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Electroconvulsive stimuli selectively affect behaviorand neuropeptide Y (NPY) and NPY Y1 receptor geneexpressions in hippocampus and hypothalamus ofFlinders Sensitive Line rat model of depression

Patricia A. Jimenez-Vasquez a, Zaida Diaz-Cabiale b,c, Laura Caberlotto d,Inmaculada Bellido b,e, David Overstreet f, Kjell Fuxe b,Aleksander A. Mathe a,*

a Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Swedenb Department of Neuroscience, Karolinska Institutet, Stockholm, Swedenc Department of Physiology, School of Medicine, University of Malaga, Spaind GlaxoSmithKline, SPA, Verona, Italye Department of Pharmacology and Clinical Therapeutics, School of Medicine, University of Malaga, Spainf Department of Psychiatry and Bowles Center for Alcohol Studies, University of North Carolina, Chapel Hill, USA

Received 28 February 2006; received in revised form 25 May 2006; accepted 27 June 2006

0924-977X/$ - see front matter D 200doi:10.1016/j.euroneuro.2006.06.011

* Corresponding author. DepartmenSweden. Tel.: +46 8 52487972; fax: +4

E-mail address: aleksander.mathe@

KEYWORDSNeuropeptide Y mRNA;NPY Y1 receptor mRNA;NPY Y1 receptorbinding;Animal model ofdepression;Electroconvulsivestimuli

Abstract Previously we reported that basal neuropeptide Y (NPY)-like immunoreactivity-(LI)in hippocampus of the bdepressedQ Flinders Sensitive Line (FSL) rats was lower compared to thecontrol Flinders Resistant Line (FRL) and that electroconvulsive stimuli (ECS) raise NPY-LI indiscrete brain regions. Here we studied NPY mRNA expression, NPY Y1 receptor (Y1) mRNAexpression and binding sites, and behavior under basal conditions (Sham) and after repeatedECS. Baseline NPYand Y1 mRNAs in the CA1—2 regions and dentate gyrus were lower while the Y1binding was higher in the FSL. ECS had larger effects on both NPY and behavior in the FSL rats.ECS increased NPY mRNA in the CA1—2, dentate gyrus and hypothalamus in FSL, but only in thedentate gyrus in FRL. ECS also increased Y1 mRNA in the CA1—2, dentate gyrus and the parietalcortex in both strains, while in the hypothalamus the increase was observed only in the FSL rats.Consistently with Y1 mRNA increase, Y1 binding was downregulated in the correspondingregions. ECS decreased FSL immobility in the Porsolt swim test. These findings suggest that NPYis involved in depressive disorder and that antidepressant effects of ECS may in part bemediated through NPY.D 2006 Elsevier B.V. and ECNP. All rights reserved.

European Neuropsychopharmacology (2007) 17, 298—308

6 Elsevier B.V. and ECNP. All rights reserved.

t of Neuroscience, Karolinska University Hospital Huddinge, Psychiatry M56, SE-14186 Stockholm,6 8 52488929.ki.se (A.A. Mathe).

Effect of ECS on neuropeptide Y in depressed rats 299

1. Introduction

Several animal models of depression have been developed inorder to study the pathophysiology of depression and to testthe antidepressant potential of new compounds. Based onthe observation that depressed patients are more sensitiveto cholinergic agonists (Janowsky and Risch, 1987) a strain ofrats, the Flinders Sensitive Line (FSL) with increasedsensitivity to diisopropylfluorophosphate (DFP), an anticho-linesterase agent, was selectively bred (Overstreet, 1986;Daws and Overstreet, 1999; Overstreet et al., 2005). TheFSL strain was proposed as a genetic animal model ofdepression, since these rats exhibit a number of behavioralsimilarities to depressed individuals, such as increasedbanhedoniaQ in response to chronic mild stress, increasedrapid eye movement (REM) sleep, reduction in REM sleeponset time, cognitive difficulties and reduced body weight.Moreover, antidepressants; e.g. fluoxetine, diminish behav-ioral symptoms of bdepressionQ in the FSL strain (Overstreetet al., 1995, 2005). The FSL rats meet reasonably wellcriteria of face, construct, and predictive validity for ananimal model of depression, thereby making it a usefulmodel to study the pathophysiology and pharmacology ofdepression.

Neuropeptide Y (NPY) is a 36 amino-acid peptide widelydistributed in the mammalian brain. The physiologicalactions of NPY in the CNS are mediated via receptors Y1,Y2, Y4, Y5 and Y6 and in fish and amphibians also Y7 (Heilig,2004; Kask et al., 2002; Kopp et al., 1999; Fredriksson et al.,2004; Dumont and Quirion, 2006; Larsson et al., 2006).Numerous studies indicate that NPY is involved in a numberof central physiological functions, such as regulation of foodintake, locomotion and sexual behavior, and plays a role inarousal, memory processing and circadian rhythms (Fuxe etal., 1990; Wahlestedt and Heilig, 1995; Kask et al., 2002;Redrobe et al., 2002; Heilig, 2004; Thorsell et al., 2006).Furthermore, clinical studies have revealed decreasedlevels of NPY-LI in the cerebrospinal fluid (CSF) and plasmaof depressed patients (Widerlov et al., 1988; Hashimoto etal., 1996; Nilsson et al., 1996; Westrin et al., 1999; Heilig etal., 2004). Alteration of NPY-LI or mRNA levels was alsofound post mortem in the prefrontal cortex of subjects thatcommitted suicide (Widdowson et al., 1992) and in bipolardisorder patients (Caberlotto and Hurd, 2001). In otherinvestigations, however, no differences in NPY-LI in CSF or inpost-mortem brain tissue were found between diagnosticgroups (Berrettini et al., 1987; Ordway et al., 1995).Additional data consistent with the NPY hypothesis are thefindings of increased NPY-LI after repeated electroconvul-sive treatment (ECT) in depressed individuals (Mathe et al.,1996; Nikisch et al., 2005) suggesting that a NPYergichypofunction could play a role in the pathophysiology ofdepression. Likewise, animals treated with electroconvul-sive stimuli (ECS) as well as lithium and antidepressantsshow selective and specific effects on brain NPY-LI and NPYmRNA expression (Mathe et al., 1990, 1994; Wahlestedt etal., 1990; Stenfors et al., 1989, 1992, 1994; Weiner et al.,1992; Zachrisson et al., 1995a,b; Madsen et al., 2000;Husum et al., 2000; Husum and Mathe, 2002). The observedchanges suggest that antidepressants may exert some oftheir therapeutic effects through upregulation of endoge-nous NPY. The hypothesis of reduced NPY neurotransmission

in depression was extended by the findings that the FlindersSensitive Line (FSL) and the Fawn Hooded rats (FH), anotherstrain considered to be a genetic model of depression, hadsignificantly decreased hippocampal NPY-LI as compared tothe two control strains, the Flinders Resistant Line (FRL) ratsand the Wistar rats, respectively (Mathe et al., 1998;Caberlotto et al., 1999; Jimenez-Vasquez et al., 2000a,b).Furthermore, a series of ECS increased NPY-LI in specificbrain regions in these rat strains (Mathe et al., 1998;Jimenez-Vasquez et al., 2000a,b). Moreover, in an environ-mental model, the early life maternal separation, NPY wasalso decreased in rat hippocampus (Jimenez-Vasquez et al.,2001; Husum and Mathe, 2002). In addition, it has beenshown that baseline hippocampal NPY mRNA expression isdecreased and Y1 receptor binding increased in the FSL ratscompared to FRL rats, and that chronic treatment withfluoxetine differentially affected NPY mRNA and Y1 receptormRNA levels in the two rat strains (Caberlotto et al., 1998,1999).

