawecki CJ Thorsell AK El Khoury A Matheacute AA Ehlers CL 2005 IncreasedCRF-like and NPY-like immunoreactivity in adult rats exposed to nicotine duringadolescence relation to anxiety-like and depressive-like behavior Neuropeptides39 369ndash377

Soslashrensen AT Nikitidou L Ledri M Lin EJ During MJ Kanter-Schlifke I et al2009 Hippocampal NPY gene transfer attenuates seizures without affectingepilepsy-induced impairment of LTP Exp Neurol 215 328ndash333

Soslashrensen G Lindberg C Woumlrtwein G Bolwig TG Woldbye DPD 2004Differential roles for neuropeptide Y Y1 and Y5 receptors in anxiety and sedationJ Neurosci Res 77 723ndash729

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Steru L Chermat R Thierry B Simon P 1985 The tail suspension test a newmethod for screening antidepressants in mice Psychopharmacology (Berl) 85367ndash370

Stogner KA Holmes PV 2000 Neuropeptide-Y exerts antidepressant-like effectsin the forced swim test in rats Eur J Pharmacol 387 R9ndashR10

Tasan RO Nguyen NK Weger S Sartori SB Singewald N Heilbronn R et al2010 The central and basolateral amygdala are critical sites of neuropeptide YY2receptor-mediated regulation of anxiety and depression J Neurosci 30 6282ndash6290

Thorsell A Michalkiewicz M Dumont Y Quirion R Caberlotto L Rimondini Ret al 2000 Behavioral insensitivity to restraint stress absent fear suppressionof behavior and impaired spatial learning in transgenic rats with hippocampalneuropeptide Y overexpression Proc Natl Acad Sci USA 97 12852ndash12857

Thorsell A Repunte-Canonigo V OrsquoDell LE Chen SA King AR Lekic D et al2007 Viral vector-induced amygdala NPY overexpression reverses increased

alcohol intake caused by repeated deprivations in Wistar rats Brain 1301330ndash1337

Tiesjema B Adan RA Luijendijk MC Kalsbeek A la Fleur SE 2007Differential effects of recombinant adeno-associated virus-mediatedneuropeptide Y overexpression in the hypothalamic paraventricularnucleus and lateral hypothalamus on feeding behavior J Neurosci 27 14139ndash14146

Verma D Tasan RO Herzog H Sperk G 2012 NPY controls fear conditioningand fear extinction by combined action on Y1 and Y2 receptors Br J Pharmacol166 1461ndash1473

Wahlestedt C Blendy JA Kellar KJ Heilig M Widerloumlv E Ekman R 1990Electroconvulsive shocks increase the concentration of neocortical andhippocampal neuropeptide Y (NPY)-like immunoreactivity in the rat Brain Res507 65ndash68

Widerloumlv E Lindstrom LH Wahlestedt C Ekman R 1988 Neuropeptide Y andpeptide YY as possible cerebrospinal fluid markers for major depression andschizophrenia respectively J Psychiatr Res 22 69ndash79

Witter M 2012 Hippocampus In Watson C Paxinos G Puelles L (Eds) TheMouse Nervous System London Academic Press

Woldbye DPD Aumlngehagen M Goslashtzsche CR Elbroslashnd-Bek H Soslashrensen ATChristiansen SH et al 2010 Adeno-associated viral vector-inducedoverexpression of neuropeptide Y Y2 receptors in the hippocampus suppressesseizures Brain 133 2778ndash2788

Wu G Feder A Wegener G Bailey C Saxena S Charney D et al 2011 Centralfunctions of neuropeptide Y in mood and anxiety disorders Expert Opin TherTargets 15 1317ndash1331

Yamamoto Y Tanahashi T Kawai T Chikahisa S Katsuura S Nishida K et al2009 Changes in behavior and gene expression induced by caloric restrictionin C57BL6 mice Physiol Genomics 39 227ndash235

ARTICLE IN PRESS

Please cite this article in press as SH Christiansen MV Olesen CR Goslashtzsche DPD Woldbye Anxiolytic-like effects after vector-mediated overexpression of neuropeptide Y inthe amygdala and hippocampus of mice Neuropeptides (2014) doi 101016jnpep201409004

10 SH Christiansen et alNeuropeptides (2014) ndash