chronic intracerebral prolactin attenuates neuronal stress circuitries in virgin rats

Chronic intracerebral prolactin attenuates neuronal stresscircuitries in virgin rats

Nina Donner, Remco Bredewold, Rodrigue Maloumby and Inga D. NeumannDepartment of Behavioural Neuroendocrinology, University of Regensburg, Regensburg, Germany

Keywords: anxiety, c-fos, corticotropin-releasing factor, paraventricular nucleus, stress

Abstract

Prolactin (PRL) has been shown to promote maternal behaviour, and to regulate neuroendocrine and emotional stress responses.These effects appear more important in the peripartum period, when the brain PRL system is highly activated. Here, we studied themechanisms that underlie the anti-stress effects of PRL. Ovariectomized, estradiol-substituted Wistar rats were implanted with anintracerebroventricular cannula and treated with ovine PRL (0.01, 0.1 or 1 lg ⁄ h; 5 days via osmotic minipumps) or vehicle, and theirresponses to acute restraint stress was assessed. Chronic PRL treatment exerted an anxiolytic effect on the elevated plus-maze, andattenuated the acute restraint-induced rise in plasma adrenocorticotropin, corticosterone and noradrenaline. At the neuronal level,in situ hybridization revealed PRL effects on the expression patterns of the immediate-early gene c-fos and corticotropin-releasingfactor (CRF). Under basal conditions, PRL significantly reduced c-fos mRNA expression within the central amygdala. In response torestraint, the expression of both c-fos mRNA and protein and of CRF mRNA was decreased in the parvocellular part of theparaventricular nucleus (PVN) of PRL-treated compared with vehicle-treated animals. In conclusion, our data demonstrate thatchronic elevation of PRL levels within the brain results in reduced neuronal activation within the hypothalamus, specifically within thePVN, in response to an acute stressor. Thus, PRL acting at various relevant brain regions exerts profound anxiolytic and anti-stresseffects, and is likely to contribute to the attenuated stress responsiveness found in the peripartum period, when brain PRL levels arephysiologically upregulated.

Introduction

Synthesized mainly in lactotrophe cells of the adenohypophysis,prolactin (PRL) exerts classical hormonal effects, including lactogen-esis (Ben-Jonathan et al., 1996). In addition, PRL synthesis (DeVitoet al., 1992; Emanuele et al., 1992; Clapp et al., 1994; Torner et al.,2002), PRL immunoreactivity (Paut-Pagano et al., 1993) and binding(Crumeyrolle-Arias et al., 1993; Chiu & Wise, 1994; Bakowska &Morrell, 1997; Pi & Grattan, 1998a; Fujikawa et al., 2004) have beendescribed in various brain regions, including the paraventricularnucleus of the hypothalamus (PVN). PRL has been demonstrated topromote maternal behaviour (Bridges et al., 1990; Lucas et al., 1998;Torner et al., 2002), grooming (Drago et al., 1983) and food intake(Noel & Woodside, 1993; Li et al., 1995). PRL has also been shown tobe locally released from hypothalamic neurons in response to relevantstimulation, including suckling and exposure to a psychologicalstressor (Torner et al., 2004), which fully qualifies it as a brainneuropeptide (Hokfelt et al., 2000).As a potential regulator of the stress responsiveness (Drago et al.,

1985; Torner et al., 2001, 2002, 2004; Fujikawa et al., 2005), acuteintracerebroventricular (i.c.v.) PRL exerts anxiolytic effects both infemale and male rats (Torner et al., 2001). Moreover, brain PRL islikely to attenuate the stress-induced secretion of adrenocorticotropin(ACTH) and corticosterone into blood. These central effects aremediated via brain PRL receptors as indicated by anti-sense-induced

receptor downregulation (Torner et al., 2001). In this context it isimportant to note that the brain PRL system is highly activated in theperipartum period, a time of significant adaptations in emotionalityand neuroendocrine stress responsiveness (Russell et al., 1999;Toufexis et al., 1999; Lightman et al., 2001; Neumann, 2001;DeWeerth & Buitelaar, 2005). Accordingly, enhanced PRL receptorexpression in the brain (Sugiyama et al., 1994; Augustine et al., 2003;Kokay & Grattan, 2005) and elevated levels of PRL receptorimmunoreactivity specifically within hypothalamic brain regions(Pi & Grattan, 1999; Grattan et al., 2001) have been demonstrated.Additionally, hypothalamic PRL expression is enhanced in pregnantand lactating animals (Torner et al., 2002). Thus, the reduced level ofanxiety, the attenuated responses of the hypothalamo–pituitary–adrenal (HPA) axis and the sympathetic nervous system (Altemuset al., 1995; Douglas et al., 2005), as well as the blunted c-fos andcorticotropin-releasing factor (CRF) mRNA expression withinhypothalamic and limbic brain regions (Da Costa et al., 1996,2001), which are characteristic for the peripartum period (Stern et al.,1973; Lightman & Young, 1989; Walker et al., 1995; Windle et al.,1997; Douglas et al., 1998; Neumann et al., 1998), may at least partlybe due to the overall activation of the brain PRL system (Torner &Neumann, 2002; Torner et al., 2002).To examine this hypothesis, we investigated the effects of chronic

PRL treatment (5 days) on behavioural, neuroendocrine and neuronalstress parameters on virgin female rats. All animals were ovariectom-ized (OVX) to avoid cycle-dependent changes in steroid hormones, andestradiol-substituted (OVXE2) to maintain low 17-b-estradiol (E2)levels, as seen in di- and proestrus rats. Chronic treatment with PRLinto the brain ventricular system was used to induce brain hyper-

Correspondence: Dr I. D. Neumann, Department of Behavioral Neuroendorinology,University of Regensburg, 93040 Regensburg, Germany.E-mail: [email protected]

Received 4 October 2006, revised 12 January 2007, accepted 16 January 2007

European Journal of Neuroscience, Vol. 25, pp. 1804–1814, 2007 doi:10.1111/j.1460-9568.2007.05416.x

ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd

prolactinaemia, a physiological state also known from the peripartumperiod in humans (Altemus et al., 2004). The anxiety-related behaviouron the elevated plus-maze (EPM), plasma ACTH, corticosterone andnoradrenaline concentrations, and the neuronal expression of both theimmediate-early gene c-fos and of the CRF gene were determinedunder basal conditions and in response to acute restraint.