In this study NPY mRNA and Y1 receptor mRNA expressionas well as Y1 receptor binding sites levels were measured inbrain regions following ECS and Sham (bbaselineQ) treat-ments in both FSL and FRL rats to clarify the followingquestions: (1) are the effects of ECS on NPY mRNA, NPY Y1mRNA and Y1 binding different in FSL vs. FRL rats, (2) does aseries of ECS produce antidepressant-like effects as assessedby the Porsolt swim test and are the behavioral effectsparallel to the effects on NPY mRNA levels, NPY Y1 mRNAlevels and Y1 binding.

2. Experimental procedures

2.1. Animals and treatment

Adult male FSL and FRL rats from breeding colonies maintained atthe Karolinska Institutet, were used. The animals were kept undercontrolled conditions of temperature (22F1 8C), relative humidity(45%—55%) and daylight cycle (12:12 h, lights on at 06:00). Rat chow(R36 Lactamin, Stockholm, Sweden) and tap water were availablead libitum. All animal experimentation was conducted in accor-dance with the Karolinska Institutet’s Guidelines for the Care andUse of Laboratory Animals. The Stockholm Ethical Committee forProtection of Animals approved the experiments. Both FSL and FRLrats were randomly divided into two subgroups (N =11/subgroup)receiving ECS or Sham. The ECS subgroups received a series of8 transauricular ECS, given every other day. The current (150 V, 50Hz, 20 ms for 1 s; the paradigm was originally tested to produce amarked tonic—clonic seizure without harming the animals and hasever since been a standard procedure in our laboratory; see Stenforset al., 1989) was administered via earclip electrodes using a Grass S-44 stimulator (Grass Inc., Quincy, MA, USA). Each stimulationelicited tonic—clonic seizures that lasted approximately 10—14 s.Controls had the earclips mounted with no current passed (Sham).After the last ECS or Sham, 5 animals/subgroup were submitted tothe Porsolt swim test, while the rest (5—6 animals/subgroup) weresacrificed 24 h later for subsequent in situ hybridization andreceptor autoradiography. Following decapitation the brains wererapidly removed and frozen in liquid isopentane (�40 8C) and storedat �80 8C until sectioned in a Leitz cryostat. Coronal sections werecut at 14 lm (Bregma, 3.80 and �2.56 mm approximately) andthaw-mounted on Fisher super Frost Plus slides for in situ experi-ments and on gelatin coated slides for receptor autoradiography andkept at �20 8C until further processing. The swim test (Porsolt etal., 1978) and biochemical measurements were performed on

P.A. Jimenez-Vasquez et al.300

different groups of animals to avoid possible effects of stress on thebiochemical measurements. Both behavioral data and biochemistryanalyses were assessed by investigators who were blind to theanimal strain and the experimental conditions.

2.2. Porsolt swim test

The procedure (Porsolt et al., 1978) was used as a model oflearned helplessness to detect the antidepressant activity of theECS. Swim sessions were conducted by placing rats in a verticalcylinder (40 cm height�18 cm in diameter) containing 25 8Cwater, 21 cm deep, so that rats could not support themselves bytouching the bottom with their feet. The swim sessions wereconducted between 9.00 and 13.00. An initial 15 min pre-testwas followed 24 h later by a 5 min test. The pre-test sessionswere performed 1 h after the last ECS or Sham. After the pre-test the rats were removed from the cylinder, dried with papertowels, placed in a heated enclosure and allowed to dry for 15min at 32 8C before they were returned to their home cages.Twenty-four hours later, the animal was placed into the cylinderand exposed again to the swim test conditions. The totalduration of active swimming behavior was measured during the5 min test session. The rat was judged to be active whenever itdid not remain floating passively upright in the water and it wasconsidered to have stopped swimming when at least three pawswere immobile. The duration of immobility is presumed toindicate a state of bdespairQ as the animal has learned thatescape is impossible.

2.3. Probe preparation and in situ hybridization

Probe preparation and in situ hybridization were carried out aspreviously described (Caberlotto et al., 1998).

2.3.1. RNA probe synthesisThe NPY rat riboprobe was made from a 508 bp cDNA of the entireNPY sequence (Hanze et al., 1991) that was subcloned into apGEMZ4 vector. The rat NPY Y1 riboprobe corresponded to a 245 bpcDNA fragment of the Y1 receptor (gene bank accession numberX95507), spanning over the 4th and 5th transmembrane regions; thiscDNAwas subcloned into a Bluescript II SK-vector. 35S labeling of theprobes: RNA probes complementary to the coding sequences weretranscribed from the linearized plasmid template with 150 ACi a-[35S]UTP (Dupont NEN, Boston, MA). T3 RNA polymerase was used forgenerating the antisense and T7 for generating the sense. Transcrip-tion occurred in the presence of 10mM dithiothreitol, 0.5 mM each ofATP, GTP, CTP and CTP, and 1 lg linearized plasmid template in a 1�transcription buffer for 60 min at 37 8C. The labelled probe was thenseparated from unincorporated nucleotides using spin columns(Amersham-Pharmacia, England).

2.3.2. In situ hybridizationThe slides were allowed to thaw and dry to room temperatureand were then fixed in 4% paraformaldehyde/1� phosphatebuffered saline (PBS) for 10 min, and washed twice for 5 minin PBS. Following acetylation in 0.1 M triethanolamine (pH 8) (toreduce background)/0.25% acetic anhydride/0.9% sodium chloridefor 5 min, the slides were washed in 2� SSC for 5 min anddehydrated through graded alcohols (70, 80, 95 and 100%,respectively) and delipidated with chloroform, and then theslides were allowed to air dry before being used or frozen at �708C until use. All aqueous solutions were pretreated with 0.1%diethylpyrocarbonate before use. The sections were hybridizedwith a prehybridization buffer (0.5 mg/ml sheared ss DNA, 250Ag/ml Yeast tRNA, 1� Denhardt’s solution, 50% deionizedformamide, and 4� standard saline citrate (SSC)). Before the

hybridization the labelled 35S antisense RNA probe or sense RNAprobe for the control sections (concentration 1�106 c.p.m. perAl) were added to the hybridization cocktail. Volumes of 100 Al ofdiluted probe were applied to the brain sections and the slideswere covered with coverslips (to prevent evaporation) and wereincubated for hybridization overnight at 55 8C in a humidifiedchamber. Incubation was followed by washing in graded series ofSSC (2�, 1�, 0.5�, 0.5�/50% formamide, 0.1�) containing 1 mMDTT, all at room temperature except for 0.5�/50% formamide (488C) and 0.1� SSC (53 8C). The sections were finally dehydrated ingraded ethanol solutions containing 300 mM ammonium acetate,air dried and then exposed to B-Max Hyperfilm (Amersham, UK)for 3—5 weeks.

2.3.3. Analysis of labelingThe film autoradiograms from the in situ hybridization experimentswere semi-quantitatively analyzed bymeasuring the grey values usinga computer-assisted image analyzer system (Avanzati, Milano, Italy).Regions of interest were defined by anatomical landmarks accordingto Paxinos et al. brain atlas (Paxinos et al., 1986).

w?>Three measurements were performed for each region: (a) thetotal value, i.e.measurements of the region in the sections hybridizedwith a [35S]UTP-labeled antisense RNA, (b) the unspecific value, i.e.measurements of the corresponding region in the control sectionshybridizedwitha [35S]UTP-labeled senseRNA, and (c) the backgroundvalue,e.g.measurementsof thefilmbackgroundoutsidethesections.The transmittance percentage values (T%) of specific and unspecificlabelingwereobtainedasdescribedbyBenfenatietal.(1986).FromtheT% values the optical density could be obtained (O.D.=� logT%). Thedataareexpressedasopticaldensity.

2.4. Receptor autoradiography

2.4.1. In vitro receptor autoradiographyThe slides were pre-incubated for 1 h at room temperature in aKrebs Ringer Phosphate buffer (KRP), pH 7.4. Subsequently, theslides were incubated for 2 h in the same buffer supplementedwith 0.1% BSA, 0.05% bacitracin (Sigma), and 25 pM [125I][31Pro34]—PYY, respectively (non saturated conditions) close to the Kd value(Dumont et al., 1995), a state in which change could reflectaffinity or number of binding sites. To determine the non-specificbinding, 1 AM unlabeled NPY was added to the incubation solution.Following the incubation, the slides were washed 4 times for 2 minin ice-cold KRP buffer, and rinsed in deionized water to remove thesalt. The sections were air dried, and exposed to AmershamHyperfilm for 5 days together with 125I microscales (AmershamInternational) as reference standards.

2.4.2. Analysis of labeling for NPY Y1 receptor

autoradiographyThe semi-quantification was done as described previously (Benfe-nati et al., 1986) using a computer assisted image analysis systemdeveloped by Imaging Research (Avanzati, Milan, Italy). Prefabri-cated 125I labelled polymer strips (Amersham microscale, UK) wereused to convert the grey values into fmol/mg protein values.

2.5. Data analysis

The ANOVA was performed using the Macintosh Super ANOVApackage. In all statistical analyses, p V0.05 was consideredsignificant. A two-way analysis of variance (ANOVA) was used forthe swim test, where treatment (Sham/ECS) and strain (FRL/FSL)were used as main factors, followed by Bonferroni post-hoc test.The NPY mRNA, Y1 receptor mRNA as well as Y1 receptor bindingdata were analyzed for each brain region by a two-way indepen-dent ANOVA followed by Tukey—Kramer post-hoc and Bonferroni’scorrection.

Effect of ECS on neuropeptide Y in depressed rats 301

3. Results

3.1. NPY mRNA expression

In accordance with previous studies NPY mRNA levels wereexpressed inter alia in the cerebral cortex, the hippocampalformation and the hypothalamus (Fig. 1) (Morris, 1989).

3.2. Basal levels (Sham-treated animals)

3.2.1. CA1—2 regions and dentate gyrusAs shown in Figs. 1 and 2, NPY mRNA levels were significantlylower in the bdepressedQ FSL as compared to the control FRLrats in both hippocampal regions, the reductions reaching60% in the CA1—2 region ( F1,19=33.5; p b0.0001) and 27% inthe dentate gyrus ( F1,19=4.2 p b0.05) (Figs. 1 and 2).

3.2.2. Parietal cortex, medial amygdala andhypothalamusThere were no significant strain differences in basal NPYmRNA levels (Figs. 1 and 2).

3.3. Effects of ECS treatment

3.3.1. CA1—2 regions, dentate gyrus and hypothalamusFollowing ECS treatment, a significant increase of NPYmRNA levels was observed in FSL with a 125% increase(vs. FSL Sham) in the CA1—2 regions ( F1,19 =40,5;p b0.0001) and a 27% increase (vs. FSL Sham) in thehypothalamus ( F1,19=8.2; p b0.01) (Figs. 1 and 2). In thedentate gyrus the NPY mRNA levels were significantlyincreased in both FRL and FSL rats ( F1,19 = 40,4;p b0.0001), by 13% (vs. FRL Sham) and 66% (vs. FSL Sham),respectively (Figs. 1 and 2). In addition, a significant

Figure 1 Representative autoradiograms of NPY mRNA expressioFlinders Resistant Line (FRL) control rat and Flinders Sensitive Lintreatment. The NPY mRNA expression was studied in the parietal comedial amygdala (MA) and the hypothalamus (HT). Scale=2 mm.

strain� treatment interaction was observed in the CA1—2regions ( F1,19=20.2; p b0.001) and in the dentate gyrus( F1,19=14,2; p b0.001), indicating that ECS treatment dif-ferentially affected NPY mRNA levels in these regions in thetwo strains (Fig 2).

3.3.2. Parietal cortex and medial amygdalaECS had no significant effects on NPY mRNA levels in theseregions (Figs. 1 and 2).

3.4. NPY Y1 receptor mRNA expression

Y1 mRNA expression was in line with previous results (Fig. 3)(Larsen et al., 1993; Caberlotto et al., 1998) with high levelsin the cerebral cortex, the hippocampal formation, thethalamus and several hypothalamic nuclei.

3.5. Basal levels (Sham-treated animals)

3.5.1. CA1—2 regions, dentate gyrus and parietal cortexThe FSL had significantly lower Y1 mRNA levels compared tothe FRL rats, the reductions reaching 22% in the CA1—2regions ( F1,19=25.5; p b0.0001), 22% in the dentate gyrus( F1,19=24.7; p b0.0001) and 23% in the parietal cortex( F1,19=13.2; p b0.002) (Figs. 3 and 4).

3.5.2. Medial amygdala and hypothalamusNo significant strain differences in Y1 mRNA levels werefound in these regions (Figs. 3 and 4).

3.6. Effects of ECS treatment

3.6.1. CA1—2 regions, dentate gyrus and parietal cortexECS increased Y1 mRNA expression in both FRL and FSL ratsin the CA1—2 regions ( F1,19=32.5; p b0.0001) by 39% (vs.

n (coronal sections approximately �2.8 mm from Bregma) in ae (FSL) bdepressedQ rat under Sham conditions and after ECSrtex (PC), the CA 1—2 regions (CA), the dentate gyrus (DG), the

Figure 2 Effects of ECS and Sham treatments on NPY mRNA expression in brain tissue of the control FRL rats and the bdepressedQFSL rats. Results are presented as meanFS.D. based on 5—6 animals per group. Statistical analysis was performed by means of two-way ANOVA followed by Tukey—Kramer post-hoc tests and Bonferroni’s correction ( p’sb0.05). : = Significantly lower than FRL Sham;U = Significant difference between FRL Sham and FRL ECS; UU = Significant difference between FSL Sham and FSL ECS.