Materials and methods

Animals

All surgical, sampling and behavioural protocols were approved by theCommittee on Animal Health and Care of the local governmentaladministration, and are in accordance with the guide for the care anduse of laboratory animals by the NIH. Virgin female Wistar rats[200–210 g body weight (BW) at the beginning of the experiments;Charles River, Sulzfeld, Germany] were kept under standard labor-atory conditions (12 : 12 h light : dark cycle, lights on at 06.00 h,22 �C, 60% humidity, and free access to water and standard rat chow).BW was measured on Day 1 before OVX and on Day 8 afterbehavioural testing on the EPM.

Experimental protocol

Under isoflurane anaesthesia, virgin female rats underwent OVX andreceived a subcutane E2 pellet at the same time. Four days later, anosmotic minipump-driven i.c.v. cannula was implanted under deepisoflurane anaesthesia, in order to deliver ovine PRL (oPRL) orvehicle into the right lateral ventricle for 5 days. After 4 days of oPRLtreatment, the anxiety-related behaviour of the rats was tested on theEPM between 09.00 and 12.00 h. The next day, rats were exposed torestraint for 30 min (Plexiglas tube, 30 cm long, 10 cm diameter, withsmall holes for oxygen support) between 08.00 and 10.00 h, andbrains were removed after decapitation at varying time pointsthereafter. In order to study hypothalamic CRF mRNA expression,the first set of animals was returned to their home cages anddecapitated 180 min after termination of restraint, as CRF mRNAlevels have been shown to peak 180 min after exposure to restraint(Rivest et al., 1995). In order to study c-fos mRNA expression, asecond set of animals was killed immediately (within 1 min) aftertermination of 30-min restraint without returning the animals to theirhome cages. The trunk blood of all killed animals was collected intoEDTA-coated tubes (10 lL Trasylol, Sarstedt AG, Numbrecht,Germany) and, after centrifugation (5 min, 5000 rpm, 4 �C), plasmasamples were stored at ) 20 �C for the detection of ACTH,corticosterone and noradrenaline. All animals were killed between09.00 and 11.00 h.

Brains were frozen in dry ice-prechilled n-methylbutane and storedat ) 20 �C until sectioning. Adrenal glands were cleaned fromsurrounding tissue, stored in isotonic saline on ice and weighed.Respective control animals remained unstressed. Some animalswithout stereotaxic surgery and i.c.v. treatment were included as anadditional control group (OVXE2-group, unstressed controls: n ¼ 4:killed immediately after restraint: n ¼ 4) to ensure that brain surgeryper se did not cause changes in neuronal gene expression.

Surgical procedures

All surgical procedures were performed under deep isofluraneanaesthesia (Baxter GmbH, Unterschleissheim, Germany) and semi-sterile conditions.

OVX and E2-replacement

Because ovarian steroid variations during the estrous cycle mayinfluence hypophyseal PRL secretion (Neill et al., 1971), PRLreceptor expression (Shamgochian et al., 1995; Pi & Grattan, 1998b)and also c-fos reaction to stress (Figueiredo et al., 2002), each ratunderwent bilateral OVX via the dorsal approach and simultaneousimplantation of a SILASTIC capsule (1.5 mm ID, 3.5 mm OD, DowCorning Midland, MI, USA; 10 mm length ⁄ 100 g body weight)containing vegetable oil with 150 lg ⁄ mL E2 (Sigma-Aldrich, Schn-elldorf, Germany). This mimics E2 concentrations within the normalproestrus range (Goodman, 1978), and should result in a constant,high level expression of PRL receptors in the brain (Mustafa et al.,1995; Shamgochian et al., 1995).

Chronic i.c.v. PRL administration

Osmotic minipumps (model 1007D; Alzet, Palo Alto, CA, USA,purchased from Charles River) were used to continuously infuse oPRLinto the right lateral ventricle over 5 days and to mimic the elevatedPRL levels in the brain peripartum. This method of administrationobviates additional animal handling and repeated infusions during thetreatment period. Minipumps were filled with concentrations of oPRLcalculated to deliver doses of 0.01 lg ⁄ h (n ¼ 11), 0.1 lg ⁄ h (n ¼ 12)and 1 lg ⁄ h (n ¼ 34) or vehicle (5% 0.01 m NaOH ⁄ 95% Ringersolution, n ¼ 33) at a delivery rate of 0.5 lL ⁄ h. The pump wasconnected to the i.c.v. cannula via polyethylene tubing, stored inisotonic saline overnight at room temperature and allowed toequilibrate to 37 �C for 2 h before implantation. Four days afterOVX, rats were anaesthetised with isoflurane, and the i.c.v. cannulawas stereotaxically positioned in the lateral ventricle. The minipumpwas placed subcutaneously between the scapulae, and the braininfusion device was secured to the skull using dental cement andstainless steel screws.Following surgery, rats received a depot injection of antibiotic

(30 lL subcutaneously, Baytril�, Bayer Leverkusen, Germany). Ratswere housed singly and handled carefully to reduce non-specific stressresponses during the experiment.

EPM

In order to study chronic effects of PRL on anxiety-related behaviour,rats were tested on the EPM. In brief, on the EPM, a conflict situationis created between the rat’s exploratory drive and its innate fear ofopen and exposed areas (Pellow et al., 1985). The EPM is built of anelevated (80 cm) plus-shaped platform with two closed and two openarms, connected at the centre by a neutral zone (10 · 10 cm). The ratwas placed in the neutral zone with its head facing a closed arm.During the 5-min exposure, the following parameters for anxiety-related behaviour were recorded with a video ⁄ computer system (Plus-maze V2.0, Ernst Fricke, Germany, 1993): the percentage of entriesinto the open arms, the percentage of time spent on the open arms, thelatency until first entry into an open arm and the number of full entriesinto the open arms. The number of entries into the closed arms wasrecorded as a parameter for locomotor activity.