P.A. Jimenez-Vasquez et al.302

FRL Sham) and 35% (vs. FSL Sham), in the dentate gyrus( F1,19=85.8; p b0.0001) by 63% (vs. FRL Sham) and 56% (vs.FSL Sham) and in the parietal cortex ( F1,19 = 27.3;p b0.0001) by 22% (vs. FRL Sham) and 42% (vs. FSL Sham)(Figs. 3 and 4).

3.6.2. HypothalamusThe Y1 mRNA expression was increased by 26% (vs. FSL Sham)only in the FSL rats ( F1,19=15.9; p b0.001) (Figs. 3 and 4).

3.6.3. Medial amygdalaECS treatment did not to modulate the Y1 mRNA levels ineither strain (Figs. 3 and 4). There was no significantstrain� treatment interaction in any of the brain regionsanalyzed.

3.7. NPY Y1 receptor binding

In vitro receptor autoradiography using [125I][Leu31Pro34]—PYY showed a characteristic pattern of Y1 receptordistribution similar to that previously described (Caber-lotto et al., 1999; Dumont et al., 1998) with abundantbinding inter alia in the superficial layers of the cerebralcortex, the amygdala, the dentate gyrus and the thalamus(Fig. 5).

3.8. Basal binding (Sham-treated animals)

3.8.1. CA1—2 regions and dentate gyrusHigher levels of Y1 receptor binding were found in the FSLcompared to the FRL rats in the CA1—2 regions ( F1,18=30.5;p b0.0001) and the dentate gyrus ( F1,18=8.2; p b0.01), thelevels being increased by 62% and 26%, respectively (Figs. 5and 6).

3.8.2. Parietal cortex, medial amygdala and hypothalamusNo significant changes were seen.

3.9. Effects of ECS treatment

3.9.1. CA1—2 regions and dentate gyrusECS reduced the Y1 receptor binding in both FRL andFSL rats in these hippocampal regions. In the CA1—2regions the reduction was 50% (vs. FRL Sham) and61% (vs. FSL Sham) ( F1,18=115.1 p b0.0001). In thedentate gyrus the reduction was 66% (vs. FRL Sham)and 80% (vs. FSL Sham) ( F1,18=823.4; p b0.0001) (Figs.5 and 6).

A significant strain� treatment interaction was observedin the CA1—2 regions ( F1,18=11.9; p b0.003) and in thedentate gyrus ( F1,18=34.4; p b0.0001), indicating that ECStreatment differentially affected Y1 receptor binding levelsin the two strains (Fig 6).

3.9.2. Parietal cortex and hypothalamusECS reduced Y1 receptor binding only in the depressed FSLrats. In parietal cortex the reduction was 22% (vs. FSLSham) ( F1,18=5.5; p b0.05), and in the hypothalamus thereduction was 35% (vs. FSL Sham) ( F1,18=5.1; p b0.05)(Figs. 5 and 6).

3.9.3. Medial amygdalaNo treatment effects were found in either FSL or FRL rats inthis region (Figs. 5 and 6).

3.10. Porsolt swim test

The Sham treated FSL were immobile for longer timesthan the control FRL rats ( p b0.01) (Fig. 7). ECS

Figure 3 Representative autoradiograms of Y1 mRNA expression (coronal sections approximately �2.8 mm posterior from Bregma)in an FRL control rat and FSL bdepressedQ rat under Sham conditions and after ECS treatment. The NPY mRNA expression was studiedin the parietal cortex (PC), the CA1—2 regions (CA), the dentate gyrus (DG), the medial amygdala (MA) and the hypothalamus (HT).Scale=2 mm.

Effect of ECS on neuropeptide Y in depressed rats 303

diminished the immobility time in both strains. In the FRLrats it was reduced by 56% (vs. FRL Sham) and in the FSLrats by 43% (vs. FSL Sham) ( p’sb0.05 and 0.001,respectively) (Fig. 7). Following ECS treatment theimmobility time in the FSL strain was similar to that ofSham treated FRL rats.

Figure 4 Effects of ECS and Sham treatments on Y1 mRNAbdepressedQ FSL rats. Results are presented as meanFS.D. basedby means of two-way ANOVA followed by Tukey—Kramer post-hoclower than FRL Sham; U = Significant difference between FRL SSham and FSL ECS.

4. Discussion

4.1. Baseline levels

In the present study the bdepressedQ FSL rats showeddecreased basal expression of NPY mRNA, especially in the

expression in brain tissue of the control FRL rats and theon 5—6 animals per group. Statistical analysis was performedtests and Bonferroni’s correction ( p’sb0.05). : = Significantlyham and FRL ECS; UU = Significant difference between FSL

Figure 5 Representative autoradiograms of [125I][Leu31Pro34]—PYY binding (coronal sections approximately �2.8 mm posteriorfrom Bregma) in an FRL control rat and an FSL bdepressedQ rat under Sham conditions and after ECS treatment. The NPY mRNAexpression was studied in the parietal cortex (PC), the CA1—2 regions (CA), the dentate gyrus (DG), the medial amygdala (MA) andthe hypothalamus (HT). Scale=2 mm.

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hippocampal CA1—2 regions and the dentate gyrus. Inaddition, the Y1 receptor mRNA levels in the CA region, thedentate gyrus, and the parietal cortex were also de-creased, while the Y1 receptor binding was increased inthe CA1—2 regions and dentate gyrus compared to thecontrol FRL rats. The results are in agreement with

Figure 6 Effects of ECS and Sham treatment on NPY Y1 recebdepressedQ FSL rats. Results are presented as meanFS.D. based omeans of two-way ANOVA followed by Tukey—Kramer post-hoc telower than FRL Sham; U = Significant difference between FRL Shamand FSL ECS.

previous findings in the FSL and FRL rats (Caberlotto etal., 1998, 1999) and are likely due to decreased synthesis/release of NPY leading to adaptive upregulation of Y1receptor binding. The mechanism of the reduction of NPYmRNA and Y1 mRNA remains, however, to be clarified.Significantly, in another animal model of depression, the

ptor binding in brain tissue of the control FRL rats and then 5—6 animals per group. Statistical analysis was performed bysts and Bonferroni’s correction, ( p’sb0.05). : = Significantlyand FRL ECS; UU = Significant difference between FSL Sham

Figure 7 Effects of ECS and Sham treatment in the FRLcontrol rats and the FSL bdepressedQ rats on immobility in theswim test. The values are presented as meanFS.D. based onfive animals per group. Statistical analysis was performed bymeans of two-way ANOVA, followed by a Bonferroni post-hoctest. : = Significantly lower than FRL Sham; U = Significantdifference between FRL Sham and FRL ECS; UU = Significantdifference between FSL Sham and FSL ECS.