Hormone analysis

In order to evaluate the effects of elevated brain PRL levels on the(re)activity of the HPA axis and the sympathetic nervous system, theplasma hormone concentrations of ACTH, corticosterone and norad-renaline were estimated radioimmunologically in trunk blood from

Prolactin attenuates neuronal stress circuitries in virgin rats 1805

ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 25, 1804–1814

unstressed rats and stressed rats immediately after exposure to 30 minof restraint. Plasma ACTH and corticosterone concentrations weremeasured using commercially available kits (ICN Biomedicals, CostaMesa, CA, USA) with a sensitivity of 10 pg ⁄ mL and 25 ng ⁄ mL,respectively. Plasma noradrenaline was measured using a commercialkit with a detection limit of 0.3 ng ⁄ mL (IBL Immuno BiologicalLaboratories, Hamburg, Germany).In order to exclude significant diffusion of i.c.v. PRL into blood,

plasma PRL concentration was measured via a non-species-specific(human, rat, mouse, sheep) enzyme-amplified sensitivity immunoas-say (EASIA; BioSource Europe S.A., Belgium) with a sensitivity of11 lIU ⁄ mL (lIU ¼ micro international units, with 53 lIU equivalentto 2.5 ng PRL). To confirm OVX and E2 replacement, plasma E2

concentration was estimated using a radioimmunoassay with asensitivity of 4.7 pg ⁄ mL (Diagnostic Systems Laboratories, Webster,Texas, USA).

Tissue preparation and cryosectioning

Series of coronal cryostat sections (16 lm) from relevantforebrain regions (Cullinan et al., 1996; Windle et al., 2004) betweenbregma ) 0.3 mm and ) 4.9 mm (Paxinos & Watson, 1998) weremounted onto superfrost slides for in situ hybridization. Sections wereplaced in dry boxes with silica gel and stored at ) 80 �C until analysis.For analysis of c-fos mRNA expression, brain areas of interest were

chosen based on the localization of PRL receptor expression (Grattanet al., 2001) and the sites of restraint-induced increases in c-fosexpression (Kononen et al., 1992; Mohammad et al., 2000; Tan &Nagata, 2002; Fujioka et al., 2003; Windle et al., 2004). Sectionsincluded the following brain regions: piriform cortex, central (CeA)and medial (MeA) amygdala, bed nucleus of the stria terminalis(BNST), lateral septum, habenula, medial preoptic area (MPOA),supraoptic nucleus (SON), PVN, arcuate nucleus, paraventricularthalamus, anterodorsal and anteroventral thalamus (AD ⁄ AV), centro-medial and paracentral thalamus, dorsomedial hypothalamus, ventro-medial hypothalamus, dentate gyrus and pyramidal projection areas(CA1, CA2, CA3) of the dorsal and ventral hippocampus.For analysis of CRF mRNA expression, sections from the

hypothalamic PVN, the main site of CRF synthesis, were analysed(bregma ) 1.0 mm to ) 2.2 mm).

In situ hybridization of CRF and c-fos mRNA

In order to detect chronic PRL effects on basal and restraint-inducedCRF mRNA expression, a highly specific, 48-mer 35S-labelledoligonucleotide probe (5¢-GGC-CCG-CGG-CGC-TCC-AGA-GAC-GGA-TCC-CCT-GCT-CAG-CAG-GGC-CCT-GCA-3¢) (Bosch et al.,2006), complementary to bases 64–111, was used. Two sets of slides,each carrying six PVN sections per animal, were processed. For c-fosmRNA detection, a 45-mer 35S-labelled oligonucleotide (5¢-GCA-GCG-GGA-GGA-TGA-CGC-CTC-GTA-GTC-CGC-GTT-GAA-ACC-CGA-GAA-3¢) (Erdtmann-Vourliotis et al., 1999), complement-ary to bases 141–185, was used. One set of slides with six brainsections per slide and per animal, targeting the brain areas of interest,was used.In situ hybridization was performed using an established protocol

(DeVries et al., 1994). Briefly, sections were postfixed in 4%paraformaldehyde, washed in phosphate-buffered saline, acetylatedand dehydrated through a series of graded ethanol. Followingchloroform fixation and another ascending ethanol row, slides wereair-dried before prehybridization in hybridization buffer (50% w ⁄ v

deionized formamide, 20% w ⁄ v dextran sulphate, 1 · Denhardt’s,8 mm dithiothreitol and 2 mg ⁄ mL transfer RNA) for 2 h at 50 �C.Slides were then washed twice in 2 · sodium chloride ⁄ sodium citrate-buffer (SSC) and dehydrated in ethanol. The 35S-labelled oligonucle-otide was applied to each section at a concentration of 106 cpm perslide in 200 lL hybridization solution in which transfer RNAconcentration was reduced to 500 lg ⁄ mL. After overnight hybridiza-tion at 50 �C, the slides were washed three times for 15 min in1 · SSC at 50 �C and once in 1 · SSC at room temperature. Slideswere further processed when cooled down to room temperature anddehydrated with ethanol. Hybridized and processed sections wereexposed to Biomax MR film (Kodak, Rochester, NY, USA). Underoptimized conditions, the duration of exposure amounted to 7 days forCRF and 18 days for c-fos. Using matrices in the shape of the brainregions of interest, mRNA expression levels were determined bymeasuring the light transmittance of the film in pictures ofanatomically matched sections. This was accomplished by thesoftware ImageJ (version 1.31, National Institute of Health,http://rsb.info.nih.gov/ij/), which automatically converts the measuredtransmittance into the more convenient unit of optical density (OD).To yield values for specific binding, background activity wassubtracted. A minimum of three OD values constituted the averagevalue per brain region and individual animal.

Statistical analysis

Experimental subjects were included in the statistics only if thelocalization of the i.c.v. cannula was verified during cryosectioning.All data are expressed as the group mean ± SEM, and were eitheranalysed by one-way anova (factor treatment) followed by Tukey’spost hoc test (behavioural data, plasma PRL, plasma E2, delta BW,adrenal weight, plasma corticosterone ⁄ adrenal weight ratio) or bytwo-way anova (factor restraint, factor treatment) followed byBonferroni’s post hoc test (plasma ACTH, corticosterone, noradre-naline, data from in situ hybridization) where appropriate. Inexceptions, the non-parametric Mann–Whitney-U-test was used forpairwise data comparison where indicated. Results were consideredsignificant at P ¼ 0.05. All statistics were performed using thecomputer software SPSS 12.0 for Windows.

Results

Effects of i.c.v. PRL on anxiety-related behaviour

As revealed on the EPM, chronic i.c.v. PRL significantly reduced theanxiety-related behaviour in a dose-dependent manner (F3,85 ¼ 3.65;P ¼ 0.02; Fig. 1), as reflected by an increase in the percentage of timespent on the open arms of the EPM in rats chronically i.c.v. treatedwith the highest dose of PRL (1 lg ⁄ h). Importantly, i.c.v. PRL did notalter the locomotor activity of the animals (entries into closed arms;Fig. 1).

Effects of i.c.v. PRL on BW, adrenal weight and basal plasmacorticosterone

Dose-independently, chronic PRL treatment caused a significantincrease in BW gain compared with vehicle-treated and OVXE2

animals (F4,91 ¼ 3.32, P ¼ 0.01; Table 1). Consequently, the relativeweight of the adrenal glands (mg tissue ⁄ g BW) was reduced in PRL-treated rats (F4,91 ¼ 2.05, P ¼ 0.09), but a significant differencebetween rats treated with the highest dose of PRL (1 lg ⁄ h) and

1806 N. Donner et al.


vehicle-treated animals could only be revealed using non-parametricstatistics (MWU: P ¼ 0.03; Table 1). Also, chronic i.c.v. PRL resultedin an upregulation of adrenal corticosterone secretion under basalconditions, as reflected by an elevated ratio of plasma corticosteroneconcentration and absolute adrenal glands weight (F3,66 ¼ 3.05,P ¼ 0.03; Table 1).

Effects of i.c.v. PRL on hormonal responses to restraint

Chronic i.c.v. PRL treatment, to varying degrees, generally tended toreduce the neuroendocrine response of relevant systems including theHPA axis (ACTH, corticosterone) and the sympathetic nervous system(noradrenaline) in response to restraint. In detail, compared withunstressed rats, plasma ACTH concentrations were found to beelevated at 30 min of restraint in both vehicle and PRL (1 lg ⁄ h)-treated rats (factor restraint: F1,35 ¼ 169; P < 0.0001; Fig. 2A).Although PRL treatment did not significantly alter absolute plasma

ACTH levels, the stress-induced percentage increase in ACTHsecretion was blunted in PRL-treated rats (vehicle: 713 ± 71.0%;PRL: 418 ± 41.5%; P ¼ 0.008). In parallel to the ACTH response,plasma corticosterone was elevated by restraint in all groups ofvehicle- and PRL-treated rats (F1,34 ¼ 48.6; P < 0.0001; Fig. 2B).Again, PRL treatment did not alter absolute corticosterone concentra-tions, but lowered the percentage increase in corticosterone secretionafter restraint (vehicle: 386 ± 41.1%; PRL: 223 ± 12.7%; P ¼ 0.001).Plasma noradrenaline was significantly elevated at 30 min of

restraint in vehicle-treated rats only (F1,37 ¼ 10.5; P ¼ 0.002). Incontrast, the stress response of noradrenaline was not significant inPRL-treated animals (Fig. 2C).In all rats that were killed 180 min after termination of restraint,

plasma concentrations of ACTH, corticosterone and noradrenaline hadreturned to basal levels equal to those of the unstressed controlanimals.Chronic infusion of PRL at varying doses into the right lateral

ventricle did not alter plasma PRL concentrations measured underbasal, unstressed conditions (F4,93 ¼ 1.81, P ¼ 0.13, Table 1),excluding the possibility of diffusion of significant amounts of i.c.v.PRL into the blood circulation. Moreover, as PRL and PRL receptorexpression are sensitive to changes in E2 concentration (Neill et al.,1971; Shamgochian et al., 1995; Pi & Grattan, 1998b), we quantifiedplasma E2 concentrations in all groups. OVX and E2 replacementresulted in similar E2 concentrations resembling physiological levels(Table 1) of cycling proestrus rats (Neill et al., 1971) throughtout allgroups.

Effects of i.c.v. PRL on c-fos mRNA expression

Under basal non-stressed conditions, a low level of c-fos mRNAexpression (grey density units between 0 and 10) was found in allregions of both vehicle- and PRL-treated rats, except the piriformcortex (grey density: 15.3), the AD ⁄ AV (14.2), and the pyramidal celllayers CA1 (12.0) and CA2 (10.4) of the ventral hippocampus.Chronic PRL treatment (1 lg ⁄ h) reduced the basal level of c-fosmRNA expression in some regions, including the CA1 region of theventral hippocampus (P ¼ 0.03) and the CeA (P ¼ 0.03), comparedwith vehicle treatment (Table 2, Fig. 3). In contrast, higher basal levelsof c-fos mRNA expression were found in the SON after PRL treatment(P ¼ 0.04; Table 2, Fig. 3).In vehicle-treated animals exposed to 30-min restraint, a significant

increase in c-fos mRNA expression was found in various brain regions

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Fig. 1. Effect of i.c.v. prolactin (PRL) infusion on behavioural parameters onthe EPM. All rats were virgin, ovariectomized and OVXE2. Groups were eitherchronically i.c.v. treated with PRL (0.01, 0.1 or 1.0 lg ⁄ 0.5 lL ⁄ h) or vehicleover 5 days via osmotic minipumps. Chronic i.c.v. PRL decreased the anxiety-related behaviour in a dose-dependent manner as shown by the increasedpercentage of time spent on the open arms of the EPM. The number of entriesinto the closed arms representing locomotor activity was not altered by i.c.v.PRL. Data are means + SEM. *P < 0.05 vs. vehicle. Numbers in parenthesesindicate group size.

Table 1. Effects of chronic i.c.v. treatment with oPRL on physiological parameters

OVXE2

(n ¼ 8)Vehicle(n ¼ 31 or 32)

PRL

0.01lg/h (n ¼ 11) 0.1lg/h (n ¼ 12) 1.0lg/h (n ¼ 30–34)

Plasma-E2 (pg/mL) 65.0 ± 4.8 57.3 ± 3.9 57.2 ± 11.6 58.1 ± 4.5 61.8 ± 3.6Plasma-PRL (lIU/mL) 50.8 ± 3.2 55.8 ± 1.3 56.2 ± 2.6 52.2 ± 1.8 52.2 ± 1.1DBW (g) 4.5 ± 3.5 3.5 ± 1.7 12.3 ± 2.1* 11.7 ± 2.6* 9.3 ± 1.6*Adrenal glands (mg/g BW) 0.33 ± 0.01 0.32 ± 0.01 0.33 ± 0.02 0.30 ± 0.02 0.29 ± 0.02*MWU.