Effect of ECS on neuropeptide Y in depressed rats 305

FH rats, NPY-LI in hippocampus was decreased compared tothe control Wistar and Sprague—Dawley rats (Mathe et al.,1998). Furthermore, we consistently found reduced hippo-campal NPY-LI also in a behavioral model of depressionbearly life maternal separationQ (Jimenez-Vasquez et al.,2001; Husum and Mathe, 2002). Consequently, presentresults together with the above mentioned findings supportthe hypothesis that decreased hippocampal NPY plays arole in animal models of bdepressionQ and possibly also indepression in humans, and that one of the mechanismsinvolved is altered Y1 receptor mediated NPY transmission(Caberlotto et al., 1998, 1999; Mathe et al., 1998; Mathe,1999). The observed increase in Y1 receptor binding sitemay reflect and enhanced affinity, and/or a reducedinternalization due to the presumed reduced agonistactivation of the Y1 receptor based on the decreased NPYgene expression and protein levels (Mathe et al., 1998;Caberlotto et al., 1998, 1999; Jimenez-Vasquez et al.,2000a,b). An increased Y1 receptor synthesis is an unlikelyexplanation of the present findings, since the Y1 mRNAbaseline levels were in fact reduced. In line with thesefindings, decreased NPY-LI concentrations in CSF andplasma of depressed patients, as well as mRNA in brainsof suicide victims have been reported (Widdowson et al.,1992; Mathe et al., 1996; Hashimoto et al., 1996; Nilssonet al., 1996; Heilig et al., 2004). In humans, the Y1receptor was not altered in the prefrontal cortex ofsubjects afflicted with major depression or bipolar disor-der; unfortunately, the CA1—2 and the dentate gyrus werenot explored (Caberlotto and Hurd, 2001). Thus, thepresent findings suggest Y1 involvement in affectivedisorders. This assumption is strengthened by findings thatblockade of Y1 receptor inhibits the antidepressive effect

of NPY given centrally to rodents (Redrobe et al., 2002;Mathe and Gruber, 2004; Heilig, 2004; Thorsell et al.,2006). It is also conceivable that changes in Y4 and Y5receptors have in part contributed to our findings since theligand used ([Leu31Pro34]—PYY) also exhibits some affinityfor the Y4 and Y5 receptors. However, the affinity for Y4and Y5 is low compared to Y1 and, moreover, the Y4 and Y5constitute only a very small proportion of the total numberof NPY receptors in rat brain (Dumont et al., 1998; Dumontand Quirion, 2006). Thus further investigations with moreselective Y1, Y4, and Y5 agonists and antagonists arenecessary to elucidate if and to what extent the Y4 andY5 receptor subtypes might also be changed in affectivedisorders.

A link between hippocampal NPY and memory processinghas been shown in animal experiments. Thus, intracerebro-ventricular injections of NPY improve memory retention inmice (Flood et al., 1989). An improvement in memoryretention was observed when NPY was injected into eitherdorsal hippocampus or septum, while administration into theamygdala or ventral hippocampus induced amnesia (Flood andMorley, 1989; Morley and Flood, 1990). NPY can also reversethe amnesia observed following administration of scopolamineor protein synthesis inhibitors (Flood et al., 1987). Theseeffects havebeen shown tomainly involve Y2 receptors (Morleyand Flood, 1990). The reduction of NPY mRNA in the FSLhippocampal formation reported here together with previousfindings of decreased NPY-LI in the hippocampus of FH and FSLrats (Mathe et al., 1998; Caberlotto et al., 1999; Jimenez-Vasquez et al., 2000a,b), raise the possibility that the memorydeficits observed in depressed subjects may, in part, besecondary to reduced NPY.

4.2. Effects of ECS

To the best of our knowledge this is the first study to explorethe effects of repeated ECS treatment using a combinedanalysis of NPY mRNA and Y1 receptor mRNA levels as well asof Y1 receptor binding in specific brain areas of a geneticanimal model of depression. We found that repeated ECSincrease NPY mRNA and Y1 receptor mRNA levels in specificareas in both FSL and FRL rats, especially in the hippocampalformation. Consistent with these data, are the findings ofdecreased Y1 receptor binding after ECS. Thus our results inbdepressedQ rats are in line with and extend the reportedchanges in both Y1 and Y2 receptors in normal rat brainfollowing ECS (Madsen et al., 2000). Significantly, effects ofECS on brain NPYand behavior were larger in the bdepressedQFSL strain compared to the control FRL indicating that thesevariables reflect one of the therapeutic mechanisms of ECTaction. Lithium also enhances NPY-LI release in vivo and NPY-LI and NPY mRNA levels in the hippocampus (Husum et al.,2000; Mathe et al., 1990; Weiner et al., 1992; Zachrisson etal., 1995b), thereby lending support to the hypothesis thatone of the common denominators of antidepressive treat-ment modalities is upregulation of the NPY system (Mathe,1999; Mathe et al., 2003).

The observations that ECS increased NPY mRNA and Y1mRNA levels and decreased Y1 receptor binding in thehypothalamus only in the bdepressedQ FSL animals areconsistent with the findings that ECS increases NPY-LI inthis region in the FSL (Jimenez-Vasquez et al., 2000a,b).

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Since it is assumed that mood changes and changes inappetite and food intake in major depression may in partreflect pathophysiological processes in hippocampus andhypothalamus, the elevation of NPY mRNA levels anddecreased Y1 binding sites in these two regions by ECS arealso in line with the hypothesis that one of the mechanismsof action of ECT in depression is an increased bioavailabilityof NPY, in particular in the hippocampus. As expected, therewere no FSL—FRL differences in the amygdala in the NPYparameters measured under the basal conditions; moreover,ECS also had no apparent effects. This is consistent with thenotion that amygdala are primarily but not exclusivelyinvolved in fear and anxiety, whereas hippocampus is mainlyinvolved in memory function and depression. Previous workshowing decreased NPY in hippocampus and effects of ECS inthis region is also in line with such reasoning (Stenfors et al.,1989; Mathe, 1999; Jimenez-Vasquez et al., 2000a,b, 2001)and so are the results from an environmental depressionmodel bthe early maternal separationQ, where NPY-LI wasdecreased in the hippocampus but not in the amygdala(Jimenez-Vasquez et al., 2001; Husum and Mathe, 2002).Furthermore, NPY-LI was also decreased in the hippocam-pus, but not in the amygdala of an alcohol preferring ratmodel (Ehlers et al., 1998), findings that led to thehypothesis that a dysregulation of the NPY system mayconstitute a core mechanism of disturbed emotionality(Mathe et al., 2003). Since a series of ECS results inincreased synthesis/release of NPY (Stenfors et al., 1989;Jimenez-Vasquez et al., 2000a,b; Husum et al., 2000) andNPY Y1 receptor and an inverse relationship between NPYmRNA levels and Y1 receptor binding has been reported innormal rats (Mikkelsen et al., 1994; Greisen et al., 1997;Madsen et al., 2000) it seems reasonable that increasedagonist activation of the Y1 receptors leads to decreased Y1binding and that this occurs via a reduction of affinity and/or an increased internalization and breakdown of the Y1receptor.