Corticosterone (ng/mL)/ – 4.21 ± 0.8 2.57 ± 1.1 4.34 ± 1.4 6.89 ± 1.0*mg adrenal glands – (n ¼ 22) (n ¼ 10) (n ¼ 11) (n ¼ 27)

Virgin female rats were ovariectomized, E2-substituted (OVXE2) and treated with vehicle or prolactin (PRL) at three different doses (0.01, 0.1 or 1.0 lg/0.5 lL/h)via an osmotic minipump connected to an i.c.v. infusion cannula over 5 days. The OVXE2 control group did neither receive stereotaxic surgery nor i.c.v. treatment.Except for the corticosterone synthesis rate, data from unstressed and stressed animals were pooled, as no restraint effect was seen. Plasma E2 and PRL werequantified in trunk blood collected 5 days after i.c.v. treatment. Body weight (BW) was taken on the day of OVX and 4 days after the onset of i.c.v. treatment (afterthe EPM test). The weight of both adrenal glands was estimated 5 days after i.c.v. treatment and is presented in relation to the BW. Plasma corticosteroneconcentrations of unstressed rats are presented in relation to absolute adrenal gland tissue weight, thus indicating adrenal production or synthesis rate. Data aremeansSEM. *P < 0.05 vs. vehicle. Numbers in parentheses indicate group size. MWU, Mann–Whitney U-test.



listed in Table 2. Among the brain areas with the strongest signals(P < 0.0001) were the BNST, the lateral septum, the MPOA, the SON(Fig. 3), the PVN (Figs 3 and 4), and the paraventricular and AD ⁄ AVthalamus.In PRL-treated rats, the restraint-induced rise in c-fos mRNA

expression was found to be reduced within the PVN (P < 0.0001) andthe AD ⁄ AV thalamus (P ¼ 0.01) compared with vehicle-treatedrestraint rats (Table 2, Figs 3 and 4). Moreover, within the dentategyrus and the CA3 of the dorsal hippocampus, the c-fos signal foundin vehicle-treated rats was absent in the PRL group. In contrast, asignificant stress-induced c-fos expression within all pyramidalprojection layers (CA1, CA2 and CA3) of the ventral hippocampuswas exclusively found in PRL-treated, but not in vehicle-treated,animals (for statistical details, see Table 2).All anova comparisons of c-fos mRNA expression presented in

Table 2 and Fig. 3 include the OVXE2-group that received neitherstereotaxic surgery nor i.c.v. treatment. OVXE2 animals in thisadditional control group did not differ from corresponding i.c.v.vehicle-treated animals, confirming that the surgical interventionper se did not alter c-fos expression.

Effects of i.c.v. PRL on CRF mRNA expression within the PVN

Exposure to restraint significantly altered the expression of CRFmRNA within the hypothalamic PVN, an effect that was found todepend on i.c.v. treatment (factors restraint · treatment: F1,36 ¼ 5.52,P ¼ 0.02; Fig. 5). In detail, in vehicle-treated rats, restraint signifi-cantly elevated CRF mRNA expression within the PVN, whereas thiseffect was lost after PRL treatment. Consequently, the restraint-induced rise in hypothalamic CRF mRNA expression was signifi-cantly blunted in PRL-treated (1 lg ⁄ h) compared with vehicle-treatedrats (P < 0.0001). The basal expression of CRF mRNA did not differbetween treatment groups.

Discussion

The present study revealed that a state of induced hyperprolactinaemiawithin the brain reduced the general stress responsiveness in virgin

female, OVX rats. Central i.c.v. infusion of PRL over 4–5 daysreduced anxiety-related behaviour on the EPM. Moreover, in responseto restraint, CRF mRNA and c-fos mRNA expression was signifi-cantly attenuated within the hypothalamic PVN of PRL-treated rats.With respect to hormonal parameters, the blunted stress responsive-ness after chronic PRL was reflected by a reduced restraint-inducedrise in plasma noradrenaline, whereas plasma ACTH and corticoster-one responses were only marginally altered. These findings provideevidence that central PRL is a significant regulator of relevant stresssystems of the brain. Moreover, activation of the brain PRL system,resulting in elevated brain PRL, is likely to be involved in theinhibition of neuroendocrine and neuronal stress circuitries as seen inthe peripartum period.PRL has recently been shown to exert acute anxiolytic actions both

in male, virgin female and lactating rats (Torner et al., 2001, 2002).Here, we demonstrate that chronic i.c.v. PRL treatment reducedanxiety of virgin rats in a dose-dependent manner. Interestingly, thereduced level of anxiety of PRL-treated rats was accompanied byreduced c-fos mRNA levels within the CeA under basal conditions.Therefore, it is likely that central PRL inhibited brain regions thatparticipate in the generation and regulation of fear and anxietyresponses, such as the CeA (Davis, 1992; Liebsch et al., 1995;LeDoux, 2000; Bale et al., 2001; Dayas et al., 2001; Blair et al., 2005;Wand, 2005).Moreover, changes in plasma PRL concentrations have been

repeatedly discussed in the context of emotionality (Baumgartneret al., 1988; Reavley et al., 1997; Steimer et al., 1997; Landgraf et al.,1999), and it has been shown that pathological hyperprolactinaemia isoften found in depressed patients (Mendlewicsz et al., 1980; Favaet al., 1981; Thienhaus & Hartford, 1986; Reavley et al., 1997). Theextent to which systemic PRL contributes to the regulation ofemotionality including anxiety-related behaviour remains to be shown,as selective uptake of PRL from blood into the brain has beendescribed (Walsh et al., 1987).Chronic PRL treatment attenuated neuronal responses to restraint in

a regionally specific manner. Within the PVN, chronic PRLsuppressed the restraint-induced expression of both c-fos and CRFmRNA. The expression patterns of c-fos and CRF are linked as c-Fos-protein acts as a nuclear transcription factor within the parvocellular

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Fig. 2. Effects of chronic i.c.v. prolactin (PRL) infusion on (a) plasma adrenocorticotropin (ACTH), (b) corticosterone and (c) noradrenaline concentrationsunder basal control conditions (C) and at 30 min after 30-min exposure to restraint stress (S). OVXE2 rats were chronically i.c.v. treated with PRL(1.0 lg ⁄ 0.5 lL ⁄ h, black bars) or vehicle (open bars) via osmotic minipumps over 5 days. The significant restraint-induced rise in plasma noradrenaline found invehicle-treated rats was absent after PRL treatment. Data are means + SEM. Numbers in parentheses indicate group size. ##P < 0.01 vs. unstressed controls (anova,followed by Bonferroni’s post hoc test).



neurons of the PVN, activating CRF mRNA expression (Viau &Sawchenko, 2002). C-fos mRNA was exclusively detected within theparvocellular part of the PVN and thus matched the neuroanatomicalexpression pattern of CRF. The lower c-fos mRNA expression withinthe PVN was also confirmed by fewer restraint-induced c-Fos-immunopositive nuclei in PRL-treated animals of the same experiment(data not shown).

As a marker for early neuronal activation (Sheng & Greenberg,1990; Smith et al., 1992; Hoffmann et al., 1993) and in support ofrecent studies (Kononen et al., 1992; Melia et al., 1994; Chowdhuryet al., 2000; Mohammad et al., 2000; Tan & Nagata, 2002; Fujiokaet al., 2003; Windle et al., 2004), c-fos was also found to be expressedin various telencephalic, diencephalic and hippocampal brain regionsin response to restraint (Table 2). Besides the PVN, a highlysignificant restraint-induced c-fos expression was found in the lateralseptum, the MPOA, the SON and the paraventricular and AD ⁄ AVthalamus. However, with respect to neuronal activation within theamygdala, we could not confirm the discriminative nature ofsubnuclear amygdala responses to psychological and physical stimuli,as described by Chen & Herbert (1995), Dayas et al. (2001) andWindle et al. (2004). In contrast, we found stress-related neuronalsignals within both the CeA and the MeA. Accordingly, we alsodetected a strong restraint-induced c-fos signal within the BNST, anarea through which amygdala signals are relayed to the hypothalamus.

The effects of PRL on restraint-induced neuronal activation werefound to be regionally dependent. While some brain regions continu-

ously showed a similar c-fos response despite reduced hypothalamicCRF and hormonal stress responses (e.g. piriform cortex, BNST, lateralseptum), a parallel reduction of the c-fos response was found, forexample, in the AD ⁄ AV thalamus. This indicates that PRL may inhibitthalamic relay nuclei, which integrate signals of stress perceptionbefore they are transmitted to layers of upper sensory cortex. Moreover,restraint-induced neuronal activation within the CA3 and the dentategyrus of the dorsal hippocampus was blocked by PRL. Those regionsnot affected by central PRL were also recently found to show robustand non-discriminative stress responses (Da Costa et al., 1996; Windleet al., 2004). It is therefore suggested that such regions are locatedupstream of PRL-sensitive gating mechanisms.The reduced c-fos expression in the ventral hippocampus (CA1)

found under basal, unstressed conditions suggests modulation ofinputs to the hippocampus by PRL. Surprisingly, all CA areas of theventro-hippocampal pyramidal projection layers showed a stressresponse after PRL treatment, but not after vehicle treatment. Furtherexperiments are necessary to show whether this relates to potentialeffects of PRL on memory consolidation of stressful stimuli and onstress-related behavioural parameters within the hippocampus (Shingoet al., 2003). The reduced basal c-fos expression within the CeA ofPRL-treated animals also suggests an influence of central PRL onother emotional behaviours such as fear and aggression (Davis, 1992;Liebsch et al., 1995; Haller et al., 2006).Central PRL infusion also altered the secretion of ACTH and

corticosterone. Interestingly, basal secretion tended to be elevated after

Table 2. P-values from the statistical analysis of the effects of chronic central oPRL on acute restraint-induced c-fos mRNA expression in forebrain areas

Vehicle andrestraint

PRL

Peptide (basal) Restraint Interaction

TelencephalonPiriform cortex 0.005(F1,28 ¼ 9.49) NS 0.002(F1,28 ¼ 12.1) –Central amygdala 0.033(F1,27 ¼ 5.22) 0.032 ()) (F1,27 ¼ 5.30) 0.008(F1,27 ¼ 8.52) –Medial amygdala 0.012(F1,28 ¼ 7.38) NS <0.0001(F1,28 ¼ 19.0) –BNST <0.0001(F1,28 ¼ 48.4) NS <0.0001(F1,28 ¼ 19.2) –Lateral septum <0.0001 (F1,28 ¼ 16.4) NS 0.001(F1,28 ¼ 14.2) –

DiencephalonHabenula 0.017(F1,27 ¼ 6.54) NS 0.05 (F1,27 ¼ 4.04) –MPOA <0.0001(F1,28 ¼ 31.3) NS <0.0001(F1,28 ¼ 25.9) –SON <0.0001F1,29 ¼ 43.2) MWU: 0.043(+) <0.0001(F1,29 ¼ 35.1) –PVN <0.0001(F1,29 ¼ 144) NS <0.0001(F1,29 ¼ 32.9) <0.0001 ()) (F1,29 ¼ 15.3)Arcuate nucleus 0.003(F1,28 ¼ 10.4) NS 0.001(F1,28 ¼ 12.8) –Paraventricular thalamus <0.0001(F1,28 ¼ 25.8) NS 0.016(F1,28 ¼ 6.54) –AD/AV thalamus <0.0001(F1,29 ¼ 29.7) NS 0.046(F1,29 ¼ 4.34) 0.009 ()) (F1,29 ¼ 5.22)Centromedial and paracentral thalamus 0.002(F1,28 ¼ 12.1) NS 0.003(F1,28 ¼ 10.7) –Dorsomedial hypothalamus 0.001(F1,28 ¼ 12.6) NS 0.005(F1,28 ¼ 9.06) –Ventromedial hypothalamus 0.012(F1,28 ¼ 7.13) NS 0.005(F1,28 ¼ 9.28) –