In another study, a 2 week fluoxetine (3 mg/kg)treatment increased NPY mRNA expression in the CA1—2regions and dentate gyrus of the FSL, but not the FRLrats (Caberlotto et al., 1999). Moreover, no significantchanges in the hippocampal Y1 receptor mRNA or bindingwere observed. These differences could be due either tothe facts that a relatively low fluoxetine dose was usedand it was given once daily (although an antidepressantaction was demonstrated behaviorally; Caberlotto et al.,1998, 1999) or to different profiles of ECS and antide-pressant drug action. The latter assumption is consistentwith our previous findings that although ECS, lithium andcitalopram, all affected the NPY system, the effects werenot parallel, that is ECS and lithium’s effects weresimilar but not identical to those of citalopram (Husumet al., 2000).

The baseline FSL—FRL differences observed in thePorsolt swim test are a good indicator that FSL rats area reasonable model of depression, while the finding thatECS reversed the immobility shown by the FSL in the swimtest is in line with its antidepressant properties. In thiscontext, it is noteworthy that intracerebroventricularinjections of NPY, in particular in the FSL strain (Matheand Gruber, 2004) produce antidepressant like effects,similar to those produced by SSRIs in the swim test (Lucki,

1997; Caberlotto et al., 1999; Stogner and Holmes, 2000).These results have been confirmed and extended in themouse version of this test; e.g. icv administration of[Leu31Pro34]—PYY reduced immobility time in a dosedependent manner (Redrobe et al., 2002). These datalead to suggestion that increases in NPY gene expressionand release, proposed to be one of the ECS mechanisms ofaction (Mathe, 1999), are mediated via increased NPY Y1transmission.

In the parietal cortex of both FSL and FRL rats, ECSdid not change the NPY mRNA but increased Y1 mRNA (Y1receptor binding was reduced but only in FSL). Thesedata are similar to other studies, where ECS increasesboth NPY mRNA and NPY-LI in cortical areas in normalrats (Wahlestedt et al., 1990; Mikkelsen et al., 1994;Mathe, 1999). The profile of changes is similar to that inthe hippocampal formation and the hypothalamus, sug-gesting that also in this region enhancement of NPY Y1receptor mediated transmission occurs after ECT. On theother hand, SSRIs and tricyclics do not result inconsistent NPY-LI changes in cortical areas; increases,decreases, or no changes have been reported (Bellmannand Sperk, 1993; Heilig et al., 1988; Smialowska andLegutko, 1991; Widdowson and Halaris, 1991; Heilig,2004; Thorsell et al., 2006). In addition to being anefficient antidepressant, ECT is also efficient in therapyof psychosis, acute mania and psychotic features indepression (Mathe, 1999). Consequently, and as previouslysuggested (Mathe et al., 1998; Mathe, 1999), the effectsof ECS on NPY in the cortical areas may constitute one ofits mechanisms of action in psychosis.

In summary, NPY mRNA and Y1 receptor mRNA weredecreased while Y1 receptor binding was increased in thebdepressedQ FSL compared to the control FRL rats; thealterations were most marked in the CA1—2 regions and thedentate gyrus. ECS counteracted these changes and hadsignificantly larger effects in the FSL. The presumed resto-ration of NPY Y1 transmission by ECS in the FSL rats suggeststhat these effects may constitute one of the therapeuticmechanisms of ECT action and focuses interest on NPY Y1mediated hippocampal transmission in depressive disorders.

Acknowledgments

The authors would like to thank Annika Andersson andAnders Knall for technical assistance. This work wassupported by funds from the Karolinska Institutet, theSwedish Medical Research Council (grants 10414 to A.A.Mathe and 04x-715 to K. Fuxe), and the G. and Z. CostakisSwedish Foundation for Medical Research.

References

Bellmann, R., Sperk, G., 1993. Effects of antidepressant drugtreatment on levels of NPY or prepro-NPY-mRNA in the rat brain.Neurochem. Int. 22, 183–187.

Benfenati, F., Cimino, M., Agnati, L.F., Fuxe, K., 1986. Quantitativeautoradiography of central neurotransmitter receptors: meth-odological and statistical aspects with special reference tocomputer-assisted image analysis. Acta Physiol. Scand. 128,129–146.

Effect of ECS on neuropeptide Y in depressed rats 307

Berrettini, W.H., Doran, A.R., Kelsoe, J., Roy, A., Pickar, D., 1987.Cerebrospinal fluid neuropeptide Y in depression and schizo-phrenia. Neuropsychopharmacology 1, 81–83.

Caberlotto, L., Hurd, Y.L., 2001. Neuropeptide Y Y(1) and Y(2)receptor mRNA expression in the prefrontal cortex of psychiatricsubjects. Relationship of Y(2) subtype to suicidal behavior.Neuropsychopharmacology 25, 91–97.

Caberlotto, L., Fuxe, K., Overstreet, D.H., Gerrard, P., Hurd, Y.L.,1998. Alterations in neuropeptide Y and Y1 receptor mRNAexpression in brains from an animal model of depression: regionspecific adaptation after fluoxetine treatment. Mol. Brain Res.59, 58–65.

Caberlotto, L., Jimenez, P., Overstreet, D.H., Hurd, Y.L., Mathe,A.A., Fuxe, K., 1999. Alterations in neuropeptide Y levels and Y1binding sites in the Flinders Sensitive rats, a genetic animalmodel of depression. Neurosci. Lett. 263, 191–194.

Daws, L.C., Overstreet, D.H., 1999. Ontogeny of muscariniccholinergic supersensitivity in the Flinders Sensitive Line rat.Pharmacol. Biochem. Behav. 62, 367–380.

Dumont, Y., Quirion, R., 2006. An overview of neuropeptide Y:pharmacology to molecular biology and receptor localization.EXS 95, 7–33.

Dumont, Y., Fournier, A., St-Pierre, S., Quirion, R., 1995. Charac-terization of neuropeptide Y binding sites in rat brain membranepreparations using [125I][Leu31Pro34]peptide YY and [125I]peptideYY3-36 as selective Y1 and Y2 radioligands. J. Pharmacol. Exp.Ther. 272, 673–680.

Dumont, Y., Fournier, A., Quirion, R., 1998. Expression andcharacterization of the neuropeptide Y Y5 receptor subtype inthe rat brain. J. Neurosci. 18, 5565–5574.

Ehlers, C.L., Li, T.K., Lumeng, L., Hwang, B.H., Somes, C.,Jimenez, P., Mathe, A.A., 1998. Neuropeptide Y levels inethanol-naive alcohol-preferring and nonpreferring rats and inWistar rats after ethanol exposure. Alcohol., Clin. Exp. Res. 22,1778–1782.

Flood, J.F., Morley, J.E., 1989. Dissociation of the effects ofneuropeptide Y on feeding and memory: evidence for pre- andpostsynaptic mediation. Peptides 10, 963–966.

Flood, J.F., Hernandez, E.N., Morley, J.E., 1987. Modulation ofmemory processing by neuropeptide Y. Brain Res. 421, 280–290.