Dorsal hippocampusCA1 NS NS NS –CA2 NS NS NS –CA3 0.035(F1,28 ¼ 4.89) NS NS –Dentate gyrus 0.035(F1,28 ¼ 4.93) NS NS –

Ventral hippocampusCA1 NS 0.027 ()) (F1,25 ¼ 4.58) 0.004(F1,25 ¼ 10.1) –CA2 NS NS 0.004 (F1,25 ¼ 10.3) –CA3 NS NS 0.04(F1,25 ¼ 4.70) –

OVXE2 rats were chronically i.c.v. treated with prolactin (PRL; 1.0 lg/0.5 lL/h) or vehicle. After 5 days, controls remained in their home cages and stress groupswere exposed to 30-min restraint and decapitated immediately afterwards (within 1 min). Columns show P-values from pairwise post hoc Bonferroni tests (based ontwo-way anova; factors restraint and treatment), i.e. effects of restraint in vehicle-treated rats, and effects of PRL treatment on basal (peptide effect) or restraint-induced c-fos mRNA levels. A separate anova including the groups OVXE2-C, OVXE2-S (P-values not shown, no difference from vehicle groups), vehicle-C,vehicle-S, PRL-C and PRL-S was conducted for each brain area. AD/AV, anterodorsal/anteroventral thalamus; BNST, bed nucleus of the stria terminalis; CA, cornuammonis; MWU, Mann–Whitney U-test; PVN, paraventricular nucleus; SON, supraoptic nucleus. Expression vs. corresponding vehicle group (), decreased; +,increased). NS, not significant.



chronic PRL with the consequence that, despite similar peak values,the percentage increase in ACTH and corticosterone secretion torestraint became blunted. It is likely to conclude that the inhibitedneuronal stress responses within the PVN contributed to this effect.However, while a restraint-induced increase in CRF mRNA expres-sion within the PVN was virtually absent in PRL-treated rats, ACTHand corticosterone stress responses were still found to be significant,although attenuated. This observation could reflect a graded inhibitoryeffect of PRL on the HPA axis, and claims further investigation inorder to measure PRL effects on actual CRF peptide content andrelease, as well as a more gentle method of blood sampling to measureplasma ACTH and corticosterone responses.Chronic i.c.v. PRL also increased the basal adrenal corticosterone

production ⁄ secretion rate, calculated as the ratio of the basalcorticosterone concentration and the absolute adrenal weight.Although we cannot completely exclude a direct effect of PRL on

adrenal cells, attributable to PRL leakage from the ventricular system,a central, still unknown, mechanism is more likely. In support, plasmaPRL concentrations were almost identical in all treatment groups, andadrenal cells have been shown to be PRL receptor-negative (Bole-Feysot et al., 1998).Interestingly, the restraint stress-induced rise of plasma noradren-

aline was suppressed in PRL-treated animals. This could reflect aninhibitory effect of PRL on the sympathoadrenal axis. In this context itis of interest to note that a generally reduced activity of thesympathomedullary system has been described in pregnancy (Doug-las, 2005) and in lactation (Altemus et al., 1995). It is thus reasonableto suppose that brain PRL directly contributes to the central regulationof the sympathetic nervous system, possibly acting on hypothalamicand ⁄ or brainstem nuclei. For example, PRL could affect inhibitorysympathetic preganglionic neurons of the brainstem, e.g. those of therostroventrolateral reticular nucleus, with projections to sympathoad-

c-fo

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Fig. 3. Effects of central prolactin (PRL) infusion on c-fos mRNA expression in the paraventricular nucleus (PVN), the supraoptic nucleus (SON) and the centralamygdala (CeA) of non-stressed control animals (C) and in response to 30 min restraint (S). Animals were ovariectomized, E2 substituted (OVXE2 controls,n ¼ 4 ⁄ 4) or additionally i.c.v. treated with vehicle (n ¼ 8 ⁄ 7) or PRL (1.0 lg ⁄ 0.5 lL ⁄ h; n ¼ 6 ⁄ 6). Restraint resulted in a significant increase in c-fos mRNAexpression in all brain regions shown. PRL treatment reduced the c-fos stress response within the PVN and the basal neuronal activity within the CeA, whereas anelevated c-fos expression was found in the SON of unstressed PRL-treated rats. Data are means + SEM. #P < 0.05, ##P < 0.01, vs. respective C-group. *P < 0.05,**P < 0.01 vs. corresponding vehicle group (two-way anova followed by Bonferroni post hoc test): *aP < 0.05 vs. vehicle-C.

pm

dp

mpv

mpddpv

mpdv3V

PVN Vehicle S PRL S

3V

Fig. 4. Left side: organization of the paraventricular nucleus of the hypothalamus (PVN) with special reference to the sites of CRF synthesis within the medialparvocellular (mp) part (mpdd, dorsal mp division; mpdv, ventral mp division). Other subdivisions: posterior magnocellular part (pm), periventricular part (pv) andautonomic-related areas (dp) (as suggested by Swanson & Simmons, 1989). Right side: original images from c-fos mRNA responses to restraint within the PVN ofvehicle-treated (Vehicle S) or chronically PRL-treated animals (PRL S). No basal c-fos mRNA expression was detected in the PVN (no picture). 3V, third ventricle.Scale bar: 250 lm.



renal neurons (Sun, 1995). Also, the possibility of an indirectinhibitory effect of brain PRL on PVN neurons via noradrenergicafferents originating in the brainstem cannot be excluded. At first,however, another experimental schedule allowing more frequent bloodsampling is needed in order to thoroughly investigate PRL effects onthe rapid rise of plasma noradrenaline in response to an acute stressor.

It is important to mention that in OVXE2 rats, PRL receptors havebeen localized in those brain regions involved in stress regulation,including the hypothalamic SON, MPOA, arcuate nucleus andhippocampus (Pi & Grattan, 1998b). Expression of the long form ofthe PRL receptor was found to be transiently upregulated by acutestress in the ventromedial hypothalamus, the PVN and the choroidplexus (Fujikawa et al., 1995) and ⁄ or during female reproduction(Sugiyama et al., 1994; Grattan et al., 2001; Grattan, 2002; Augustineet al., 2003; Kokay & Grattan, 2005). Moreover, we could recentlyreveal PRL receptor-mediated activation of intracellular signallingcascades within the hypothalamus, specifically within the PVN andSON (Blume et al., 2006). It is thus likely that the PRL effects weobserved are mediated through local PRL receptors.

It is well accepted that the rat stress response as well as anxietybehaviour is regulated by gonadal steroid hormones (Dayas et al.,2000; Popeski et al., 2003; Adachi et al., 2005; Lund et al., 2005;Ueyama et al., 2006). As seen for the oxytocin system (Windle et al.,2006), it is also likely that these hormones interact with PRL effects.For this first study, we therefore utilized an established standardprotocol that excluded cycle-dependent effects, but maintainedconstant proestrus concentrations of E2. Low E2 levels were intendedto ensure constant PRL receptor expression in the brain (Mustafaet al., 1995; Shamgochian et al., 1995). Future studies will have toreveal whether PRL itself without the presence of estrogens issufficient to exert effects similar to those seen in the present study. Inrespect of our aims, however, it would have exceeded the norms ofanimal ethics to include more than two control groups.

The present demonstration of PRL-mediated suppression of hormo-nal stress responses and associated neural circuitries has significantimplications for the regulation of stress responses, particularly in theperipartum period. Indeed, the emotional changes following chronic

i.c.v. PRL infusion, including reduced anxiety, are similar to thosedescribed in pregnant or lactating animals (Picazo & Fernandez-Guasti,1993; Toufexis et al., 1999; Neumann, 2001; Torner et al., 2002) andhumans (Altemus et al., 1995; Asher et al., 1995; Heinrichs et al.,2001). Moreover, attenuated neuronal responses to an acute stressorwithin the PVN and other relevant brain regions were described bothduring lactation (Da Costa et al., 1996) and in our study followingchronic i.c.v. PRL. Likewise, the expression of CRF mRNAwithin thePVN (Johnstone et al., 2000; Da Costa et al., 2001) and, consequently,the neuroendocrine output of the HPA axis in response to acute stressare reduced in the peripartum period (Stern et al., 1973; Lightman &Young, 1989; Altemus et al., 1995; Walker, 1995; Windle et al., 1997;Neumann et al., 1998; Toufexis et al., 1999; Heinrichs et al., 2001),whereas basal plasma glucocorticoid concentrations are elevated(Walker, 1995; Neumann et al., 1998, 2001; Douglas et al., 2003).These findings were also, at least partly, confirmed after chronic PRL.Additionally, the elevated basal c-fos activity within the SON afterchronic central PRLmight have physiological relevance as the oxytocinsystem is activated peripartum. Other similarities between chronic PRLtreatment and peripartum adaptations include the blunted response ofthe noradrenergic system to stress (Altemus et al., 1995; Toufexis &Walker, 1996; Douglas et al., 2005).Based on our estimations and a comparison with PRL concentra-

tions within the cerebrospinal fluid (CSF) of pseudopregnant rats(Kishi & Kobayashi, 1984), the highest PRL dose used in this study(1 lg ⁄ h) was most likely to represent physiological peripartum levels.However, we did not monitor CSF PRL contents, nor have exactmeasurements from lactating rats been reported so far. Thus, wecannot guarantee that our protocol successfully mimicked physio-logical PRL concentrations within the CSF of peripartum rats.From our results we can conclude that upregulation of PRL levels

within the brain significantly contributes to the modulation of neuronalcircuits involved in the regulation of the stress responses. Consequently,alterations in the activity of the brain PRL system as seen, for example,peripartum are likely to contribute to neuronal, neuroendocrine andbehavioural stress adaptations, both under physiological and psycho-pathological conditions. However, the findings also raise important

Vehicle C Vehicle S

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Fig. 5. Effects of chronic i.c.v. prolactin (PRL) treatment on c-fos and corticotropin-releasing factor (CRF) mRNA expression in the PVN. Left side: CRF mRNAexpression of non-stressed control (C) and restraint (S) OVXE2 rats chronically i.c.v. treated with PRL (1.0 lg ⁄ 0.5 lL ⁄ h) or vehicle 180 min after termination of30 min restraint. Only in vehicle-treated rats, stress exposure caused a significant increase in CRF mRNA expression (##P < 0.01 vs. C), whereas PRL treatmentblunted the CRF stress response (**P < 0.01 vs. vehicle). Data are means + SEM. Numbers in parentheses indicate group size. Right side: original images fromin situ hybridization; basal (Vehicle C, PRL C) CRF mRNA expression within the PVN vs. responses to restraint (Vehicle S, PRL S). Scale bar: 250 lm.



questions whether similar PRL effects can be seen inmales, andwhethertargeting the brain PRL systemwould be a potential therapeutic strategyfor the treatment of anxiety- or stress-related disorders.

Acknowledgements

The authors are grateful to Drs Annegret Blume and Nicolas Singewald forscientific advice (Fos-immunocytochemistry), and to Mrs Gabi Schindler andLuxiola Gonzales for excellent technical help. This study was supported byDFG (I.D.N.), Volkswagen-Stiftung (I.D.N.) and Studienstiftung des deutschenVolkes (N.D.).

Abbreviations

ACTH, adrenocorticotropin; AD ⁄ AV, anterodorsal and anteroventral (thal-amus); BNST, bed nucleus of the stria terminalis; BW, body weight; CA(1–3),cornu ammonis (pyramidal projection areas of the hippocampus); CeA, centralamygdala; CRF, corticotropin-releasing factor; CSF, cerebrospinal fluid; E2, 17-b-estradiol; EPM, elevated plus-maze; HPA, hypothalamo–pituitary–adrenal;i.c.v., intracerebroventricular; MeA, medial amygdala; MPOA, medial preopticarea; OD, optical density; oPRL, ovine prolactin; OVX, ovariectomy; OVXE2,ovariectomized, estradiol-substituted; PRL, prolactin; PVN, paraventricularnucleus of the hypothalamus; SON, supraoptic nucleus of the hypothalamus;SSC, sodium chloride ⁄ sodium citrate-buffer.

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chronic intracerebral prolactin attenuates neuronal stress circuitries in virgin rats

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