Flood, J.F., Baker, M.L., Hernandez, E.N., Morley, J.E., 1989.Modulation of memory processing by neuropeptide Y varies withbrain injection site. Brain Res. 503, 73–82.

Fredriksson, R., Larson, E.T., Yan, Y-L., Postlethwait, J.H., Larham-mar, D., 2004. Novel neuropeptide Y Y2-like receptor subtype inzebrafish and frogs supports early vertebrate chromosomeduplications. J. Mol. Evol. 58, 106–114.

Fuxe, K., Aguirre, J.A., Agnati, L.F., von Euler, G., Hedlund, P.,Covenas, R., Zoli, M., Bjelke, B., Eneroth, P., 1990. Neuropep-tide Y and central cardiovascular regulation. Focus on its role asa cotransmitter in cardiovascular adrenergic neurons. Ann. N. Y.Acad. Sci. 611, 111–132.

Greisen, M.H., Sheikh, S.P., Bolwig, T.G., Mikkelsen, J.D., 1997.Reduction of neuropeptide Y (NPY) binding sites in the rathippocampus after electroconvulsive stimulations. Brain Res.776, 105–110.

Hanze, J., Kummer, W., Haass, M., Lang, R.E., 1991. Neuropeptide YmRNA regulation in rat sympathetic ganglia: effect of reserpine.Neurosci. Lett. 124, 119–121.

Hashimoto, H., Onishi, H., Koide, S., Kai, T., Yamagami, S., 1996.Plasma neuropeptide Y in patients with major depressivedisorder. Neurosci. Lett. 216, 57–60.

Heilig, M., 2004. The NPY system in stress, anxiety and depression.Neuropeptides 38, 213–224.

Heilig, M., Wahlestedt, C., Ekman, R., Widerlov, E., 1988. Antide-pressant drugs increase the concentration of neuropeptide y(NPY)-like immunoreactivity in the rat brain. Eur. J. Pharmacol.147, 465–467.

Heilig, M., Zachrisson, O., Thorsell, A., Ehnvall, A., Mottagui-Tabar,S., Sjogren, M., Asberg, M., Ekman, R., Wahlestedt, C., Agren,H., 2004. Decreased cerebrospinal fluid neuropeptide Y (NPY) inpatients with treatment refractory unipolar major depression:preliminary evidence for association with preproNPY genepolymorphism. J. Psychiatr. Res. 38, 113–121.

Husum, H., Mikkelsen, J.D., Hogg, S., Mathe, A.A., Mork, A., 2000.Involvement of hippocampal neuropeptide Y in mediating thechronic actions of lithium, electroconvulsive stimulation andcitalopram. Neuropharmacology 39, 1463–1473.

Husum, H., Mathe, A.A., 2002. Early life stress changes concentra-tions of neuropeptide Y and corticotropin-releasing hormone inadult rat brain, lithium treatment modifies these changes.Neuropsychopharmacology. 27, 756–764.

Janowsky, D.S., Risch, S.C., 1987. Acetylcholine mechanism inaffective disorders. In: Meltzer, H.T. (Ed.), Psychopharmacology.The Third Generation of Progress. Raven Press, New York,pp. 527–534.

Jimenez-Vasquez, P.A., Salmi, P., Ahlenius, A., Mathe, A.A., 2000a.Neuropeptide-Y in brains of Flinders Sensitive Line rat, a modelof depression. Effects of electroconvulsive stimuli and d-amphetamine on peptide concentrations and locomotion. Behav.Brain Res. 111, 115–123.

Jimenez-Vasquez, P.A., Overstreet, D.H., Mathe, A.A., 2000b.Neuropeptide Y in male and female brains of Flinders SensitiveLine, a rat model of depression. Effects of electroconvulsivestimuli. J. Psychiatr. Res. 34, 405–412.

Jimenez-Vasquez, P.A., Mathe, A.A., Thomas, J.D., Riley, E.P.,Ehlers, C.L., 2001. Early maternal separation alters neuropep-tide Y concentrations in selected brain regions in adult rats.Brain Res. Dev. Brain Res. 131, 149–152.

Kask, A., Harro, J., von Horsten, S., Redrobe, J.P., Dumont, Y.,Quirion, R., 2002. The neurocircuitry and receptor subtypesmediating anxiolytic-like effects of neuropeptide Y. Neurosci.Biobehav. Rev. 26, 259–283.

Kopp, J., Nanobashvili, A., Kokaia, Z., Lindvall, O., Hokfelt, T.,1999. Differential regulation of mRNAs for neuropeptide Yand itsreceptor subtypes in widespread areas of the rat limbic systemduring kindling epileptogenesis. Mol. Brain Res. 72, 17–29.

Larsen, P.J., Sheikh, S.P., Jakobsen, C.R., Schwartz, T.W.,Mikkelsen, J.D., 1993. Regional distribution of putative NPYY1 receptors and neurons expressing Y1 mRNA in forebrainareas of the rat central nervous system. Eur. J. Neurosci. 5,1622–1637.

Larsson, T.A., Larson, E.T., Fredriksson, R., Conlon, J.M., Larhammar,D., 2006. Characterization of NPY receptor subtypes Y2 and Y7 inrainbow trout Oncorhynchus mykiss. Peptides 27, 1320—1327.

Lucki, I., 1997. The forced swimming test as a model for core andcomponent behavioral effects of antidepressant drugs. Behav.Pharmacol. 8, 523–532.

Madsen, T.M., Woldbye, D.P., Larsen, P.J., Bolwig, T.G., Mikkelsen,J.D., 2000. Electroconvulsive treatment enhance both neuro-peptide Y receptor Y1 and Y2 messenger RNA expressionand levels of binding in the rat hippocampus. Neuroscience 98,33–39.

Mathe, A.A., 1999. Neuropeptides and electroconvulsive treatment.J. ECT 15, 60–75.

Mathe, A.A., Gruber, S.H.M., 2004. Neuropeptide Y has markedantidepressant properties in a rat model of depression. Neurop-sychopharmacology 29, 154–155.

Mathe, A.A., Jousisto-Hanson, J., Stenfors, C., Theodorsson, E.,1990. Effect of lithium on tachykinins, calcitonin gene-relatedpeptide, and neuropeptide Y in rat brain. J. Neurosci. Res. 26,233–237.

Mathe, A.A., Norstedt-Wikner, B., Stenfors, C., Theodorsson, E.,1994. Effects of lithium on neuropeptide Y, neurokin A, andsubstance P in brain and peripheral tissues of rat. Lithium 5,241–247.

P.A. Jimenez-Vasquez et al.308

Mathe, A.A., Rudorfer, W.V., Stenfors, C., Manji, H.K., Potter, W.Z.,Theodorsson, E., 1996. Effects of electroconvulsive treatmenton somatostatin, neuropeptide Y, endothelin, and neurokinin Aconcentrations in cerebrospinal fluid of depressed patients.Depression 3, 250–256.

Mathe, A.A., Jimenez, P.A., Theodorsson, E., Stenfors, C., 1998.Neuropeptide Y, neurokinin A and neurotensin in brain regions ofFawn Hooded bdepressedQ, Wistar, and Sprague Dawley rats.Effects of electroconvulsive stimuli. Prog. Neuro-psychopharma-col. Biol. Psychiatry 22, 529–546.

Mathe, A.A., Husum, H., Jimenez-Vasquez, P.J., Bolwig, T.G.,Overstreet, D.H., Li, T.-K., Riley, E.P., Slawecki, C.J., Heilig,M., Ehlers, C.L., 2003. Neuropeptide Y is the marker of mooddisorders and risk for alcohol dependence. Biol. Psychiatry 53,68.

Mikkelsen, J.D., Woldbye, D., Kragh, J., Larsen, P., Bolwig, T.G.,1994. Electroconvulsive shocks increase the expression ofneuropeptide Y (NPY) mRNA in the piriform cortex and thedentate gyrus. Mol. Brain Res. 23, 317–322.

Morley, J.E., Flood, J.F., 1990. Neuropeptide Y and memoryprocessing. Ann. N. Y. Acad. Sci. 611, 226–231.

Morris, B.J., 1989. Neuronal localisation of neuropeptide Y geneexpression in rat brain. J. Comp. Neurol. 290, 358–368.

Nikisch, G., Agren, H., Eap, C.B., Czernik, A., Baumann, P., Mathe,A.A., 2005. Neuropeptide Y and corticotropin-releasing hormonein CSF mark response to antidepressive treatment with citalo-pram. Int. J. Neuropsychopharmacol. 8, 403–410.

Nilsson, C., Karlsson, G., Blennow, K., Heilig, M., Ekman, R., 1996.Differences in the neuropeptide Y-like immunoreactivity of theplasma and platelets of human volunteers and depressedpatients. Peptides 17, 359–362.

Ordway, G.A., Stockmeier, C.A., Meltzer, H.Y., Overholser, J.C.,Jaconetta, S., Widdowson, P.S., 1995. Neuropeptide Y in frontalcortex is not altered in major depression. J. Neurochem. 65,1646–1650.

Overstreet, D.H., 1986. Selective breeding for increased cholinergicfunction: development of a new animal model of depression.Biol. Psychiatry 21, 49–58.

Overstreet, D.H., Pucilowski, O., Rezvani, A.H., Janowsky, D.S.,1995. Administration of antidepressants, diazepam and psycho-motor stimulants further confirms the utility of FlindersSensitive Line rats as an animal model of depression. Psycho-pharmacology (Berl.) 121, 27–37.

Overstreet, D.H., Friedman, E., Mathe, A.A., Yadid, G., 2005.The Flinders Sensitive Line rat: a selectively bred putativeanimal model of depression. Neurosci. Biobehav. Rev. 9,739–759.

Paxinos, G., Palacios, J.M., Mengod, G., 1986. The Rat Brain inStereotaxic Coordinates. Academic Press, Sydney.

Porsolt, R.D., Anton, G., Blavet, N., Jalfre, M., 1978. Behavioraldespair in rats: a new model sensitive to antidepressanttreatments. Eur. J. Pharmacol. 47, 379–391.

Redrobe, J.P., Dumont, Y., Fournier, A., Quirion, R., 2002. Theneuropeptide Y (NPY) Y1 receptor subtype mediates NPY-inducedantidepressant-like activity in the mouse forced swimming test.Neuropsychopharmacology 5, 615–624.

Smialowska, M., Legutko, B., 1991. Influence of imipramine onneuropeptide Y immunoreactivity in the rat brain. Neuroscience41, 767–771.

Stenfors, C., Theodorsson, E., Mathe, A.A., 1989. Effect of repeatedelectroconvulsive treatment on regional concentrations oftachykinins, neurotensin, vasoactive intestinal polypeptide,neuropeptide Y, and galanin in rat brain. J. Neurosci. Res. 24,445–450.

Stenfors, C., Srinivasan, G.R., Theodorsson, E., Mathe, A.A., 1992.Electroconvulsive stimuli and brain peptides: effect of modifi-cation of seizure duration on neuropeptide Y, neurokinin A,substance P and neurotensin. Brain Res. 596, 251–258.

Stenfors, C., Mathe, A.A., Theodorsson, E., 1994. Repeatedelectroconvulsive stimuli: changes in neuropeptide Y, neuroten-sin and tachykinin concentrations in time. Prog. Neuro-psycho-pharmacol. Biol. Psychiatry 18, 201–209.

Stogner, K.A., Holmes, P.V., 2000. Neuropeptide-Y exerts antide-pressant-like effects in the forced swim test in rats. Eur. J.Pharmacol. 387, 9 –10.

Thorsell, A., Karlsson, M.R., Heilig, M., 2006. NPY in alcoholism andpsychiatric disorders. EXS 95, 183–192.

Wahlestedt, C., Heilig, M., 1995. Neuropeptide Y and RelatedPeptides. In: Bloom, F.E., Kupfer, D.J. (Eds.), Psychopharmacol-ogy: The Fourth Generation of Progress. Raven Press, New York,pp. 543–551.

Wahlestedt, C., Blendy, J.A., Kellar, K.J., Heilig, M., Widerlov, E.,Ekman, R., 1990. Electroconvulsive shocks increase the concen-tration of neocortical and hippocampal neuropeptide Y (NPY)-like immunoreactivity in the rat. Brain Res. 507, 65–68.

Weiner, E.D., Mallat, A.M., Papolos, D.F., Lachman, H.M., 1992.Acute lithium treatment enhances neuropeptide Y gene expres-sion in rat hippocampus. Mol. Brain Res. 12, 209–214.

Westrin, A., Ekman, R., Traskman-Bendz, L., 1999. Alterations ofcorticotropin releasing hormone (CRH) and neuropeptide Y (NPY)plasma levels in mood disorder patients with a recent suicideattempt. Eur. Neuropsychopharmacol. 9, 205–211.

Widdowson, P.S., Halaris, A.E., 1991. Chronic desipramine treat-ment reduces regional neuropeptide Y binding to Y2-typereceptors in rat brain. Brain Res. 25 (539), 196–202.

Widdowson, P.S., Ordway, G.A., Halaris, A.E., 1992. Reducedneuropeptide Y concentrations in suicide brain. J. Neurochem.59, 73–80.

Widerlov, E., Heilig, M., Ekman, R., Wahlestedt, C., 1988. Possiblerelationship between neuropeptide Y (NPY) and major depres-sion — evidence from human and animal studies. Nord. Psykiatr.Tidsskr. 42, 131–137.

Zachrisson, O., Mathe, A.A., Stenfors, C., Lindefors, N., 1995a.Limbic effects of repeated electroconvulsive stimulation onneuropeptide Y and somatostatin mRNA expression in the ratbrain. Mol. Brain Res. 31, 71–85.

Zachrisson, O., Mathe, A.A., Stenfors, C., Lindefors, N., 1995b.Region-specific effects of chronic lithium administration onneuropeptide Y and somatostatin mRNA expression in the ratbrain. Neurosci. Lett. 194, 89–92.